i

SURVEILLANCE OF IN ENVIRONMENTAL ICE AND WATER SAMPLES

Gang Zhang

A dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

December 2007

Committee:

Scott Rogers, Advisor

Robert Midden Graduate Faculty Representative

Carmen Fioravanti

George Bullerjahn

John Castello ii

ABSTRACT

Scott O. Rogers, Advisor

Influenza A virus (IAV) is an important human pathogen. IAV infects humans

and also a variety of other warm-blooded animals, including various domestic and wild

fowl, many domestic and wild mammals. In wild aquatic birds, IAVs primarily are

enteric viruses. IAV may have reached evolutionary stasis in birds. As the primary

reservoir of all IAV subtypes, wild aquatic fowl play an important role in the ecology of

IAV, by maintaining various subtypes of IAV and continuously transmitting genes and

viral strains to other host species. Among wild aquatic birds, transmission of IAV occurs

through the “oral-fecal” pathway. Environmental ice is a good reservoir for preserving

microorganisms alive for long periods of time. In this study, we hypothesized that

environmental ice was a good abiotic reservoir for preserving IAV virions shed by wild

birds. IAV H1 gene sequences were detected in ice and water samples collected from

northeastern Siberian ( Park, Lake Edoma). After cloning and sequencing, 83

unique sequences were derived from a Lake Park ice sample and 1 unique sequence was

derived from a Lake Edoma water sample. Phylogenetic analysis indicated that the

sequences were heterogenous. The sequences shared similarities to IAVs isolated from

humans in the 1930s in and in the 1960s in . Through this study, a

procedure for developing high sensitivity PCR-based methods for virological surveillance of IAV was established. Although no evidence on the viability of the IAV contained in environmental ice and water was obtained, our results indicated that IAV could be preserved alive in the lake ice. This is supported by the fact that IAV RNA fragments of iii more than 600 bp were found in the ice. Therefore, environmental ice might act as the abiotic reservoir for infectious IAV, and possibly many other waterborne viruses.

iv

ACKNOWLEDGMENT

I want to express my sincere appreciation to my major advisor, Dr. Scott O.

Rogers, for his guidance, kindness, and patience. What I learned from him is more than doing research. The days I spend in his lab are an inerasable memory in my life. I also want to express my gratitude to my committee members. Thank you for arranging time to attend my presentations and give valuable suggestions to my research. Thanks for Dr.

Fioravanti for using his equipment. I appreciate Dr. John Castello for his very careful

reading of the dissertation, and a lot of detailed revisions and critical comments. Thanks to my friends and labmates, Seung-Geuk Shin, Vincent Theraisnathan, Ram Satish

Veerapaneni, Tom D’Elia, Lorena Harris, Zeynep Kocer, Farida Sidiq, and America

Vicol for their friendship and the good time we spend together. Special thanks to Seung-

Geuk Shin for the help he gave me when I just joined the lab.

I want to thank my parents for the everlasting love and support. I want to express

my sincere appreciation to my girlfriend Chia-Jui Tsai for her love and support and

understanding.

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TABLE OF CONTENTS

Page

CHAPTER I. LITERATURE REVIEW...... 1

An introduction to IAV...... 3

The structure of an IAV virion...... 4

The life cycle of IAV …...... 16

Influenza pandemics ...... 20

The ecology of IAV ...... 21

Human IAV: appearance, disappearance and reappearance...... 23

The perpetuation of IAV in environmental ice and water ...... 25

The evolution of IAVs ...... 26

Predict the evolutionary trend and upcoming pandemics...... 33

Methods in IAV surveillance...... 34

Phylogenetic analysis methods ...... 35

Literature cited…. …….. …….. ……...... 41

CHAPTER II. SURVEILLANCE OF INFLUENZA A VIRUSES IN

ENVIRONMENTAL ICE AND WATER SAMPLES ...... 58

INTRODUCTION………………...... 59

MATERIALS AND METHODS………………...... 62

Primer design ...... 62

IAV strains ……………………………………………………………………...…. 63

Measurement of primer sensitivity ...... 63 vi

Ice and water samples ...... 63

Processing of samples…………………………………………………………...... 64

RT-PCR, semi-nested/nested-PCR, and sequencing …………………………...... 65

RESULTS………………...... 68

Primer selection………………...... 68

Assays on environmental ice and water samples………………...... 68

DISCUSSION………………...... 72

LITERATURE CITED………………...... 76

CHAPTER III. EVIDENCE OF INFLUENZA A VIRUS RNA IN SIBERIAN LAKE

ICE………………………...... 81

INTRODUCTION……… ...... ……… 82

MATERIALS AND METHODS…...... ……… 84

Ice and water samples… ...... ……… 84

Sample processing…...... ……… 87

Molecular assays…...... ……… 88

Data analysis ...... 90

RESULTS …………...... ……… 91

DISCUSSION……… ………...... ….. 97

LITERATURE CITED…..… ...... ….. 101

vii

CHAPTER IV. SUPPLEMENT………………………...... 106

LITERATURE CITED ...... 110

APPENDIX A. GENBANK ACCESSION NUMBERS.

APPENDIX B. Zhang, G., D. Shoham, D. Gilichinsky, S. Davydov, J. D. Castello, and S.

O. Rogers.2006. Evidence of Influenza A Virus RNA in Siberian Lake Ice. J. Virol.

80(24), 12229-12235.

viii

LIST OF FIGURES

Figure Page

CHAPTER I

1 Diagram of an IAV virion...... 5

2 Conserved ends of vRNA segments ...... 5

3 Schematic representation of the bicistronic mRNA for PB1 and PB1-F2 protein .... 8

4 Schematic representation of HA protein and the cleavage site...... 8

5 Schematic representation for the mRNA and coding region of influenza segment

7…………………………………………………………………………………….. 13

6 Schematic representation for the mRNA and coding region of influenza NS1 and

NS2 proteins ...... 15

CHAPTER III

1 Locations of lakes assayed…………………………………………...... 85

2 Neighbor-joining phylogram of the influenza virus hemagglutinin H1 gene

sequences from Lake Park ice (collected in March 2002) and Lake Edoma water

(collected in September 2001)…… ...... 92

3 Maximum parsimony phylogram of a wide selection of hemagglutinin H1 gene

sequences………...... 94

ix

LIST OF TABLES

Table Page

CHAPTER II

1 Primers selected for detection of IAV genes……...... 67

2 RT-PCR assays on northeastern Siberian lake ice and lake water samples...... 69

3 RT-PCR assays on glacial ice samples and water samples collected in ...... 70

CHAPTER III

1 assayed or to be assayed for the presence of influenza A virus...... 86

CHAPTER IV

1 Sample collected from the lakes in north-eastern (Kolyma lowland)……. .. 107

1

CHAPTER I LITERATURE REVIEW

Influenza A virus (IAV) is the causative agent of the “flu”. Seasonal influenza

epidemics often take place in the fall and winter seasons in different locations worldwide,

affecting 10-20% of the human population (WHO, 2003) and threatening the

immunologically naïve patients. Each year in the USA alone, IAV infects 5-20% of the

total population, hospitalizes more than 200,000 and kills about 36,000. If an

unpredictable IAV pandemic emerges, up to millions of people could be killed and huge

economic loss could be induced. In the 20th century, human society was attacked by 4 serious global flu pandemics. The first one and the most notorious one, the “Spanish” flu,

arose in 1918. Nearly 20% of the world’s total population were infected during this

pandemic; and up to 50 million people were killed within months (Niall et al. 2002). The

“Spanish” flu was later determined to be an H1N1 strain (containing an H1 subtype of the

hemagglutinin gene and an N1 subtype of the neuraminidase gene) (Reid et al. 1999;

Reid et al. 2000). Following that, human society was attacked by the “Asian” flu (H2N2)

in 1957, the “Hong Kong” flu (H3N2) in 1968, and the “Russian” flu (H1N1) in 1977.

The first isolation of IAV from humans was made in 1933. As an important

human pathogen and a relatively simple virus, IAV has received extensive studies. Much

information about the virus has been revealed, including the structure, genome, life-cycle,

ecology, and pathogenicity. However, there are mysteries about this virus waiting to be

solved. It is still unknown as to why some influenza viruses are more virulent than others,

how an IAV pandemic becomes established, and why and how some influenza viruses

reenter the human population after many years of disappearance. Answering these 2

questions relies on further extensive studies (Peter Palese, 2004; Fodor, Devenish et al.

1999).

Previous studies have shown that environmental ice is a good reservoir for various

microorganisms, including fungi, bacteria and viruses, keeping them alive for extended

periods of time. Viable microbes, including pathogenic ones entrapped in environmental

ice, could reenter the contemporary microbial population once they were released from

the ice, and they might confer certain selective advantages because modern hosts are

immunologically naïve to them Rogers et al. (2004). Smith et al. (2004) proposed that

environmental ice might have acted as repositories in the recycling of human pathogenic

viruses, specifically caliciviruses, influenza viruses, and enteroviruses.

This dissertation presents results of studies of the surveillance of influenza A

virus in environmental ice and water samples. These studies probe into the question of

whether IAV could be preserved in various environmental ices for extended periods of

time. Specifically, ice and water from lakes in the Kolyma lowland in northeastern

Siberia were sampled. Those lakes are visited annually by large quantities of migratory

wild aquatic birds. Through the oral-fecal transmission route, wild birds infect others

with IAV they are carrying, and deposit numerous viral particles into the waters. IAV

virions are capable of persisting long in cold water and ice, and are resistant to freeze- and-thaw effects. It is not yet clear if these viruses can survive the periods of time when there are no susceptible hosts present. If they can, then when new flocks of avian hosts become available, the viruses have a chance to reenter the biotic cycle. If so, these lakes have acted as abiotic reservoirs for IAV. Additionally, ancient glacial ice collected from

Greenland and , as well as ice and water collected from a pond (41.383333, - 3

83.618056) in Bowling Green, Ohio, were assayed in this study. I hypothesize that ice acts as an abiotic reservoir (long-term or short-term) for preservation of influenza viruses.

Through this study, I wanted to address the following questions:

1) Can influenza genomic RNA sequences be retrieved from environmental ice and water

samples?

2) If yes, then what subtypes of IAV are present?

3) What is the relationship between the isolated sequences and other influenza sequences?

An introduction to IAV

IAV is an RNA virus belonging to the family. The viral family of Orthomyxoviridae contains 5 genera of viruses. Among them, three are termed influenza viruses, specifically IAV, influenza B virus (IBV), and influenza C virus (ICV).

The 3 types of viruses have significant differences in their genomes, host ranges, pathogenicity, and evolutionary patterns, although they are evolved from one common ancestor. IAV infects a wide range of warm-blooded animals, including humans, pigs, horses, many sea mammals, feline and canine animals, domestic fowl, and various wild birds (Webster et al. 1992). IBV and ICV are mainly found in human beings, with rare infections in other animals. IAV generally causes diseases of severe symptoms in its

hosts, and is capable of causing epidemics/pandemics occasionally. In contrast, the

diseases caused by IBV and ICV generally are much milder. The evolution of IAV genes is very active, characterized by high evolutionary rates and diverged evolutionary patterns. Differently, IBV and ICV genes are evolving at much slower rates (Buonagurio,

1985;Yoko, 2000). 4

The structure of an IAV virion

The virions of IAV exhibit a variety of morphologies under different conditions.

At initial isolation from their natural hosts, some IAV virions are filamentous. After culture, they become almost spherical particles with a diameter of 80-120 nanometers.

IAV is an enveloped virus, having a lipid bilayer envelope derived from the host cell plasma membrane (Fig 1). There are two major surface glycoproteins, hemagglutinin and neuraminidase that reside in the viral membrane and mediate important biological roles in the viral life cycle. Also present in the viral membrane are M2 proteins, functioning as proton channels. Beneath the viral envelope is a thick shell composed mainly of the M1 matrix protein, the most abundant protein in an IAV virion. Located in the core of a virion is the nucleocapsid, which is composed of the viral genome, the associated viral

RNA polymerase complexes and nucleoproteins. The genome of IAV consists of 8 segments of single-stranded negative sense RNA, designated as segments 1 through 8 based on their mobility on polyacrylamide gels, with segment 1 being the slowest migrating fragment (highest molecular weight) and segment 8 being the fastest moving fragment (lowest molecular weight). It should be noted that the length of each fragment can vary in different viral strains. All of the RNA segments have the same conserved 3’- ends of 12 nucleotides and conserved 5’-ends of 13 nucleotides. See figure 2 for more details. The 8 RNA segments are known to encode at least 11 proteins (Lamb,

1989;Webster et al. 1992;Chen, 2001). Most of these proteins are components of 5

Fig 1. Diagram of an IAV virion (not drawn proportionally). On the viral surface, the number of HA

(hemagglutinin protein) molecules is 3-4 times that of NA (neuraminidase protein) molecules. Inside the virion, there are 30-60 molecules of RNA polymerase complexes, associated with some nucleoprotein (NP) and viral RNA segments.

vRNA (-) (3’) UCGCUUUCGUCC------GGAACAAGAUGAppp (5’) G

Fig 2. Conserved ends of vRNA segments. For each of the 8 segments of IAV genome, both the 3’ and the 5’ ends are conserved by 12 nucleotides. 6 influenza virions, while 1 (in some cases 2) of them only appear in the infection stage, and will not be incorporated into IAV virions. In the following paragraphs, a brief introduction to each of the RNA segments is made, with emphasis on their protein(s) functions. Since these topics have been extensively reviewed by Lamb et al. (1989) and Krug et al. (1989), the following review is partly based on their reviews (individual citations are thus not given) and supplemented with discoveries made in recent years. If not specified, the lengths of genomic RNA segments and viral proteins are those of IAV strain A/Puerto Rico/8/34.

RNA segment 1

As the longest fragment in the influenza genome, RNA segment 1 is composed of

2341 nucleotides (nt) and encodes the 759 amino-acid-long protein PB2 (Polymerase

Basic Protein 2). PB2 proteins form a kind of multifunctional polymerase complex with equal proportions of viral RNA polymerases: PB1 and PA (Huang, Palese et al. 1990).

By estimation, there are around 30-60 molecules of the polymerase complex in one IAV virion. The polymerase complex always works as an entity in catalyzing the synthesis of viral RNA species. PB2 protein is essential for the synthesis of viral mRNAs (Lee et al.

2002). Viral mRNA synthesis is primer-dependent. When the polymerase complex binds to the 5’-terminal sequence of vRNA (viral genomic RNA), PB2 is activated to bind to the cap-1 structure of the cellular pre-mRNAs and cut the 5’-end of the cellular mRNAs to serve as primers for viral mRNA synthesis (Shi, 1995). However, it is still arguable whether PB2 or PB1 is the endonuclease in cap cleavage (Li, 2001). Recent studies indicate that PB2 also is essential for vRNA synthesis, probably through facilitating the assembly of polymerase complexes by inducing conformational changes and interactions 7 of PB1 and PA, in the process of synthesizing cRNA (complementary RNA) from vRNA templates and vRNA from cRNA templates (Lee, 2002;Perales, 1997). PB2 may down- regulate the expression of PB2, PB1 and PA genes, while up-regulating the expression of the HA gene (Mukaigawa et al. 1991). Some mutations in PB2 inhibit the virus replicating efficiently in humans, probably because these mutations decrease the functions of the polymerase complexes (Massin et al. 2001).

RNA segment 2

This RNA segment is 2341 nt, encoding the PB1 (Polymerase Basic Protein 1,

Figure 3) polymerase, a 757-amino acid polypeptide. In the viral polymerase complex, the N-terminal region of PB1 binds to PA and the C-terminal region binds to PB2

(Gonzalez et al. 1996). PB1 might have endonuclease ability in deriving the host mRNA cap, as discussed above (Li, 2001). It catalyzes the elongation of the nascent RNA chains during the synthesis of viral RNA species: viral mRNA, cRNA (RNA complementary to vRNA) and vRNA (González and Ortín, 1999). At the presence of IAV nucleoprotein,

PB1 can synthesize cRNA and polyA-RNA without the PB2 and PA subunits (Nakagawa et al. 1996).

By using an alternative reading frame of the PB1 genes, some IAV strains can encode a conserved 87-aa polypeptide, PB1-F2 (Chen, 2001). Compared with other IAV proteins, PB1-F2 is unique in several aspects. Firstly, a considerable portion of this protein locates within the inner mitochondrial membrane. Secondly, not all IAV strains can encode this polypeptide. Thirdly, its expression levels vary in different host cells.

Fourthly and importantly, PB1-F2 can induce apoptosis in infected cells, especially in immune cells. Further studies indicate PB1-F2 can damage mitochondrial function and 8

Fig 3. Schematic representation of the bicistronic mRNA for PB1 and PB1-F2 proteins (Adapted from

Lamb et al. 2001). The thin lines represent transcription products from vRNA. The black bar at the 5’ end of the mRNA represent the primer derived from host mRNAs, the red bars represent the coding region. A single bicistronic mRNA is used for translation of the 2 proteins, using two open reading frames.

Fig 4. Schematic representation of HA protein and the cleavage site. 9 cellular viability by forming apoptotic pores in the mitochondrial outer membrane (Chen et al. 2001;Gibbs et al. 2003;Chanturiya et al. 2004; Lamb & Takeda, 2001).

RNA segment 3

This 2,233 nt segment encodes the PA (Polymerase Acidic) polymerase, a 716- amino acid polypeptide. As a subunit of the viral polymerase complex, PA is believed to act as a protein kinase with helix-unwinding ability. PA is necessary for vRNA synthesis, perhaps by switching cRNA synthesis to vRNA synthesis through inducing conformational changes in the PB1 subunit (Nakagawa et al. 1996). Fodor et al. (2002) found that a single amino acid change in the PA protein could result in the inhibition of endonucleolytic cleavage of capped mRNAs by the polymerase complex. Studies also suggest that PA is a phosphoprotein, which is subjected to post-translational modification by cellular activity (Sanz-Ezquerro et al. 1998). Expression of PA reduces the half-lives of co-expressed proteins (both IAV protein and non-IAV proteins), probably because of the proteolytic ability of PA (Sanz-Ezquerro et al. 1995; Sanz-Ezquerro et al. 1996).

RNA segment 4 RNA segment 4 is 1,778 nts in length, encoding the hemagglutinin protein (HA) of 566 amino acids (Figure 4). HA is an integral membrane glycoprotein and the most important IAV antigen. The initial translational product of the H gene is a single polypeptide termed HA0. After a series of post-translational modifications, HA0 molecules are assembled into homotrimers and trafficked to host cell surface. When a newly produced IAV virion is budding off the host cell surface, a piece of the cell membrane studded with HA0 trimers surrounds the virion and separates from the cell. To 10

be functionally mature, HA0 needs to be cleaved by host cell proteases at a cleavage site

to remove one or a few amino acids, producing two subunits, HA1 (319-326 amino acids)

and HA2 (221-222 amino acids), linked by one or more disulfide bonds. HA proteins are

essential for IAV to initiate an infection. At the beginning, HA molecules on the viral surface bind to sialic acid-containing receptors on the host cell surface. After being taken into the host cell by endosomes, HA molecules undergo conformational changes, which mediate the fusion of the viral membrane with the endosomal membrane and result in the release of the ribonucleoprotein (RNP) complexes into the cytoplasm. RNP complexes are composed of nucleoprotein molecules and the nucleoproteins encapsidated viral genomic RNA fragments and viral RNA polymerase complexes (Bullido et al. 2001).

RNA segment 5

The fifth segment is 1565 nt, encoding the 498-amino acids nucleoproteins (NP).

In mature IAV virions, NP molecules encapsidate the viral RNA segments and the polymerase complexes to form the RNP particles. NP molecules interact with polymerase subunit PB1 and PB2, but not with PA (González et al. 1996). More than just a structural protein, NP also facilitates viral RNA transcription, replication and packaging through its interactions with other viral molecules. NP proteins have interactions with a variety of host cell molecules, with potential functions in RNP complexes trafficking (Portela et al.

2002).

RNA segment 6 11

The 1,413-nt RNA segment 6 encodes the 454-amino acid neuraminidase protein

(NA). NA is another integral membrane glycoprotein and an important influenza antigen.

Mature NA molecules are mushroom-shaped tetramers studded on the viral membrane.

Functional NA molecules are required to cut the α-glycosidic linkages between HA molecules and sialic acids, to release progeny virions from host cell surface. No evidence that NA molecules are involved in the entry, replication, assembly or budding of the virus

(Liu et al. 1995). Goto et al. (1998) presented that the NA molecules of IAV strain

A/WSN/33 (H1N1) could mediate the cleavage of HA0, by binding to plasminogen molecules and sequestering them for cleavage activation. As has been discussed above, the cleavage of HA0 by cellular proteases is essential for viral infectivity. The availability of cellular proteases is thus one of the key determinants of the viral pantropicity. Plasmin is a kind of serine protease that can cleave HA0 into HA1 and HA2. However, plasmin is usually not available because it is often associated with its inhibitors in the form of plasminogen. The IAV strain A/WSN/33 is capable of infecting various cultured cell lines without the addition of trypsin, making it significantly different from other human

IAV strains. Genetic studies have shown that NA is necessary for HA cleavage in

A/WSN/33 (Schulman et al. 1977). Goto et al. (1998) found that the NA of A/WSN/33 can bind to plasminogen through a lysine residue at the C-terminus of the NA to increase the local concentration of plasminogen near the virion surface. After activation by plasminogen activator, plasmin of enzymatic activity cleaves HA0 into HA1 and HA2

(Goto et al. 1998). It is worthwhile to note that not every NA of each IAV strain have this ability. This important finding helps to explain why some IAV strains are more virulent and pantropic than others (Goto et al. 1998;Taubenberger, 1998). 12

RNA segment 7

RNA segment 7 is a 1,027-nt long segment, which is translated into two proteins, the 252-amino acid M1 (Matrix Protein 1) and the 97-amino acid M2 (matrix protein, Fig

5). M1 is a highly conserved protein, encoded by the unspliced collinear transcription product from RNA segment 7. Less than 5% differences are observed among the

sequences of a wide variety of IAVs (Reid et al. 2002). In an IAV virion, M1 molecules form a thick protein shell beneath the viral lipid envelope and surround the viral RNP. In infected cells, M1 proteins are present in both the cytoplasm and the nucleus. By stopping vRNA synthesis and exporting viral RNPs from the nucleus to the cell membrane, M1 facilitates the assembly of progeny virions (Chen, 2001). More functions of M1 include mediating the transport of RNPs into the nucleus upon infection and preventing newly synthesized RNP from reentering the nucleus.

M2 is translated from the alternative splicing product of the collinear RNA

transcript. As an integral membrane protein, M2 tetramers embed in the viral membrane

acting as ion channels, which pump protons into the interior of the viral core to disassemble the strongly associated M1 proteins and RNP. M2 is required for the fusion

of endosomal membrane with viral membrane to uncoat the influenza nucleocapsid

(Pinto, 1992;Reid et al. 2002). 13

Fig 5. Schematic representation for the mRNA and coding region of influenza segment 7 (adapted from Lamb, 1989). The thin lines represent transcription products from vRNA. The black bar at the 5’ end of the mRNA represent the primer derived from host mRNAs, the red bars represent the coding region and the green bars indicate the introns to be removed in M2 mRNA. M1 mRNA is the original transcript of

RNA segment 8; the coding region starts at nucleotides 26-28 and terminates at 782-784. M2 mRNA shares the first 51 nt with M1 mRNA and continues from 740 to 1,004 nt after a flanking intron. Mature M2 mRNA is the splicing product of the linear transcript. 14

RNA segment 8

This 890 nt RNA segment encodes two proteins: the 230-amino acid NS1 (Non-

Structural protein 1) and the 121-amino acid NS2 (Non-Structural protein 2 or nuclear

export protein, fig 6). The two proteins were termed as non-structural proteins since they were not found in mature IAV virions. However, the term is not accurate since it has been confirmed that NS1 is present in IAV virions, where it interacts with IAV M1 proteins

(Richardson, 1991;Yasuda et al. 1993). NS1 protein is translated from the unspliced

transcripts of the vRNA segment, and NS2 protein is translated from mRNAs generated

by alternative splicing of the transcript. The two proteins share the first 10 residues of the

N-termini. NS1 protein is a RNA-binding protein with multiple functions. In infected cells, NS1 primarily is found in the nucleus. NS1 protein regulates host and viral protein expression at both transcriptional and translational levels (Marion et al. 1997;De la et al.

1995;Enami et al. 1994). NS1 dimers bind to polyA-binding protein II of the host cellular

3’-end processing machinery, inhibiting host cell mRNA polyadenylation and the export

of polyA containing mRNAs, to suppress host cell protein synthesis (Qiu & Krug, 1994;

Chen, 1999). Increased concentration of NS1 triggers the transition from mRNA

synthesis to vRNA synthesis. By binding to double-stranded RNA, NS1 inhibits the

activation of the dsRNA-induced antiviral pathway as well as the synthesis of IFN-α/β

(Talon et al. 2000;Wang et al. 2000). NS1 may disable the host’s innate defenses against viruses, probably by blocking signals for summoning immune cells.

NS2 proteins primarily are found in the cytoplasm. NS2 can down-regulate viral

RNA replication. Bullido et al. (2001) proposed that the inhibitory function of NS2 was 15

Fig 6. Schematic representation for the mRNA and coding region of influenza NS1 and NS2 proteins

(adapted from Lamb, 1989). The black bar at the 5’ end of the mRNA represent the primer derived from host mRNAs. The red bars represent the coding region. The green bar shows the intron to be removed in

NS2 mRNA. NS1 mRNA is the original transcript of RNA segment 8. The coding region starts around 0.05 and terminates around 0.75 unit of the mRNA map. NS2 mRNA needs splicing of the transcript. The coding region stars at the same position as NS1 and has the same 56 nt at the beginning, then continues from nucleotide 529 to the end. 16

achieved by exporting RNP particles from the infected cell nucleus into the cytosol to

reduce the number of the viral synthesis machinery, RNP particles, in the nucleus. Later

in infection, NS2, together with M1 proteins, facilitates the production of progeny virions,

by promoting the export of RNP particles into the cytosol and finally to the plasma

membrane (Chen, 2001).

The life cycle of IAV

Entry of the virus

The replication of IAV has been extensively reviewed in several publications

(Krug, 1989;Whittaker, 2001). When an IAV virion approaches a host cell, the HA

molecules spreading on the surface membrane of the virus bind to sialic acid-containing

receptors on the host cell membrane. Multiple endocytic pathways take the virus into the

cell, as has been reviewed by Lakadamyali et al. (2004). Electron microscopy and real-

time imaging revealed that a clathrin-mediated pathway is the main mechanism for the

endocytosis and is responsible for nearly two-thirds of viral internalization, and that

clathrin-independent pathways also can internalize IAVs (Lakadamyali et al. 2004). The internalized virus is transported along the endocytic pathway to an acidic late endosome

(Lakadamyali et al. 2004). HA molecules on the viral surface undergo a conformational change, which releases a hydrophobic fusion domain into the endosomal membrane, triggering the fusion between the endosomal membrane and the viral membrane (Li et al.

2005). At the same time, M2 ion channels pump protons into the viral interior. As a result of this acidification, the M1 shell dissociates from the RNP particle. The RNP is thus released into the cytoplasm. After being imported into the nucleus, RNP starts gene expression and replication. 17

The synthesis of viral RNA species

The replication and transcription of IAV takes place in the nucleus of the host cell

(Krug, 1989). During the initial stage, viral mRNAs are synthesized. The synthesis of

viral mRNA requires primers. Capped RNA fragments of about 10-13nt are cleaved from

host cell transcripts to serve as primers for viral mRNA synthesis. The first step of

transcription is the addition of a G residue onto the 3’ end of the primer, and pairing with

a C residue on the vRNA template. PB1 protein catalyzes the elongation of nascent

mRNAs, which stop at a stretch of 5-7 U residues near the 3’ end of the vRNA template.

Finally, polyA tails are added to the 3’ ends of mRNAs. Mature viral mRNAs are exported from inside the nucleus into the cytoplasm to be translated by ribosomes. This

process is facilitated by host cell machinery and is controlled by viral NS1 proteins (Chen

& Krug, 2000).

Influenza cRNA functions as the template for vRNA replication. Synthesis of

cRNA does not need capped RNA primers, but does need a switch from mRNA synthesis

to cRNA synthesis, achieved by accumulated NP molecules. To be completely

complementary to vRNA segments, cRNA elongation must overwhelm the termination

site of the stretch of U residues. Free NP molecules are believed to the responsible for the

anti-termination function. The partially complementary 5’ and 3’ vRNA termini form a

panhandle, which blocks the termination site. Then free NP molecules can dissemble the

panhandle permitting the cRNA elongation through the termination site. After synthesis,

viral cRNAs are not capped or polyadenylated. They stay in the nucleus to serve as templates for vRNA synthesis (Whittaker, 2001).

18

Viral protein synthesis and traffic

Viral protein synthesis varies in different infection stages. Early in an infection,

NP and NS1 are the primary translational products and they are soon transported into the

nucleus. The prioritized synthesis is likely because of their essential roles in regulating

the synthesis of different viral RNAs, inhibiting host cell antiviral activities, and blocking

host protein synthesis. Later in the infection, M1, HA and NA molecules are mainly

produced. After posttranslational processing, HA trimers and NA tetramers migrate to the

host cell surface, forming patches of viral proteins on the plasma membrane.

The transportation of viral glycoproteins HA and NA to the cell surface is through

a common pathway (Roth, 1989). Following the synthesis by ribosomes on the rough

endoplasmic reticulum (RER), nascent polypeptides enter the exocytic pathway.

Complicated co-translational activities occur at this stage, including the addition of

oligosaccharides to specific asparagine residues, and the folding of secondary structures.

Post-translational processing takes place in the endoplasmic reticulum (ER), such as the folding and trimerization of HA molecules (or tetramerization of NA proteins) within the

smooth endoplasmic reticulum (SER). Proteins processed in the SER are taken into

transport vesicles. After passing through the Golgi bodies, these proteins enter small

vesicles destined for the plasma membrane with different distribution patterns. HA

molecules are diffusely distributed over the cell surface, while NA molecules cluster in

patches. All of the viral envelope proteins are independently transported to the assembly

site at the plasma membrane (Whittaker, 2001).

The assembly, budding and release of IAV virions 19

The maturation of IAV particles has been reviewed by Whittaker (2001). The

virus assembly takes place on the plasma membrane. Every viral component is

transported to the correct position and processed. M1 molecules are critical to the

assembly of subviral particles which are composed of the M1 proteins and the vRNPs. By

binding and encasing vRNPs, M1 protein forms a shell beneath the viral membrane. This

protein shell has complex interactions with the plasma membrane. It is generally believed

that the binding of M1 to membranes involves hydrophobic, electrostatic and specific

protein interactions. Envelope proteins HA, NA and M2 are transported to host cell

surface independently, as has been confirmed in different experiments.

The budding sites are specific regions of the plasma membrane known as

detergent-insoluble glycolipid-enriched domains (Scheiffele et al. 1999;Zhang et al.

2000). The budding of virus at these sites also is likely correlated with the presence of

HA and NA cytoplasmic tails (Whittaker, 2001). The morphology of progeny virions,

spherical or filamentous, is determined by glycoproteins HA and NA (Jin et al. 1997),

M1 and M2 (Lamb et al. 1998), as well as by host cell phenotypes and the integrity of the

actin microfilament network (Roberts & Compans, 1998).

The release of progeny viruses from host cells is mediated by the proteolytic

activity of NA, which cuts the linkages between HA molecules and host cell receptors. If this enzymatic activity is not functional or is deficient, new virus particles cannot be

released into the extracellular space. Finally, there is proteolysis by cellular proteases to

cleave HA0 molecules (in the form of HA0 homotrimers) into HA1 and HA2 subunits

linked together by disulfide bonds. For most viral strains, this cleavage is extracellular.

However, some avian IAV strains contain multiple basic amino acids at their cleavage 20

sites, which cause cleavage to take place intracellularly. In avian hosts, the cleavage of

HA0 might occur in a new host’s enteric tract, since the fecal-oral transmission route

requires the virions to pass through the very acidic stomach while cleaved HA molecules are not stable at low pH and only virions with uncleaved HA0 can reach the enteric tissues with undamaged HA molecules.

Influenza pandemics

In the 20th century, at least 4 global IAV pandemics afflicted human populations

(Palese, 2004). In 1918, the “Spanish” flu made its appearance in humans and infected

nearly 20% of the total population and killed 30-50 million worldwide (Johnson &

Mueller, 2002). For most of the 20th century, little was known about this deadly influenza.

Taubenberger and his colleagues successfully reconstructed the entire genome of the

“Spanish” flu, using victims’ tissues preserved in permafrost (Reid et al. 1999;Reid et al.

2002;Reid, Fanning et al. 2000;Taubenberger et al. 1997; Taubenberger, 2005). These

studies confirmed that the virus was an H1N1 subtype as indicated previously by

archaeological methods. Even with the complete genome of the “Spanish” flu, however it

has been difficult to determine the origin of that viral strain (Reid & Taubenberger, 2003).

One hypothesis is that the virus had entered the human population some time between

1900 and 1918 (Webster, 1999), either through direct transmission from an avian host to

humans or through an intermediate host, i.e. swine (Reid & Taubenberger, 2003;Reid et

al. 1999;Taubenberger et al. 1997). Since the outbreak in 1918, the “Spanish” flu

continued to circulate in humans until 1957, when a new IAV subtype emerged and

replaced it. Nevertheless, the impact of this virus was beyond its circulation period from 21

1918 to 1957. Most genes of that virus were inherited by the IAV strains that circulated

in humans subsequently.

In 1957, the so-called “Asian” flu caused another severe global pandemic. Around

70,000 people in the US were killed and many more were killed worldwide in this IAV

pandemic (Lipatov et al. 2004). The virus responsible for the pandemic was determined

to be an H2N2 subtype. This virus had derived its H2, N2 and PB1 genes from an avian

IAV strain and retained the remaining 5 genes from the previously circulating H1N1

subtype in humans (Kawaoka et al. 1989;Palese, 2004). Eleven years later, an IAV strain of H3N2 subtype known as the “Hong Kong” flu caused another pandemic in 1968. In the USA alone, 34,000 excessive deaths were induced (Lipatov et al. 2004). The last IAV pandemic of the 20th century happened in 1977, caused by an H1N1 subtype, now known

as the “Russian” flu, which originated in northern . Very interestingly, this H1N1 was found to be almost identical to the H1N1 strain that had circulated in the early 1950’s

(Nakajima et al. 1978). However, little was known about the strange reappearance of this influenza strain until today.

The ecology of IAV

Among the various IAV hosts, wild aquatic birds are found to be the ultimate

reservoir of all IAVs, by both extensive virological surveillance and phylogenetic

analyses (Webster et al. 1992). All known IAV subtypes have been isolated from wild

aquatic birds, and many IAV subtypes have only been found in avian species. Different

studies have indicated that IAVs may have reached an evolutionary stasis in many wild

aquatic bird species (Hinshaw et al. 1982;Fouchier et al. 2005;Webster et al. 1992). 22

In mammalian hosts and wild aquatic bird hosts, IAV infection targets on

different tissues and organs. In mammals, IAV primarily infects the respiratory tracts. In wild aquatic birds, IAV is largely an enteric virus, infecting the intestinal tissues.

Interestingly, it is deadliest for the avian species when IAV severely attacks their respiratory tracts. Usually, IAV infection of mammals causes diseases from mild to severe symptoms. While in wild aquatic fowl, IAV infection generally causes mild or even no symptoms at all. Some IAV strains termed as HPAI (highly pathogenic ) are capable of causing infections of high morbidity and mortality in avian species (e.g. H5N1) (Tumpey et al. 2002;Masato, 2001) and H7N7 (Arjan, 2004). HPAI is responsible for the ‘fowl plague’ in domestic poultry and ‘bird flu’ in wild avian species.

The number of IAV subtypes infecting avian species is greater than the number

infecting mammalian hosts. For humans, only one or two subtypes circulate at a given

time. In horses, only two subtypes, H3N8 and H7N7, are known to have ever infected

them (Powell, 1995;Yoshihiro, 1991). Similar patterns are found in other mammalian

hosts. In wild aquatic fowl, the transmission of influenza viruses is through an unusual

fecal-oral route. Webster et al. (1978) reported that infected feral ducks shed IAV in high

8.7 titers of 1×10 mean EID50 (egg infective dose, the dose required to infect half of the

tested chicken embryos) per gram of fecal materials. Water contaminated by these fecal

materials becomes an infectious source capable of infecting vulnerable hosts drinking the

water. Independent studies have reported the isolation of viable IAVs from wild bird

droppings (Widjaja et al. 2004) and unconcentrated lake water contacted by wild birds

(Ito, 1995; Reid et al. 2002). 23

Certain host species barriers exist that prevents the direct transmission of IAV between different hosts (Webster et al. 1992). Its existence contributes at least partly to the fact that humans and other mammals are not infected by all IAV subtypes in avian species. The mechanisms underlying the host range restriction are unclear except that they are certainly multi-factorial (Massin et al. 2001; Subbarao et al. 1993). However, the barrier is obviously not absolute. Ample evidence indicates that interspecies transmission of IAV does happen frequently in nature (Kawaoka et al. 1989; Navani, 2004; Wright,

1992; Fanning et al. 2002; Laudert, 1993). In recent years, a few IAV strains of avian origins were directly transmitted from domestic fowl into human populations and have caused limited numbers of infections, specifically the H5N1 in 1997-2007 in Southeast

Asia, Europe and , H7N7 in 2003 in (Stegeman, 2004; Navani, 2004), and the H9N2 infection in 2003 in Hong Kong (Butt et al. 2005). The three subtypes originated solely from avian hosts and human-to-human transmission does not seem to have occurred with any of the cases.

Human IAV: appearance, disappearance and reappearance

In the 20th century, at least 3 subtypes of IAVs have emerged and widely spread in human populations. Each emergence was associated with a global influenza pandemic characterized by high morbidity and mortality, as has been discussed above. The 1918

H1N1 might have had swine as an intermediate host before its transmission to humans

(Reid & Taubenberger, 2003; Fanning et al, 2002; Reid et al. 2000), the H2N2 that appeared in 1957 was a reassortant of an avian IAV strain with a human strain, and the

H3N2 that appeared in 1968 also was a reassortant of an avian and a human strain 24

(Taubenberger and Morens, 2006). In recent years, a few new IAV subtypes were

isolated from human patients, specifically H5N1, H7N7 and H9N2 as described above.

Fortunately, no widespread infection has taken place. Studies indicated that these new

IAV subtypes were closely related to avian IAVs and were thus believed to be of

completely avian origins. It seemed that the recent transmission of IAV subtypes to

humans was made via different routes from those of the pandemic IAV strains.

The establishment of a new IAV subtype in humans was accompanied by the

disappearance of the previously circulating subtype. This was true in the cases of the

H2N2 replacement of the H1N1 in 1957 and of the 1968 H3N2 replacement of H2N2 in

1968. The disappearance of an old IAV subtype was presumably attributed to different

factors. The main factor probably is herd immunity in the host populations acquired in previous infections, which rendered the old strain less competitive compared with a new

IAV subtype (Webster et al. 1992). One exception was in 1977 when the H1N1 that

“Russian” flu emerged. The previously circulating H3N2 did not disappear, but rather cocirculated with the H1N1 ever since. More interestingly was that the H1N1 appeared in

1977 was almost identical to the H1N1 that circulated in the 1950s (Nakajima, 1978).

There have been different explanations for this unusual reemergence of the 1977

IAV. Nevertheless, these opinions all agreed that this virus had been preserved alive but

inactive during the 20-year disappearance. It is not possible for an RNA virus like IAV to

have undergone such little genetic change over such a long period of time, if it had been

maintained in a living host population. RNA viruses, including IAV, mutate at high rates.

If the reappeared virus was not dormant but was replicating in living hosts for decades,

significant mutations would have accumulated. It was proposed that this virus was 25

released from a frozen state into the environment 20 years later and entered the human

population successfully (Webster et al, 1992; Rogers et al. 2004; Smith et al, 2004; Ito et

al. 1995). In previous studies, various microbes have been isolated in different ice

samples aged up to hundreds of thousands of years (Castello et al. 1999; Ma et al. 2000).

Rogers et al. (2004) and Smith et al. (2004) pointed out that the huge amounts of various

forms of environmental ice could be reservoirs for many pathogenic microbes, including

IAV.

The perpetuation of IAV in environmental ice and water

The study of perpetuation of IAV in environmental ice and water is justified by

the fact that wild aquatic birds are the ultimate reservoir of IAV in nature and by the

fecal-oral transmission route of the virus. Stallknecht et al.(1990) studied the survival of several IAV strains in water of varied salinity, pH value, and temperature. From an initial

6 concentration of 10 TCID50/ml (tissue-culture infective dose), some avian IAV strains

could remain infective in distilled water for up to 207 days at 17°C and up to 102 days at

28°C, based on predictions from linear regression models. The persistence of IAV in ice

is even longer than in water (Parker and Martel, 2002). According to Gould (1999), many viruses preserved in ice at -70°C could survive decades because of their small sizes, simple structures, and absence of free water in the preservation medium. Indirect evidence indicated that IAVs could resist repeated freezing-and-thawing and remain infective. Tumpey et al. (2002) isolated avian H5N1 viruses from factory-processed duck meat samples, by inoculating embryonated chicken eggs with tissue fluid obtained by

repeated freezing-and-thawing treatments of the duck meat. In assaying water samples

collected from Alaskan lakes, Ito et al. (1995) reported high titers of IAV of up to 102.8 26

EID50/ml in some water samples by culturing in embryonated chicken eggs. One IAV

was even isolated from a water sample collected in a fall season when most waterfowls

had left the breeding lake, indicating the possibility that IAV virions deposited by

waterfowls in those lakes in high latitude areas can be preserved alive by water/ice of low

temperatures through the winter period and that these IAVs can infect susceptible birds

when the ice melts to release the entrapped viruses. Despite these many studies and

evidences, it is still not known if infectious IAV can survive in environmental ice over

extended periods of time.

The evolution of IAVs

The genome of IAV is well known for its high degree of polymorphism, a result

of long-term evolutionary processes. Evolution enables IAVs to escape hosts’ immunity

gained in previous infections. Fitch et al. (2000) depicted the process as the following.

When influenza viruses enter host cells beginning an infection, the host immune systems detect this intrusion and make antibodies to neutralize the viruses. Before being killed,

IAVs are able to replicate and mutate. Although most of those mutants are not viable, a small proportion of them is viable and can escape host immunity surveillance to initiate new infections. A new race between the viruses and the host immune system begins. The above process continues and repeats, until either the host is killed or the viruses are cleared out.

IAV genes have unusually high mutation rates, caused by replication errors in

viral RNA synthesis. The RNA-dependent RNA polymerase complex lacks a proof-

reading function, which renders the synthesis of viral RNA species error prone. The

mutation rates of IAV genes are outstanding even among RNA viruses. Parvin et al. 27

(1986) directly measured the mutation rates of IAV genes and found that the NS gene of

IAV has a mutation rate of 1.5×10-5/nt/cycle, which is 7 times higher than that of

poliovirus VP1 gene at 2.1×10-6/nt/cycle. Using a similar method, Nobusawa et al. (2006)

carried out an independent study to compare the mutation rates between the NS genes of

influenza A and influenza B viruses. The NS gene of IAV has a mutation rate of 2.0×10-

6/nt/cycle (or 2.6×10-3/nt/year) and that of influenza B virus has a rate of 0.6×10-

6/nt/cycle (Nobusawa & Sato, 2006). Nobusawa et al. (2006) pointed out that technical

problems had made the data collected by Parvin et al. (1986) inaccurate. Nevertheless,

both studies agreed that IAV genes had higher mutation rates than some other RNA

viruses. Noteworthy, the 8 segments of the IAV genome exhibits different evolutionary

rates and evolutionary patterns, caused by different selective pressures and evolutionary

constraints (Webster, 1992).

Fast evolution of IAV H and N genes

The survival of IAV in nature is likely relying on the production of new antigenic

phenotypes resulting from continuous evolution (Kilbourne et al. 1990). IAV H and N

genes encoding major surface proteins are evolving very fast, because of the strong host

immune pressure on them (Webster, 1992). Fitch et al. (1997) reported the evolutionary

rate of the HA1 domain of H1 genes was 5.7×10-3sub/nt/year in cell lines (or 4.4 ×10-

6/nt/cycle calculated following Nobusawa & Sato, 2006), which makes the H1 gene the

fastest evolving IAV gene. The diversification of H and N genes is likely contributed by the fast evolutionary rates. Presently, at least 16 serologically uncrossreactive H gene subtypes have been identified (Fouchier et al. 2005). Even within one H subtype, the 28

RNA segments can have more than 20% differences in the nucleotide sequences. A

similar evolutionary pattern also is observed in IAV N genes, with 9 serological subtypes

identified in nature. The N genes may have slower evolutionary rates than H genes, probably due to the lesser immunoselective pressure of NA antibody (Kilbourne et al,

1990). The internal genes of IAV are not evolving as fast as the H and N genes, probably because they are under relatively weaker host immune pressure. For example, human

IAV PB2 genes have an evolutionary rate of 1.82×10-6 sub/nt/year in human strains,

which is significantly lower than that of H and N genes.

Evolution of IAV genes in different hosts

Host species may contribute to the evolution of IAV genes mainly in two aspects:

1) different host species have different immune pressure on the IAV genes; 2) host species barrier and geographical isolation produce gene lineages of host-specificity.

Among all IAV hosts, humans are believed to have the strongest selection pressure on

IAV genes and wild avian species have the weakest. According to Webster et al. (1992),

IAVs have reached an evolutionary balance in wild aquatic birds so that IAVs do little damage to those hosts and the avian hosts have little selective pressure on IAVs. As a result, the same gene may be observed with different evolutionary rates on the nucleotide level and/or on the amino acid level. This opinion is well supported by the results of many studies. On the nucleotide level, the NP gene of human IAVs and that of Old World avian IAVs were observed with similar evolutionary rates (Gorman et al. 1990). However, on the amino acid level human NP genes had nearly 4 times the changes than Old World avian IAV NP genes did, which indicated an established adaptation of the NP genes to the Old World avian species (Gorman et al. 1990). On the other hand, NP genes encode 29

internal proteins and are not under strong host immune pressure so that the evolutionary

pattern of NP genes also reflects host-specific adaptation (Gorman et al. 1990). Bean

(1984) has shown that by using RNA hybridization IAV NP genes can be divided into

five groups: equine I (represented by Equine/Prague/56), equine II (recent equine), swine

and human, H13 gull, and avian, indicating the host-specific character of the NP genes.

Among these subgroups, the classic avian lineages have diverged into ,

Australia- and Old World lineages based on studies of IAV NP genes, likely

because of the geographic separation and isolation of the host species (Gorman et al.

1984).

Diversified evolution patterns of RNP genes

The influenza RNP complex, being composed of vRNA segments as well as 4

viral proteins PB2, PB1, PA and NP, is required for the transcription and replication of

viral RNAs. Protein molecules in this complex have complicated interactions. PB1 binds

to PA with the N-terminal region and to PB2 with the C-terminal region (González et al,

1996). NP protein molecules have direct interaction with the polymerase complex

subunits PB1 and PB2, and are also responsible for encapsidating vRNA segments and

polymerase complexes to form RNP particles (González et al. 1996). Gorman et al. (1990)

systematically studied the 4 RNP protein genes and concluded that these genes of the

complex exhibited different evolutionary patterns and were not coevolving as a unit, even

though their gene products were working coordinately like an entity.

The NP genes of IAV have evolved into 5 lineages of host-specificity as discussed previously, with a maximum nucleotide difference of 18.5% and a maximum amino acid difference of 10.8% (Gorman et al. 1990). Okazaki et al. (1989) reported that 30

IAV PA genes had evolved into at least 5 lineages, similar to that of IAV NP genes. That

NP genes and PA genes exhibit parallel host-specific evolutionary pathways suggests

these proteins are coevolving in response to host-specific factors (Gorman et al. 1990).

However, the evolution of PB1 genes and PB2 genes is less correlated with host-specific

factors than the NP and PA genes, i.e., there are fewer host-specific lineages and their

lineages are less diverged (Gorman et al. 1990). Kawaoka et al. (1989) reported the

evolutionary pattern of PB1 genes were distinct from that of PA and NP genes. Gorman et al. (1990) suggested that IAV PB2 genes also had evolved into 4 major lineages. On the amino acid level, the evolutionary rates of PB1 and PB2 are slower than that of NP and PA, suggesting functional constraints on the two genes (Gorman et al. 1990).

Evolution of IAV bicistronic genes

IAV RNA segments 7 and 8 each encode 2 proteins by using alternative splicing,

with parts of the two proteins’ coding regions overlapping. According to Ito et al. (1991),

the evolution of one protein of a bicistronic gene can affect the evolution of the other protein. In RNA segment 7, significantly different evolutionary patterns were observed in the two genes, i.e. M1 and M2. Phylogenetic analysis indicated that M genes have evolved into at least 4 major lineages of host-specificity, similar to that of NP and PB2 genes. However, compared with IAV NP and PB2 genes, the M1 and M2 genes are evolving relatively more slowly, especially the M1 genes (Ito et al. 1991). It was reported that the M1 protein sequences were observed with changes at 24.6% of the total positions and the M2 protein sequences were observed with changes at 48.5% of the total positions

(Ito et al. 1991). There was no change observed in the first 9 amino acids shared by M1 31

and M2 proteins. While in the overlapping reading frame, M2 proteins showed high

variability with amino acid changes observed at 10 of 13 positions. A great majority of

those changes resulted from changes at the second-codon positions, which were the third-

codon positions in the M1 genes. Apparently, the conservation of the M1 protein places

constraints on M1 gene evolution, and in turn, affects the evolution of the M2 gene and

its product. Interestingly, the structurally similar NS genes do not have the same

evolutionary pattern.

The NS gene, i.e. RNA segment 8, encodes two proteins: NS1 and NS2. A major

function of NS1 is to inhibit the nuclear export of mRNA (Qiu & Krug, 1994). NS2

protein interacts with the M1 protein in IAV virions (Richardson, 1991;Yasuda et al.

1993). The NS genes have two distinct gene pools, designated group A and group B

(Baez et al. 1981;Treanor et al. 1989). Group A NS genes included NS genes from

humans, horses, pigs and birds, while group B NS genes are completely from birds

(Kawaoka et al. 1998). The NS genes are relatively conserved IAV genes. Within group

A and group B, nucleotide sequence similarities were about 90% or above; however, only around 70% similarity is achieved between the most similar sequences from group A and group B NS genes (Kawaoka et al, 1998;Treanor et al. 1989). NS1 and NS2 proteins also are fairly conserved. Kawaoka et al. (1998) reported that the NS1 protein had a similarity

of at least 71.3% and 96.5% in group A and group B respectively; NS2 protein had

similarity of 84.3% and 95% in group A and B respectively. Similar to that in M genes,

the coding region shared by NS1 and NS2 proteins is evolving slower than other regions,

which is presumably to maintain the structure and function of each protein, indicating

that the evolution of each protein is affected by the other (Kawaoka et al. 1998). The 32

evolution of NS1 and NS2 proteins are dependent on the lineages. It is different in M

genes that the evolutionary constraint from M1 universally affects the evolution in M2,

and is not dependent on lineages (Kawaoka et al. 1991). Buonagurio et al. (1986) studied

the evolution of human IAV NS genes and found that these genes had a consistent evolutionary rate of 2×10-3 sub/nt/year. Buonagurio et al. (1986) also noticed that the

evolution of the NS gene was under positive selection. In the phylogenetic tree of NS

genes, branches other than the one succeeded in surviving for the longest time died out in

only 3 years, which was a pattern very similar to that of human IAV H genes

(Buonagurio et al. 1986;Bush et al. 1999).

Different opinions on the evolution of IAV genes

Recently, Chen and Holmes (2006) reported that the evolutionary rates of avian

IAV genes (1.8-8.4×10-3 sub/nt/year) are similar to that of the human H3 HA1 domain

(5.7×10-3 sub/nt/year) (Fitch et al. 1997), to the equine M gene (5.4×10-4 sub/nt/year) and

NS gene (5.1×10-4 sub/nt/year) (Lindstrom et al. 1998), and to that of the swine M gene

(1.30×10-3 sub/nt/year) (Lindstrom et al. 1998). Chen et al. (2006) reported that the

evolutionary rates of all avian IAV genes were high. According to this research, avian

IAV genes were not conserved at all, and the ratios of nonsynonymous changes to

synonymous changes were high, indicating no evolutionary stasis was reached between

avian IAVs and their avian hosts (Chen et al. 2006). In other words, there were no

significant differences in the evolutionary rates, on both the nucleotide level and amino

acid level, between the genes of avian IAVs and non-avian IAVs. Since Webster et al.

(1992) proposed that influenza viruses carried by wild aquatic birds had reached an 33

evolutionary stasis, this theory has been central to the studies of avian IAV evolution

(Chen & Holmes, 2006), and is congruent with many study results. These contradictions

await resolution.

Predicting evolutionary trends and upcoming pandemics

Predicting the evolution of IAV can enable the medical community to prepare for

upcoming influenza epidemics. Currently, flu vaccines are made with attenuated viral

strains, two IAVs and one IBV, which represent the viral strains that are most likely to

circulate in the coming year. The vaccine can be more effective if one can accurately

predict the evolution of the viruses. However, the continuous and fast evolution of IAVs

makes it extremely difficult to predict their evolutionary trends. Important progress was

made by Fitch et al. (2000), who observed that in human populations only one main IAV

lineage would persist in the long run, which was reflected in a phylogenetic tree where

only one main trunk persisted while other branches died out in 2-3 years. Fitch et al.

(2000) hypothesized that the most successful virus progenitors were those carrying the

greatest number of beneficial mutations. A beneficial mutation was defined as a mutation

that had helped the virus escape from human immunity surveillance. With this model,

Fitch and his colleagues made 9 correct predictions out of 11, in predicting which H3

strains were going to survive in human populations (Fitch et al. 2002).

There have been attempts to predict future human IAV pandemics (Webster,

1997). Historical records have shown that in the 20th century, global IAV pandemics had

happened every few decades and caused deaths of millions. It would be best if one knows when, where, and what IAV is going to initiate a pandemic. In that case, preparations can

be made to reduce the losses. However, such a prediction is difficult. According to 34

Shortridge (1995) and Webster (Webster, 1997),it was impossible to predict the time and severity of the next influenza pandemic.

Methods in IAV surveillance

Among the various methods available for the surveillance of IAVs, virus culture and PCR-based methods are most popular. Each of them has its advantages and disadvantages. Virus culture is the traditional method. Originally, virus culture was used to sustain the passage of viral strains for preservation. Culture can be done in either embryonated chicken eggs or in cell lines. The application of the virus culture method is limited by its time-consuming nature, lack of universal susceptible cell lines for all IAV strains, and critical demands for facilities and personnel. Despite these, virus culture methods remain the priority choice for many researchers because of its sensitivity and the ability to produce living viral strains for further studies. PCR-based methods are relatively easier, sensitive, and time-saving. Generally, samples that can be used in virus culture can be assayed by PCR based methods without further processing. More importantly, PCR-based methods can be applied to non-living virus samples, even on samples that only contain severely damaged vRNA fragments. The important findings about the 1918 “Spanish” flu was achieved by retrieving small pieces of IAV RNA fragments from victims’ tissues, which were either formalin-fixed and paraffin-embedded or permafrost-buried (Krafft, 1997;Taubenberger et al. 1997;Reid et al. 2000;Fanning et al. 2002;Reid et al. 2002;Taubenberger, 2005). PCR-based methods have been successfully applied to both clinical samples and environmental samples, such as human throat swabs (Widjaja et al. 2004), bird droppings(Hanson, 2003), and lake water samples 35

(L'vov et al. 2006). In many studies, both virus culture and PCR-based methods are utilized at the same time to ensure the best results.

Phylogenetic analysis methods

Molecular phylogenetics employs molecular biology data and statistical techniques to study the evolutionary relationship among groups of organisms or genes (Li,

1997). Generally, a phylogenetic tree is built to display the interrelationship among the studied units. A phylogenetic tree is composed of nodes and branches. The nodes represent taxonomic entities; the branches indicate the ancestry or descent relationship between two nodes; an external node represents an extant unit, which is often termed as an Operational Taxonomic Unit (OTU). The construction of a phylogenetic tree is based on the differences and similarities of studied OTUs, and the algorithm chosen to build the tree. Initially, phylogenetic trees were built primarily from morphological data to study the evolutionary relationships between species. When molecular data, especially amino acid sequences and nucleic acid sequences became readily available and abundant, they were widely adapted in the reconstruction of phylogenetic trees. Molecular data have certain advantages over morphological data, including more regular evolutionary patterns, making it easier for quantitative treatment.

A phylogenetic tree is a hypothesis of the evolutionary route of the studied OTUs.

Although there is only one actual evolutionary route for a group of organisms, more than a single tree may be inferred in a tree reconstruction process. This means a phylogenetic tree may or may not be the same as the actual tree. Different algorithms have been used in phylogenetic tree construction for different purposes. The following briefly introduces

3 categories of these methods: the distance matrix methods, the maximum parsimony 36

methods, and the maximum likelihood methods. Additionally, the Bootstrap method,

which is used to estimate the confidence levels of phylogenetic hypotheses, is discussed.

Distance matrix methods

Distance Matrix methods reconstruct phylogenetic trees on evolutionary distances,

which generally are calculated differences of nucleotides or amino acids between each

pair of the studied units (Li, 1997). The Unweighted Pair-Group Method with Arithmetic mean (UPGMA) is a Distance Matrix method and is the simplest method for tree reconstruction. It is assumed in this method that the evolutionary rates are constant among all the different lineages so that there is an approximately linear relationship between the evolutionary distance and the divergence time. The UPGMA method uses a sequential clustering algorithm to build a tree. Specifically, two OTUs with the highest similarity are chosen and made into a composite OTU, which is the mean distance of the sum of the differences on the two branches. The composite OTU joins the remaining

OTUs to form a new group of OTUs. From this new group of OTUs, the two with the highest similarity are picked out and calculated into a new composite OTU. The same process repeats until only two OTUs are left.

Neighbor-Joining (NJ) method is another distance matrix method that is generally

used. The principle of NJ is to sequentially find OTU neighbors which give minimum

total length of the tree. Here “neighbors” refer to OTUs that are connected with a single

internal node. At first, a star-like tree is built regardless of the similarities and differences

of any OTUs. Then a pair of OTUs is selected out from the rest again the two closest

OTUs, generating a tree with one interior branch. The sum of all branch lengths (S) is calculated. For a group of n OTUs, the total number of possible pairs is C(n,2) = n(n-1)/2. 37

After surveying all possible pairs of the first OTUs, the one with the smallest S is chosen.

This pair of OTUs is regarded as a single OTU to join other OTUs forming a new distance matrix. The above procedures are repeated until all the internal branches are

found.

Maximum Parsimony (MP) method

The Maximum Parsimony (MP) method builds a tree which has the minimum

numbers of evolutionary changes to explain the observed differences among the OTUs.

Often more than one tree can be inferred with the same minimum number of changes.

These trees are said to be equally parsimonious (Graur and Li, 1999). To reconstruct a

MP tree, the first step is to identify all the informative sites. If one is analyzing nucleic acid sequences, a phylogenetically informative site refers to a nucleotide site which favors some trees over other possible trees (Graur and Li, 1999). A necessary condition for a site to be an informative site is that there must be at least two different kinds of nucleotides at the site and each nucleotide is represented in at least 2 of the sequences under study. After identifying all informative sites, the number of changes at each informative site and the total number of changes at all informative sites are calculated for each possible tree. The tree(s) with the least changes is (are) the MP tree(s). In many cases, more than one tree can be inferred from a set of data. The total number of changes throughout the analyzed sequences in a particular tree is called the tree length. In some

computations, all changes, transitions and transversions, are given equal weight. These

procedures are called unweighted parsimony. If different weights are given to different

types of changes, such as giving more weight to transversions than to transitions, these

methods are known as weighted parsimony. Searching for MP trees uses different 38 methods. If the total number of OTUs is small, the number of all possible MP trees is small. One can then examine all the possible trees and find the shortest one(s). This procedure is an exhaustive search. However, since the number of possible trees grows rapidly with the number of OTUs, heuristic search can only handle datasets with 12 or less OTUs. If more than 12 but less than 20 OTUs are involved, the branch-and-bound method is a good choice. This algorithm considers an arbitrary tree and computes the minimum numbers of substitutions, M. Then this method uses a similar procedure to that used in the exhaustive search. If a tree under examination is longer than M, then it is discarded and not used for further analysis, because adding branches can only increase the total length. This method can significantly reduce the total number of trees to be examined. Heuristic searches are generally applied when more than 20 OTUs are analyzed. In a heuristic search, an initial tree is built from a certain procedure. Then the program examines trees having similar topology to compare with the initial tree. If a tree with a shorter length is found, this tree replaces the initial tree and new searches begin from this tree. This process stops at a certain round when no shorter trees are found within the similar trees. Heuristic searches may not find the MP tree, because the most parsimonious tree(s) may not be similar enough to the intermediate trees so that they are not omitted from the examination.

Maximum likelihood method

Maximum Likelihood (ML) method tests a model of changes for a particular tree.

To a phylogenetic tree, the likelihood is the probability of observing the data in a given tree and under a specified model of character changes (Graur and Li, 1999). The likelihood for each site is calculated based on assumptions on nucleotide substitution and 39 the branch lengths, and the likelihood for all the sites is calculated as the product of the likelihoods of each individual site, assuming the changes at different sites are independent of each other (Li, 1997;Graur and Li, 1999). The algorithm examines all the possible trees and finds the one with the largest likelihood value. Different substitution models may find different ML trees, since the likelihood calculation is dependent on the substitution model. ML methods involve sophisticated statistical theory and extremely intensive calculations, so that they were not used very often in the past. Their application has increased recently with the rapid development of computational technology.

Bootstrap

The bootstrap method, a kind of resampling technique, was introduced into phylogenetic studies to estimate the confidence level of phylogenetic hypotheses (Li,

1997;Graur and Li, 1999). The procedure starts with a phylogenetic tree, which is the null hypothesis to be tested by bootstrap. In a phylogenetic tree analysis, a null hypothesis can have many subhypotheses, which are the clades and the included OTUs in each clade.

The second step is to build a number of pseudosamples, usually a few hundred to one thousand, to test the hypotheses. A pseudosample is an aligned matrix of the same size as the original sample, with each site of a pseudosample randomly chosen from the original sample. In the resampling process, each site has an equal chance to be sampled and resampled to any other site. This means some sites from the original sample can be sampled more than once and some are never sampled. Each pseudosample is used to build a tree using the same method used to build the phylogenetic tree under testing. Each subhypothesis is tested with this tree, either supported or rejected. After going through 40 the desired number of pseudosamples, each subhypothesis has a percentage of being supported, which is the bootstrap value. To get relatively accurate bootstrap values, several hundred resampling processes are needed, which is especially true in dealing with large numbers of OTUs. Bootstrap values are placed on the internal branches as confidence levels for the clades. It should be pointed out that this is not accurate (Graur and Li, 1999), since the bootstrap method may underestimate the confidence level at high bootstrap values and overestimate it at low values. 41

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Zhang, J., A. Pekosz, and R. A. Lamb. 2000. Influenza Virus Assembly and Lipid Raft

Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins. J. Virol.

74(10), 4634-4644. 58

CHAPTER II

SURVEILLANCE OF INFLUENZA A VIRUSES IN ENVIRONMENTAL ICE

AND WATER SAMPLES

ABSTRACT

Virological surveillance of influenza A virus (IAV) was conducted on glacial ice samples collected from the Antarctic and Arctic, on ice and water samples collected in northeastern Siberia lakes in spring and fall seasons of consecutive years, and on snow samples, river ice samples, and pond water samples collected locally in Ohio, USA. A total of 84 unique sequences of IAV H1 fragments were retrieved from northeastern

Siberia lake samples, specifically 83 from a lake ice sample and 1 from a lake water sample. No sequences were detected in any other samples. In this study, a PCR-based method of high sensitivity IAV detection was developed. 59

INTRODUCTION

Influenza A virus (IAV) is an important human pathogen. During any given year, influenza epidemics kill 500,000 to 1,000,000 people world-wide, and hospitalize millions more (Layne et al. 2001). Beside humans, IAV also infects other animal hosts, including swine, horses, domestic fowl, dogs, cats, many marine mammals, and numerous wild birds. Wild aquatic birds and shorebirds are the ultimate reservoir of all

IAV subtypes (Webster et al. 1992; Fouchier et al. 2005). IAVs of avian origin were not thought to directly transmit to humans because of the host species barrier. However, in the past few years direct transmission of avian IAVs to humans has been observed and recorded in Southeast (Butt et al. 2005; Hien et al. 2004) and Europe (Arjan, 2004;

Navani, 2004). Historical records indicate that the establishment of a new IAV subtype in humans was always associated with severe global IAV pandemics. Fortunately, recent transmissions have not spread widely. Besides direct transmission, avian IAVs can contribute parts of their genomes to human IAVs through genetic reassortment.

In wild aquatic birds, IAV is largely an enteric virus, infecting the intestinal tissues. Wild birds shed huge quantities of IAV virions in their fecal materials.

10 Experimentally infected ducks shed an estimation of 1×10 EID50 (50% mean egg infective dose) of AIV in 24 hours (Webster et al. 1978). Water contaminated by the fecal materials becomes an inoculum source, which can infect susceptible birds through the oral-fecal pathway. At the same time, contaminated water (or ice) becomes an abiotic reservoir. IAV can survive long periods of time in water, with a half-life of 3-6 days at

28˚C and 5-9 days at 17˚C in distilled water ( Stallknecht et al. 1990). The persistence of

IAVs in water is affected by pH, temperature, and salinity of the water (Stallknecht et al. 60

1990). Generally, the lower the temperature, the longer they persist. IAV can survive

repeated freeze-and-thaw cycles (Tumpey et al. 2002), indicating IAV virions can survive

even longer if frozen.

Surveillance of IAVs in all possible reservoirs, together with monitoring circulating IAV strains, will help health care agencies to be prepared for a possible IAV pandemic (Layne et al. 2001). Most studies to date have focused on the biotic reservoirs, especially wild birds, even though there have been a few reports on the isolation of IAV from abiotic samples, including fecal materials and wild bird-visited lake water (Ito et al.

1995). Environmental ice, however, as an important abiotic reservoir has been ignored

(Zhang et al. 2006). Environmental ice can preserve viable microbes for long periods of

time (Ma et al. 2000;Ma et al. 2005;Castello et al. 1999;Christner et al. 2005). According to Rogers et al. (2004), microorganisms entrapped in environmental ice could reenter the contemporary microbial population once they are released from the ice. This recycling is especially advantageous for pathogenic microbes, because the modern host species might be immunologically naïve to them. Smith et al. (2004) proposed that ice act as a huge repository for microbes in the recycling of human pathogenic viruses that could survive freezing and thawing, specifically caliciviruses, influenza viruses, and polioviruses.

On the earth, migratory wild birds make their annual migrations following certain migration flyways. Siberia is a major breeding site for wild birds. A few major migratory flyways overlap in Siberia, specifically the East Asian/Australian, Central Asia, Pacific,

and Black Sea/Mediterranean flyways. As the vectors and reservoirs of IAV, migratory

wild birds from different locations carry various IAV subtypes and strains. Through the

oral-fecal transmission pathway, they infect others with the IAV strains they carry and 61 pick up novel IAVs deposited by other birds. When they leave the breeding sites, IAVs are transported within the birds. Wild birds may transmit IAVs to humans indirectly, through intermediate hosts domestic fowl and possibly swine. IAV isolated from duck droppings in Siberia contained genes closely related to the IAV of H5N1 isolated from domestic fowl in Hong Kong in 1997 (Okazaki et al. 2000).

Previously, we have reported the detection of IAV RNA fragments in northeastern

Siberian lake ice and lake water samples (Zhang et al. 2006). We have extended the study by assaying more Siberian lake ice and lake water samples, collected in different seasons of 3 consecutive years. Additionally, glacial ice samples from Greenland and from the

Antarctic, as well as ice and water samples collected locally in Ohio, USA, were tested with RT-PCR. The underlying hypothesis is that glacial ice might have preserved wild- transported IAV virions. Additionally, Greenland is within the East Atlantic flyway, so that Greenland glacial ice might also contain virus particles discharged by wild birds migrating between North America and Europe (Shoham and Rogers, 2006). Antarctica is not traversed by any major avian migration flyway. However, local birds in Antarctica were infected with IAV (Baumeister et al. 2004; Wallensten et al. 2006), indicating that

Antarctic glacial ice may contain IAVs originating from local avian species. 62

MATERIALS AND METHODS

Primer design

Primers were designed for IAV genes H1 (hemagglutinin, subtype 1), N1

(neuraminidase, subtype 1), M (matrix protein), and NS (nonstructural protein), because we are only interested in those IAV subtypes that infect humans. For the H and N genes, only a few of them have ever widely circulated in the human population. They are H1,

H2, H3, N1 and N2. Our study was therefore focused on these subtypes. H1 and N1 genes represent the H1N1 subtype circulating in humans, and the subtype responsible for the 1918 pandemic. In the 8 RNA segments of the IAV genome, H and N genes are the most variable, with multiple subtypes of each. Even within one subtype, H and N genes sometimes have variability approaching 20%. M and NS genes are the most conserved

IAV genes, but still with a high degree of sequence divergence. Designing absolutely universal primers for any IAV gene is very difficult, if not impossible, even though there have been reports of using universal primers in the detection of IAV. As a result, individual sets of primers need to be designed for each H or N gene subtype.

The IAV gene sequences were retrieved from Genbank at http://www.ncbi.nlm.nih.gov/ and from the Influenza Sequence Database (ISD) at http://www.flu.lanl.gov/. Because we do not know which virus variants exist in the samples, efforts were made to design primers that cover all human variants to the extent possible. Special attention was paid to sequence collection, so that the collected sequences represent each gene of different host origins, year and location of isolation.

Collected sequences were aligned with Clustal-X 1.83 and The alignment matrixes were examined and manually adjusted, when necessary. Primers were designed from 63 conserved regions, using an online platform (http://seq.yeastgenome.org/cgi-bin/web- primer). Conserved regions were selected for primer design, with expected amplicon size ranges from 200bp to 600bp, flanked by one or two primers in the middle to serve as primers for semi-nested or nested PCR.

IAV strains

In the laboratory of S.O.R at Bowling Green State University, two viral stains,

A/WS/33 (H1N1, ATCC number VR-1520) and A/Hong Kong/8/68 (H3N2, ATCC number VR-544) were obtained from American Type Culture Collection (ATCC). In

J.D.C’s lab in State University of New York, College of Environmental Science and

Forestry, Syracuse, NY, the viral stain A/PR8/33 (H1N1) was used.

Measurement of primer sensitivity

In SOR’s lab, the concentration of the virus solutions were calculated by counting the numbers of viral particles, which were spread on a Collodion-coated 300-mesh cooper grid, negatively stained with uranyl acetate, and observed using a transmission electron microscope. In Syracuse, the concentration of virus solution was determined by a titration end-point method. Primer sets were applied to amplify serial dilutions of IAV stock solution. The sensitivity of a primer set is defined as the minimum number of virions that can be consistently (more than 50% of the time) amplified by RT-PCR.

Ice and water samples 64

From 2001 to 2003, ice and water samples were collected from 8 lakes located

along the Kolyma River in northeastern Siberia, as described in Chapter II. This region is

overlapped by a few major avian flyways. The lakes are the breeding and feeding sites of

the annually visiting migratory birds, and thus are frequently visited by them. Numerous

migratory birds breed there in the summer and migrate south in the fall. In these lakes, ice

forms early in October, and thaws early in June. Ice samples were collected in winter or

early spring, and water samples were collected in summer and fall (Table 1). Additionally,

four glacial ice samples from Antarctica and Greenland obtained from National Ice Core

Laboratory (NICL, Denver, CO). These glacial ice samples may also preserve IAVs

deposited by wild birds, because Greenland lies within the avian East Atlantic flyway.

Processing of samples

All samples were melted and assayed under strictly sterile conditions. The sterile

culture room and sterile laminar flow hood (biosafety level II) was irradiated with

germicidal UV light for 30 min before and after each work session. All instruments used were either autoclaved or decontaminated with Clorox (containing 5.25% sodium hypochlorite) and 70% ethanol. Water and ice samples collected from northeastern

Siberia lakes were melt as described in Zhang et al. (2006). Glacial ice samples were decontaminated and melted following the procedures detailed in Rogers et al. (2004,

2005).

Samples were assayed either before or after concentration. Two methods were

used to concentrate the virus. In one method, 1 ml of lake ice melt water were added into

1.7-ml centrifuge tubes and subjected to centrifugation at 13.2krpm for 10min. After 65

centrifugation, 900 μl supernatant in each tube was gently removed and the remaining

100 μl was used for molecular assays. This centrifugation cannot pellet IAV virions.

However, studies on glacial ice have found that many microorganisms tend to associate with large particles, which pellet under these conditions. The lake water samples assayed in this study contain visible algal aggregates and many bacteria (as indicated by cultivation). It is hypothesized here that the IAV virions are associated with those large particles. Alternatively, an ultracentrifuge was used to spin down the viruses. Centrifuge tubes were cleaned and autoclaved. Eight microliters aliquot of ice melt water was added into each centrifuge tube. After balancing, the tubes were loaded into a 70.1 Ti rotor and centrifuged in a Beckman L8-M for 45 min at 35 krpm. When finished, the supernatant was gently removed with a vacuum pump and 100 μl of autoclaved nanopure water was added to the pellet and mixed well.

RT-PCR, semi-nested/nested-PCR, and sequencing

RT-PCR reactions were carried out with a GeneAmp EZ rTth RNA PCR kit

(Applied Biosystems, Inc., Foster City, CA). The rTth enzyme has both RNA-dependent

DNA polymerase and DNA-dependent DNA polymerase ability. In a PCR tube, 10 μl of

sample (concentrated or unconcentrated) was added into a reaction mixture to reach a

final volume of 25 μl, containing 25 pmol of each of the forward and reverse primers

(Table 1), 10 μM Tris-HCl [pH 8.3], 300 μM of each dATP, dCTP, dGTP, and dTTP, 2.5

mM manganese acetate, 50 mM bicine, 115 mM potassium acetate, 8% [wt/vol] glycerol,

and 2.5 U rTth DNA polymerase. The reverse transcription was carried out at 60˚C for 30

min. The following PCR was performed: 94˚C for 4 min, then 35 cycles of 94˚C for 1 66

min and 60˚C for 90 s, and a final extension of 60˚C for 8 min. Negative controls were set up by substituting the 10 μl of sample with 10 μl of autoclaved purified water. From each RT-PCR reaction, 0.5 μl of the product was taken for a nested-PCR or semi-nested

PCR. PCR products were examined by electrophoresis on standard agarose gel, purified through low-melting point gel, cloned into vectors, and the insertions were sequenced as described in Chapter II, to confirm if the amplified products are IAV gene. For detecting

IAV H1 genes, primers H1_1F and H1_5R were used in the RT-PCR reaction, followed by using primers H1_2F and H1_4R for a nested PCR. While in detecting NS gene, primer I_NS_1F and I_NS_3R were used in the RT-PCR, and primers I_NS_2F and

I_NS_3R were used for the following semi-nested PCR.

67

Table 1. Primers selected for detection of IAV genes. Targeting gene Primer name Sequence H1_1F ATGCSAACAACTCAACCGACAC H1_2F1 (H1_2Fa)TCAACCTACTTGAGGACAGTCACA (H1_2Fb)TTAACCTGCTCGAAGACAGCCACA H1 H1_4R CGGGTGATGAACACCCCATAGTA H1_5R2 (H1_5Ra )GGGTTCCAGCAGAGTCCAGTAGTA (H1_5Rb )GGGTTCTAGCAAGGTCCAGTAATA I_NS_1F TTRCCTTCYCTTCCAGGACATACT3 NS I_NS_2F GGATGTCAAAAATGCARTTGG3 I_NS_3R TTAAATAAGCTGAAAYGAGAARGTTCTT3 1Primer H1_2F is an equimolar mixture of H1-2Fa and H1-2Fb. Italicized letters are nucleotides that are different in the sister primers.

2Primer H1_5R is an equimolar mixture of H1-5Ra and H1-5Rb. Italicized letters are nucleotides that are different in the sister primers.

3R stands for mixed bases of A and G; Y stands for mixed bases of C and T. 68

RESULTS

Primer selection

For the H1, M, and NS genes, several sets of primers were designed. The

sensitivity of each primer set was determined using control viral strains. One IAV H1

gene primer set had a measured sensitivity of 25-250 (closer to 25) virions per reaction. A

primer set designed for the NS gene was even more sensitive, being able to detect 2-25

(closer to 2) virions in one reaction. These two primer sets were then extensively used in

surveillance assays below. The specificity of these two primer sets was not tested,

because non-specific amplifications were never observed in any amplification reaction.

Assays on environmental ice and water samples

A total of 11 ice and water samples from 8 northeastern Siberian lakes were

assayed in this study (table 2). In total, 1,203 RT-PCR reactions were performed using

the H1 primer set. Only 20 of 640 reactions on ice sample from Lake Park collected on

03/02/2002 were positive, yielding a total of 83 unique sequences from cloned PCR

products. Only 1 of 168 reactions was positive on the Lake Edoma water sample

collected in September 2001. From this reaction, one unique sequence was derived.

Glacial ice samples from Greenland and Antarctica, and ice and water samples collected locally in Ohio were assayed with H1 and NS primer sets separately (table 3). No positive result was obtained in more than 500 reactions.

69

Table 2. RT-PCR assays on northeastern Siberian lake ice and lake water samples.

Sample site Location Date Form of H1 NS Sequences

Samples Assays1 Assays

68˚30’ N, 09/2001 Water 35 80 0

Park 161˚25’ E 03/02/2002 Ice 640 (20) 166 83

04/10/2003 Ice 40 88 0

Edoma 68˚40’ N, 09/2001 Water 168 (1) 136 1

161˚45’ E 3/10/2003 Ice 40 56 0

Shchychie 68˚40’ N, 09/2001 Water 40 200 0

161˚45’ E 01/08/2002 Ice 40 40 0

Yakytskoe 70˚00’ N, 09/2002 Water 40 120 0

160˚00’ E

Tschchie N/A 12/10/2003 Ice 40 128 0

Cape 70˚05’ N, Summer 2002 Water 40 40 0

Chukochii 159˚55’ E

Lupka N/A 03/09/2003 Ice 40 40 0

Akhmelo 68˚50’ N, Summer 2003 Water 40 40 0

161˚00’ E

Total 1203 (21) 1134 (0) 84

1Numbers in parenthesis indicates the number of positive results. 70

Table 3. RT-PCR assays on glacial ice samples and water samples collected in Ohio.

Sample Sampling Date of Samples H1 NS Sequences name a location b collection/ forms Assays Assays

Length of

glacial ice(m)

b

CC-62/84 Camp 109.010- Glacial 80/0 80/0 0

Century, 109.355 ice

Greenland

LAV- Little 104.590- Glacial 80/0 16/0 0

58/68 America, 104.900 ice

Antarctica

B68 Byrd, 388.560- Glacial 40/0 0/0 0

Antarctica 389.040 ice

MT3C- Milcent, 96.345-96.565 Glacial 40/0 16/0 0

93/100 Greenland ice

BG snow N/A 12/04/2003 Snow 0 64/0 0

Maumee N/A 02/12/2005 Ice 0 80/0 0

River

BG pond 41˚23’ N, 03/21/2004 Water 0 104/0 0

83˚37’ W

Total 200/0 360/0 0

71 a For glacial ice, sampling site means drilling site and year of operation. CC -62/84: glacial ice core drilled at Camp Century, Greenland in the year of 1962; LAV-58/68: drilled at Little America V in Antarctica;

B68: drilled at Byrd, Antarctica; MT3C: Milcent, Greenland. b N/A: information not available c Lengths are given from the top and bottom of ice core section to the upper glacial surface 72

DISCUSSION

Conventional virus culture methods and PCR-based methods are the main

methods employed for clinical diagnosis and epidemiological surveillance of IAV.

Compared with conventional virus culture methods, PCR-based methods are more

sensitive, less time-consuming, less expensive, and can detect nonliving viruses. PCR-

based methods have been widely used in assays of IAV on a variety of samples (Fouchier

et al. 2000; Herrmann et al. 2001; Ludwig et al. 1991).

Many primers for different IAV genes are available from published reports.

However, detailed examination of the primers reveals that they were not guaranteed to be of high sensitivity, since they were usually applied to IAV cultures, in which the amount of RNA template was abundant and was not an important consideration. Also, those primers often were targeted on IAV genes of limited host ranges, e.g. of wild aquatic birds and shorebirds (Widjaja et al. 2004), or of humans (Herrmann et al. 2001). Primers thus were specifically designed in this study and evaluated for sensitivity. IAV genes of different host origins from the earliest to the most recent time were collected and aligned for primer design to ensure the primers can amplify most of the sequences. According to

He et al. (1994), primers are decisive for the sensitivity of PCR reactions, and different primers targeting the same fragment could have a 1000-fold difference in terms of sensitivity. Our strategy was to design several primer sets for each of the IAV genes studied (H1 and NS). By testing with control viruses, the primer sets of the highest sensitivity were selected for subsequent assays. Results demonstrated that one H1 primer set had a sensitivity of 25-250 virions per reaction, and one NS primer set had a sensitivity of 2-25 virions per reaction. It is estimated that the minimum number of 73

virions needed to cause an infection is around 10 to 100. This means that some PCR-

based methods are more sensitive than conventional virus culture methods because the

ratio of infectious units to physical particles of IAV is low and there is lack of universally

susceptible host cells for IAV of different host origins (Fouchier et al. 2000). The

specificity of the primers was not specifically tested. However, the high specificity was

implied by the fact that no false positive result was ever obtained with the selected H1

primers and the NS primers in RT-PCR performed directly on non-purified ice-melt

water, which contained abundant microorganisms (including visible algae aggregates and

cultivable bacteria).

A total of 1,403 RT-PCR reactions using H1 primers were performed on 6

northeastern Siberian lake water samples, 6 northeastern Siberian lake ice samples, 4

glacial ice samples, 1 pond water sample (collected in Ohio, USA), and 1 river ice sample

(collected in Ohio, USA). Twenty-one positive reactions were achieved in 2 samples, the

Lake Park ice sample collected in March 2002 (with 20 positive reactions) and the Lake

Edoma water sample collected in September 2001 (with 1 positive reaction). Meanwhile,

1,494 RT-PCR reactions with the NS primers were conducted on the same samples. No

positive reaction resulted from any of these samples. At the same time, the H1 primer set

did not consistently amplify IAV genes from these samples either. It seemed that the

distributions of IAV fragments in these samples were not uniform. This is similar to the findings for other microbes in glacial ice. Previous studies have found microorganisms in glacial ice are scattered throughout the samples, with some local high concentrations, and larger areas that are devoid of microbes. Although the positive reaction rates were low, they were consistent with other studies. Okazaki et al. (2000) and Ito et al. (2000) 74

reported that only 0-3% reactions were positive in assaying for IAV in wild bird fecal

materials and wild bird-visited water, using virus culture method. In our studies, 1.5% of

the reactions were positive with the H1 primer set, while there was 0% positive for the

NS primer set.

RNA fragments may not survive if not protected in a complete virion. In our

study, the first RT-PCR amplicon of the H1 gene was more than 600 bp, indicating good

preservation of viral RNA. The successful amplification of large viral RNA fragments

(>600bp) indicates that the virions are well preserved, and possibly complete and

infectious. Jeffery Taubenberger and his colleagues previously reported great difficulties

in the reconstruction of the 1918 “Spanish” IAV genome from permafrost-preserved

victims (Reid et al. 2000; Reid et al. 2002; Taubenberger et al. 1997; Taubernberger et al.

2005). According to their studies, no RNA fragments of more than 120 bp were ever

isolated, indicating a severe decomposition of the viral RNA. The size of the fragments we were able to amplify indicates much better condition of the RNA, and it is likely that at least some complete virions were present in the ice samples.

Despite the fact that Lake Park was sampled only once a year for three

consecutive years (2001, 2002, and 2003) and Lake Edoma was sampled twice in two

years (2001 and 2003 separately), and that three lakes (Lake Park, Lake Shchychie and

Lake Edoma) were sampled for water in September 2001, IAV genes were detected only

in one Lake Park ice (March 2002) and one Lake Edoma water sample (September 2001)

were detected of IAV genes. We suspect that had we been able to test for all 16

hemagglutinin subtypes, IAV might have been detected in many of these samples. More

than one subtype may co-occur in some samples. Surveillance of IAV in migratory birds 75 demonstrated that the isolation frequency varied greatly with sampling locations and sampling time. Samples collected in the same locations in different years or collected at the same time but from different locations, yielded significantly different results.

In all our assays, reverse transcription and first round PCR did not produce visible

PCR products in ethidium bromide stained gel electrophoresis. A second round of nested-

PCR or semi-nested PCR was needed to make the PCR products visible in agarose gel electrophoresis. Clearly, the amount of IAV RNA fragments in these samples was essentially very low, just on the edge of being detectable by our methods. Nonetheless, the amounts that we were able to detect would be sufficient to infect individual birds, were they to ingest viable virions. 76

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12229-12235. 81

CHAPTER III

EVIDENCE OF INFLUENZA A VIRUS RNA IN SIBERIAN LAKE ICE

ABSTRACT

Influenza A virus (IAV) infects a large proportion of the human population annually, sometimes leading to the deaths of millions. The biotic cycles of infection are well characterized in the literature, including studies of populations of humans, poultry, swine, and migratory waterfowl. However, there are few studies of abiotic reservoirs for this virus. Here, we report the preservation of IAV genes in ice and water from high- latitude lakes that are visited by large numbers of migratory birds. The lakes are along the migratory flight paths of birds flying into Asia, North America, Europe, and Africa. The data suggest that IAV, deposited as the birds begin their autumn migration, can be preserved in lake ice. As birds return in the spring, the ice melts releasing the viruses.

Therefore, temporal gene flow is facilitated between the viruses shed during the previous year and the viruses newly acquired by birds during winter months spent in the south.

Above the Arctic Circle, the cycles of entrapment in the ice and release by melting can be variable in length, because some ice persists for several years, decades, or longer. This type of temporal gene flow might be a feature common to viruses that can survive entrapment in environmental ice and snow.

82

INTRODUCTION

IAV is infamous for its ability to cause seasonal human epidemics, affecting

approximately 10 to 20% of the world’s population every year (WHO, 2003).

Occasionally, it exhibits extreme virulence in poultry as well, bringing about unparalleled

economic losses. Pandemics of IAV in 1918 (subtype H1N1), 1957 (subtype H2N2), and

1968 (subtype H3N2) led to over half a million human deaths in the United States alone.

The World Health Organization and the Centers for Disease Control and Prevention continually plan for the next worldwide pandemic and have stressed the importance of both disease and virus surveillance. Therefore, it is important to identify all of the biotic as well as abiotic reservoirs for this virus. Ice potentially constitutes an abiotic reservoir of prime importance for influenza virus over short and long periods of time, particularly

in the Siberian region, which encompasses several migration routes of a variety of

waterfowl. Aquatic birds are the primary biotic reservoir for all influenza viruses

(Horimoto and Kawaoka, 2001). All IAV subtypes (H1 to H16 and N1 to N9) have been

isolated from birds and are considered to have descended from a primordial avian source

(Glezen, 1996; Webster, 1998). Infection in domestic and wild species is usually

asymptomatic, with occasional epizootic activity (Webster, 1997). Subclinically infected

ducks excrete enormous quantities of viral particles (up to 108 particles/gram feces).

Although the primary hosts are birds, a variety of IAV subtypes have been isolated from

mammals, including swine, horses, seals, whales, felines, canines, and humans. It is of

considerable note that H1N1, the subtype that resulted in over 40 million human deaths

during the pandemic of 1918, continues to circulate in avian species (Reid et al. 1999).

Other subtypes currently circulating (e.g., H5N1) have the potential to cause similar 83 degrees of human mortality. IAV is primarily an apathogenic enteric virus of birds and secondarily a pathogenic respiratory virus of mammals. Moreover, the spectrum of its avian hosts immeasurably exceeds that of its mammalian ones; the virus is thought to be fundamentally an archaic parasite of ancestral waterfowl. It is a prominent waterborne virus, persisting chiefly through fecal-oral circulation, and a common virus of holarctic migratory aquatic birds. The Chinese-Siberian axis populations of migratory waterfowl play a role of paramount importance in the evolutionary course and dynamics of IAV, due to the high percentage of infected individuals, the wide exchangeability of viral

RNAs, and the broad range of enduring variant viruses (both at a given point of time and throughout long eras). Those parameters are interrelated and yet independent of each other. One outcome of their coincidence is the periodical emergence of pandemic and epizootic IAV strains in the Chinese-Siberian domain. Most studies of IAV have focused on biotic modes of transmission. Abiotic sources tested have almost exclusively been aquatic. In previous studies, we documented the preservation of viruses, bacteria, and fungi in glacial ice up to 140,000 years old (Castello & Rogers, 2005; Castello et al. 2005;

Castello et al. 1999; Christner et al. 2005; Ma et al. 1999; Ma et al. 2005; Ma et al. 2005;

Starmer et al. 2005). In the present study, we report the results of testing for the presence of IAV genes in ice and water samples from three lakes in northeastern Siberia. The lakes are covered by ice for more than 6 months annually and are frequented by large populations of migratory waterfowl, some of which travel to North America and others that travel as far as southern Asia, Europe, and Africa. 84

MATERIALS AND METHODS

Ice and water samples

We sampled ice or water from three northeastern Siberian lakes in the Kolyma

River region (Fig. 1) and tested the samples for the presence of IAV using reverse transcription-PCR (RT-PCR) specific for the H1 version of the hemagglutinin gene. The lakes are approximately 100 km from the Arctic Ocean, in an area covered by hundreds of lakes. Ice forms on the lakes early in October and thaws early in June. The lakes sampled in our study do not contain permanent or semipermanent ice cover, as might probably be the case for Siberian lakes located north of 70°N. The significance of the lakes we sampled is for annual virus preservation and for demonstrating the feasibility of viral endurance in environmental ice. Maximum ice thickness ranged from 0.65 to 1.40 m.

Lake water temperatures varied from 2.0 to 8.0°C at the bottom to 22.0 to 27°C at the surface during the summer, and from 0.3 to 2.5°C at the bottom to 0.0°C immediately below the ice during winter. Lake Edoma (also called Yedoma) and Lake Shchychie (also called Shuchi) are thermokarst lakes on a Late Pleistocene fluvial plain isolated from the

Kolyma River, the primary river in the area. Lake Park also is a thermokarst lake but is within the floodplain of the Kolyma River between two residual outcrops of Late

Pleistocene fluvial plain. Periodically, Lake Park is flooded by the Kolyma River. Due to its distance from human settlements, Lake Park is often visited by birds and has large avian nesting areas. Each of the lakes is frequented by migratory birds (although the visitation and nesting frequencies vary [Table 1]). The lakes are along the flight paths of migratory waterfowl, which fly into temperate and , North America, Europe, and Africa for wintering. Samples (300 to 500 ml each) were collected in September 85

Fig. 1. Locations of lakes assayed. All of the lakes are in northeastern Siberia, near the Kolyma River

(indicated by large arrow on upper map). The lower satellite view shows the locations for each lake

(indicated by arrows). Hundreds of lakes cover the Kolyma River delta region. Most, including Lake Park, are in the flood plains of the rivers and rivulets. Some of the lakes, including Lake Edoma and Lake

Shchychie, are on an elevated area above the flood plain. 86

Table 1. List of lakes assayed or to be assayed for the presence of influenza A virus.

87

2001 (water), from Lake Edoma, and March 2002 (ice), from Lake Park and Lake

Shchychie. Water samples were collected in autumn during the beginning of mass

migration of birds. The water was collected in sterile bottles at stations that were 1.5 to

2.0 m from the lake edge, very close to areas frequented by migratory waterfowl.

Samples were kept at temperatures between 1 and 5°C during the transportation to the lab

and then were frozen at -80°C. Ice was collected during the winter at 10 to 15 m toward

the lake center from the edge (very close to areas frequented by waterfowl) to avoid the deep near-shore snow cover. Ice was removed with sterilized instruments. The ice samples were placed into a double plastic pack and melted in the lab without contact with air. Then, the meltwater was placed into sterilized bottles and frozen at -80°C.

Sample processing

In the lab, rigorous attention to avoidance of contamination was maintained

throughout the procedures (Ma et al. 1999; Ma et al. 2005; Ma et al. 2000; Rogers et al.

2004; Rogers et al. 2005). The sterile culture room and sterile biosafety laminar flow

hood were bathed with germicidal UV radiation for at least 30 min prior to each work

session (as well as 30 min after each session). All laboratory benches were cleaned with

undiluted Clorox (5.25% sodium hypochlorite) and 70% ethanol prior to, and following, a work session. The ice and frozen water samples were melted at room temperature in the sterile laminar flow hood. The meltwater was distributed into sterile 1.5-ml microfuge tubes.

88

Molecular assays

Meltwater from 5 tubes for each sample was assayed immediately (as described

below) while the other samples were frozen and stored at -80°C until needed. Aliquots of

10 μl per sample were subjected to RT-PCR amplification. Primers (forward, H1-1f

[ATGCSAACAACTCAACCGACAC]) and reverse (an equimolar mixture of H1-5ra

[GGGTTCCAGCAGAGTCCAGTAGTA] and H1-5rb

[GGGTTCTAGCAAGGTCCAGTAATA]) were used at 25 pmol each, in 25-μl reaction mixtures. Reverse transcription was performed with a GeneAmp EZ rTth RNA PCR kit

(Applied Biosystems, Inc., Foster City, CA) using the reaction mix provided (10 μM

Tris-HCl [pH 8.3]; 300 μM [each] dATP, dCTP, dGTP, and dTTP; 2.5 mM manganese acetate; 50 mM bicine; 115 mM potassium acetate; 8% [wt/vol] glycerol; and 2.5 U rTth

DNA polymerase) at 60°C for 30 min. This was followed by PCR (with the same reaction mix and enzyme) using the following temperature regime: 94°C for 4 min and

35 cycles of 94°C for 1 min and 60°C for 90 s, followed by a final extension at 60°C for

8 min. Next, nested PCR was performed using 0.5 μl of the RT-PCR mixtures described above. The nested primers consisted of a forward primer (an equimolar mixture of H1-2fa

[TCAACCTACTTGAGGACAGTCACA] and H1-2fb [TTAACCTGCTCGAAGACAG-

CCACA]) and a reverse primer (H1-4r [CGGGTGATGAACACCCCATAGTA]) specific for the influenza A virus hemagglutinin H1 gene (25 pmol each). The following temperature regime was used: 94°C for 5 min; then 45 cycles of 94°C for 1 min, 54°C for

1 min, a 0.3°C-per-s increase to 72°C, and 72°C for 1 min; followed by 72°C for 8 min.

The PCR products were subjected to electrophoresis on 1.5% standard agarose (Bio-Rad

Laboratories, Hercules, CA) gels in TBE (89 mM Tris base, 89 mM borate, 2 mM EDTA, 89 pH 8.0, with 0.5 μg/ml ethidium bromide). Following electrophoresis at 5 V/cm, the gels were viewed and photographed under 300 nm UV light. In total, 373 amplification attempts were performed using meltwater from Lake Park ice. An additional 40 amplification attempts were made using meltwater from Lake Shchychie ice. A total of

161 amplification attempts were performed with water from Lake Edoma. Multiple positive and negative controls were included for every set of RT-PCR experiments. When amplification bands of the expected molecular weight were observed, another aliquot of the reaction mixture was subjected to electrophoresis on a 1.0% low-melting-point agarose gel (NuSieve GTG; FMC, Rockville, ME) in TBE. The bands then were eluted from the gels (Tautz & Renz, 1983; Thuring et al. 1975) and rehydrated in water. The eluted amplicons were then ligated into plasmid vectors (pCR2.1-TOPO; Invitrogen

Corp., Carlsbad, CA). Following transformation of host bacteria, the recombinant clones were identified by growth on selective medium. The recombinant plasmids were extracted from the bacterial host cells and purified, and the inserts were amplified using

M13 primers (forward primer, CAGGAAACAGCTATGAC; reverse primer,

GTAAAACGACGGCCAG). The amplicons were gel purified (as described above) and then used in DNA sequencing reactions. For sequencing, approximately 30 ng of DNA was added to a reaction with the Terminator Ready Reaction kit, version 3.0 (ABI, Foster

City, CA). The cycling program was as follows: 1 min at 94°C and then 30 cycles of

94°C for 10 s, 50°C for 30 s, and 60°C for 4 min. The amplified DNA was precipitated by adding ethanol to a final concentration of 65% and incubating at room temperature for

15 min. Then the DNA was subjected to centrifugation in a microfuge (13,200 rpm) for

20 min. The pellets were washed with 80% ethanol and then dried under a vacuum. The 90

dried pellets were rehydrated with template suppression reagent (ABI, Foster City, CA),

denatured at 94°C for 4 min, and chilled on ice. Then the solutions were loaded into an

ABI (Foster City, CA) 310 automated DNA sequencer for determination of the influenza

A virus H1 gene sequences.

Data analysis

The sequences were compared to those in NCBI databases by using BLAST

searches (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences exhibiting the highest

similarity to influenza A virus H1 sequences (a total of 84) were aligned using

CLUSTALW (http://searchlauncher.bcm.tcm.edu/multi-align/multi-align.html). A broad selection of H1 sequences from the NCBI database also was included in the alignment.

Following alignment, the sequences were examined, and small manual adjustments were made as needed. Phylogenetic analyses were performed with PAUP (phylogenetic analysis using parsimony) using neighbor-joining and maximum parsimony. Consistency, homoplasy, retention, and rescaled consistency indices were calculated in the parsimony analysis. 91

RESULTS

The highest frequencies of IAV H1 genes were found in ice from Lake Park.

Twenty of the 373 RT-PCRs using Lake Park ice meltwater yielded amplification bands of the expected size. After cloning, a total of 83 unique sequences resulted from these 20 positive reactions. Lake Park has the highest concentration of birds, including cranes, ducks, geese, gulls, loons, sandpipers, swans, and terns (Table 1). In 40 attempts to amplify (by RT-PCR) H1 genes from Lake Shchychie ice, no amplification was evident.

This lake had the lowest bird visitation rate (including no observed visits by geese) of the three lakes (Table 1). Only 1 of the 161 attempts to amplify the H1 gene from Lake

Edoma water yielded an amplicon of the expected molecular weight. A single sequence resulted. This lake is only occasionally visited by geese and other birds (Table 1). In

BLAST searches of NCBI databases, the H1 gene amplicons were most similar to those previously isolated with neuraminidase gene subtypes N1, N2, and N5. Phylogenetic analyses indicated that while the viruses exhibit genetic diversity (Fig. 2), they form a monophyletic cluster (Fig. 3) in contrast to other H1 sequences. Comparison with a wide variety of H1 gene sequences indicates that the population in the Lake Park ice is most closely related to subtypes that were isolated from both avian and porcine hosts in the

1930s and 1960s (Fig. 3). They are distantly related to the H1 subtype from the 1918 pandemic. Our results indicate the following: (i) the highest frequencies of detection of influenza A virus RNAs are in the lakes with the highest concentrations of migratory waterfowl; (ii) influenza A virus RNA is preserved in higher concentrations in lake ice than in lake water (also of note is that the fragment we consistently were able to amplify was 610 nucleotides in length, indicating good preservation of the RNA, which implies 92

Fig 2. Neighbor-joining phylogram of the influenza virus hemagglutinin H1 gene sequences isolated from Lake Park ice (collected in March 2002) and Lake Edoma water (collected in September 2001).

Numbers indicate sequences from clones derived from nested RT-PCR mixtures. The number before the decimal point indicates the RT-PCR number, while the number after the decimal point indicates each unique clone from the reaction. Sequences from the control virus (A/WS/33, clone p1.9) and from the 93

Brevig Mission, Alaska, subtype H1 (accession number AF116575), also are shown. The Brevig Mission sequence was used as the outgroup. LPI indicates cloned sequences from Lake Park ice, while LEW indicates the one clone from Lake Edoma water. 94

Fig 3. Maximum parsimony phylogram of a wide selection of hemagglutinin H1 gene sequences, including selected sequences from this study. Gaps were scored as a fifth base. There were more than

2,000 most-parsimonious trees, with placement of several of the sequences varying. Primarily, very closely related sequences shifted relative to one another. However, the relationships of the influenza A virus H1 sequences from this study with the other sequences were consistent in all of the trees. The tree shown has

1,128 steps, with consistency, homoplasy, retention, and rescaled consistency indices equal to 0.5417,

0.4583, 0.8562, and 0.4638, respectively. The H1 genes from Lake Park ice and Lake Edoma water are closest to those from avian strains isolated in Asia in 1933 and 1967. Also, they were related to strains from 95

1938 and 1939 isolated from swine in the . These are embedded within a large clade that includes a wide range of H1 sequences isolated from humans from the 1930s to the present. There is a more distant relationship with the H1 gene from the 1918 H1 influenza A virus (upper clade) and avian strains from 1976 through 1985 (lower ingroup clade). Sequences from H6 influenza virus strains were used as representatives of the outgroup. Abbreviations for sources: Av, avian; Hu, human; Sw, swine. 96 good preservation of the virus); (iii) the H1 gene population in the lakes is genetically heterogeneous; (iv) the single H1 gene found in Lake Edoma is similar to the H1 genes in

Lake Park, indicating that this gene likely is from the same population of virions; (v) the

H1 sequences in this study are closest to those found in Europe during the 1930s and in

Asia during the 1960s; and (vi) the H1 sequence from an H1N1 specimen (Brevig

Mission, Alaska, 1918) (Reid et al. 1999) is distantly related to all of the H1 genes from

Lake Park and Lake Edoma that were characterized in this study. 97

DISCUSSION

This is the first report of the persistence of influenza A virus in lake ice, as reflected by enduring genes. It indicates a potential long-term survival mechanism for the virus. Ice may act as a reservoir for influenza A viruses, preserving them for later release and infection of animals, including migratory waterfowl and humans. Surveillance of

Arctic and subarctic lakes for influenza virus may aid health professionals to improve prediction of influenza virus subtypes that are circulating at particular points in time, thus facilitating long-term vaccination strategies. Furthermore, surveillance may shed some light on a fundamental apparatus allowing for abiotic long-term perpetuation of multiform IAV strains. Cold temperatures and freezing preserve most types of viruses, including influenza virus (Parker & Martel, 2002; Rogers et al. 2005; Rogers et al. 2004;

Shoham, 2005). Experimentally, the feasibility of IAV endurance in the frozen state has been demonstrated, implying its survivability in frozen lakes. Inactivation of 99% of a virus population occurs in approximately 1 week when water temperatures are between

22 and 25°C. However, 10 weeks is required to inactivate the same proportion of the virus population if the temperature is between 3 and 5°C. The rate of virus degradation slows down to an even greater extent at and below freezing, and it continues to decrease as the temperature is lowered. This trend continues to below -80°C. We have found that viruses and bacteriophage (as prophage) frozen in glaciers can be preserved for well over

100,000 years (Castello et al. 2005; Castello et al. 1999). We previously reported on viral

RNA preserved in ice that was approximately 140,000 years old (Castello et al. 1999) and have additional unpublished data supporting preservation to many times this age.

Therefore, glaciers and ice-covered lakes may be an unrecognized major reservoir of 98 microbes. For pathogens, this is advantageous, since upon reemergence specific genotypes may interact with host populations that may lack resistance or immunity. Some influenza virus strains have appeared, disappeared, and then reemerged decades later virtually unchanged (Nakajima et al. 1978; Scholtissek et al. 1978), indicating the presence of an abiotic mode of preservation. For example, the Russian influenza virus subtype (H1N1) that caused an epidemic in 1977 was nearly identical to the subtype

(H1N1) that caused an epidemic in 1950. Other strains, most notably specific genotypes of H2N2 and H3N2 and several H1 variants, have made similar returns. Since influenza virus is an RNA virus, the rates of mutation should have been rapid if the viruses had been reproducing in biotic hosts during those years. A possible explanation for the slow rates of mutation is that the strains may have been preserved in some way during the decades between the epidemics. Preservation in ice is a possible explanation (Castello &

Rogers, 2005; Parker & Martel, 2002; Rogers et al. 2004; Shoham, 2005; Smith et al.

2004). Ice and ice-covered lakes (as well as glaciers) may act as huge reservoirs of preserved viruses. Therefore, annually and perennially frozen lakes (some are frozen continuously for decades or longer) along the paths of waterfowl migration routes have the potential for being major sources of viruses that cause pandemics and epizootics in birds and other animals. Virological surveillance of these lakes is needed in order to assess the relationships between the prevalence of current as well as earlier influenza A virus subtypes (including sequence characteristics) and endemic and epidemic occurrence of disease. This probably relates to other diseases as well, but this awaits thorough examination. One expectation in relation to this phenomenon would be an increased rate of release of these microbes during times of global (or local) warming events and a 99 decrease during cooler periods. Bird populations maintain extensive, long-term contact with the most northerly bodies of water, particularly in remote Siberian lakes, which represent perennial freezing-thawing periodicities of high variability, reaching, at the maximum, intervals of decades or longer. This means that influenza A viruses may be preserved in those lakes for years or perhaps much longer (e.g., the viability of microbes encased in ice for hundreds of thousands of years has been demonstrated in many studies

(Abyzov et al. 2005; Castello et al. 2005; Castello et al. 1999; Christner et al. 2005;

Faizutdinova et a. 2005; Ivanushkina et al. 2005; Ma et al. 1999; Ma et al. 2000; Ma et al.

2005; Rivkina et al. 2005; Rogers et al. 2005; Starmer et al. 2005; Vishnivetskaya et al.

2005). Thawing releases entrapped viruses of various age and thus seeds the water with concurrent strains regularly harbored by nearby sojourning birds. Until refreezing takes place, viruses of both present and past strains may be contracted by the waterfowl, whereas the remaining viruses would again be encapsulated by the subsequent formation of ice. Conceivably, such ongoing perpetual mechanisms have been operating cyclically throughout the virus’s evolution, enabling recurrent emergence of past genes and genomes. Most of the birds that carry IAV are migratory, such that the disease readily moves within the bird population from one locale to another. During the spring they move northward as the frozen lakes thaw. Starting in fall they move southward as the lakes freeze. The Kolyma River lowland birds travel along major migration paths to

Southeast Asia, North America, or the northwestern Pacific Ocean, while some travel to

Europe and North Africa. As the birds visit lakes along their paths they shed viruses into the lakes and onto the ice (when present) and drink water containing viruses discharged by other birds or released from the ice by thawing. Therefore, these lakes become abiotic 100 mixing pools for the viruses, while the birds are the biotic vessels where mixing occurs

(including replication and recombination). Since there are susceptible hosts along their migration path, they may pass the viruses to other birds as well as to swine, humans, or other animals. Our results support the hypothesis that ice acts as a long-term abiotic storage matrix for influenza virus and other microbes, including pathogens (Rogers et al.

2004; Shoham, 1993; Shoham, 2005; Smith et al. 2004). Furthermore, the cold lake water also is capable of preserving the virus, although presumably for shorter time periods.

Although the findings of this study are limited to the testing of three lakes, they point to a principal mechanism that may underlie a wider natural apparatus of abiotic long-term preservation of avian influenza viruses. The prevalence and extent of such a mechanism, which may bear a wealth of implications, should be further demonstrated through additional studies, including the exploration of more geographical sites, assays for related genes, and examination of viral endurance. At present, we describe the feasibility of the mechanism and supportive evidence at the level of gene recovery and analysis. 101

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Smith, A. W., D. E. Skilling, J. D. Castello, and S. O. Rogers. 2004. Ice as a reservoir for pathogenic animal viruses. Med. Hypoth. 63:560–566.

Starmer, W. T., J. W. Fell, C. M. Catranis, V. Aberdeen, L. J. Ma, S. Zhou, and S. O.

Rogers. 2005. Yeasts in the genus Rhodotorula recovered from the Greenland ice sheet, p.

181–195. In J. D. Castello and S. O. Rogers (ed.), Life in ancient ice. Princeton

University Press, Princeton, N.J. 105

Swofford, D. 2001. PAUP: phylogenetic analysis using parsimony, version 4. Sinauer

Academic Publishers, Sunderland, Mass.

Tautz, D., and M. Renz. 1983. An optimized freeze-squeeze method for the recovery of

DNA fragments from agarose gels. Anal. Biochem. 132:14–19.

Thuring, R. W. J., J. Sanders, and P. Borst. 1975. A freeze-squeeze method for

recovering long DNA from agarose gels. Anal. Biochem. 66:213–220.

Vishnivetskaya, T. A., L. G. Erokhina, E. V. Spirina, A. V. Shatilovich, E. A. Vorobyova,

A. I. Tsapin, and D. A. Gilichinsky. 2005. Viable phototrophs: cyanobacteria and green algae from the permafrost darkness, p. 140–158. In J. D. Castello and S. O. Rogers (ed.),

Life in ancient ice. Princeton University Press, Princeton, N.J.

Webster, R. G. 1997. Influenza virus: transmission between species and relevance to emergence of the next human pandemic. Arch. Virol. 13(Suppl.):105–113.

Webster, R. G. 1998. Influenza: an emerging microbial pathogen, p. 275–300. In R. M.

Krause (ed.), Emerging infections. Academic Press, New York, N.Y.

WHO. 2003. Influenza: report by the secretariat to the 56th World Health Assembly, 17

March 2003. A56/23. World Health Organization, Geneva, . 106

CHAPTER IV

SUPPLEMENT

My study indicated that large influenza A virus RNA fragments (610+ nt) were preserved in lake ice and lake water samples collected from lakes in Northeastern Siberia.

This is the first report of finding human pathogenic viruses from environmental ice

samples, which supports the hypothesis of ice can act as a reservoir for pathogenic animal

viruses (Smith et al 2004). Rogers et al. (2004) pointed out that the recycling of

pathogenic viruses through survival by ice could give these parasites advantages of being able to infect immunological naïve hosts when they are released from ice. If influenza A virus can do such, humans are facing threats of ancient influenza A virus from perennial ice, or other kinds of ancient ice. Our study has made a pioneering study in this field.

Nevertheless, with relatively small amount of data, it is hard to draw a very confirmative conclusion as to whether environmental ice entrapped influenza A virus will threaten humans. However, it is undoubtedly that further studies should be carried out, to accumulate more data, and to elucidate questions as to the abundance and viability of the virus in environmental ice. The following are a few suggestions and anticipations to the future work.

The location of a lake have direct associated with the freezing and thawing time of the ice in the lake. Table 1 lists the position, size, location of each lake, as well as the collectors of the samples.

107

Table 1. Sample collected from the lakes in north-eastern Siberia (Kolyma lowland).

108

Study by Stallknecht et al. (1990) indicated that the persistence of

influenza A virus in water was affected by the interactive effects of salinity, pH, and temperatures. Specifically, in a range of 6.2 to 8.2, the infectivity increased with pH, but decreased with salinity. Thus, the pH value and salinity of each sample to be tested should be assayed. From our study, we found that the distribution of influenza A virus

RNA (or possibly virions) in the samples were heterogeneous. For accuracy and consistency, one might need to sample a lake at different locations, as well as different depths. Additionally, a future study includes both environmental samples from Northeast

Siberia and Alaska might be very interesting. Alaska, similar to the Northeastern Siberia, locates in high latitude areas, contains major breeding sites for numerous migratory birds

(Ito et al. 1995), and is covered by a same avian migration pathway the “Asian-

Australian” pathway. It would be very interesting to see what subtypes of viruses are preserved in samples from each of the two sites.

Because of our limited samples and the heterogeneous distribution of influenza A virus in them, our study yielded limited amount of data. However, this does not mean the amount of influenza A virus RNA fragment (or influenza A virions) are in small amount.

Actually, in some assays, we found that the influenza A virus RNA fragments were exceeding 5,000 molecules per milliliter, which indicates the existence of more than

5,000 virions in 1 ml of ice. Considering the huge amount of lake ice, the preserved influenza A virus could be unimaginable. Even only a small proportion of the virus is alive, their release are going to affect the ecology of influenza A virus significantly, and more dangerously, impact humans health. Presently, we have no definite answer to the exact concentrations of influenza A virus RNA molecules (or influenza A virions) as well 109

as their viability. It is suggested here that viability assays on the environmental samples

should be conducted immediately. Although wild aquatic birds are the ultimate hosts of

all influenza A virus subtypes, the majority of our assays were focused on influenza A virus of H1 subtype, which generally only constitutes a small proportion of all influenza

A virus in wild aquatic birds. The outbreaks of avian flu have demonstrated that any

subtypes of influenza A virus could gain the ability to directly infect humans, without any

intermediate hosts. Thus, future surveillance of influenza A virus should not be focused

only on H1 subtypes, but should be expanded to all subtypes.

110

LITERATURE CITED

Ito, T., K. Okazaki, Y. Kawaoka, A. Takada, R. G. Webster, and H. Kida. 1995.

Perpetuation of influenza A viruses in Alaskan waterfowl reservoirs. Archives of

virology 140(7), 1163-72.

Rogers, S. O., W. T. Starmer, and J. D. Castello. 2004. Recycling of pathogenic microbes

through survival in ice. Medical Hypotheses 63(5), 773-7.

Smith, A.W., D.E. Skilling, J.D. Castello, and S.O. Rogers. 2004. Ice as a reservoir for

pathogenic animal viruses. Med. Hypoth., 63:560-566.

Stallknecht D. E., M. T. Kearney, S. M. Shane, P. J. Zwank. 1990. Effects of pH, temperature, and salinity on persistence of avian influenza viruses in water. Avian

Diseases 34(2), 412-8. 111

APPENDIX A Genbank accession numbers:

EF599681-EF599756

EF660192- EF660197

Eighty-one sequences in total.

JOURNAL OF VIROLOGY, Dec. 2006, p. 12229–12235 Vol. 80, No. 24 0022-538X/06/$08.00ϩ0 doi:10.1128/JVI.00986-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evidence of Influenza A Virus RNA in Siberian Lake Iceᰔ Gang Zhang,1 Dany Shoham,2 David Gilichinsky,3 Sergei Davydov,4 John D. Castello,5 and Scott O. Rogers1* Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 434031; Begin-Sadat Center for Strategic Studies, Bar-Ilan University, Ramat-Gan, Israel2; Soil Cryology Laboratory, Institute for Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia3; Pacific Institute of Geography, Russian Academy of Sciences, 678830 Cherskii, Republic of Sakha (Yakutia), Russia4; and Environmental and Forest Biology, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210

Received 12 May 2006/Accepted 28 September 2006

Influenza A virus infects a large proportion of the human population annually, sometimes leading to the deaths of millions. The biotic cycles of infection are well characterized in the literature, including in studies of populations of humans, poultry, swine, and migratory waterfowl. However, there are few studies of abiotic reservoirs for this virus. Here, we report the preservation of influenza A virus genes in ice and water from high-latitude lakes that are visited by large numbers of migratory birds. The lakes are along the migratory flight paths of birds flying into Asia, North America, Europe, and Africa. The data suggest that influenza A virus, deposited as the birds begin their autumn migration, can be preserved in lake ice. As birds return in the Downloaded from spring, the ice melts, releasing the viruses. Therefore, temporal gene flow is facilitated between the viruses shed during the previous year and the viruses newly acquired by birds during winter months spent in the south. Above the Arctic Circle, the cycles of entrapment in the ice and release by melting can be variable in length, because some ice persists for several years, decades, or longer. This type of temporal gene flow might be a feature common to viruses that can survive entrapment in environmental ice and snow. jvi.asm.org

Influenza A virus is infamous for its ability to cause seasonal whales, felines, canines, and humans. It is of considerable note human epidemics; it affects approximately 10 to 20% of the that H1N1, the subtype that resulted in over 40 million human world’s population every year (31). Occasionally, it exhibits deaths during the pandemic of 1918, continues to circulate in by on October 22, 2007 extreme virulence in poultry as well, bringing about unparal- avian species (15). Other subtypes currently circulating (e.g., leled economic losses. Pandemics of influenza A virus in 1918 H5N1) have the potential to cause similar degrees of human (subtype H1N1), 1957 (subtype H2N2), and 1968 (subtype mortality. H3N2) led to over half a million human deaths in the United Influenza A virus is primarily an apathogenic enteric virus of States alone. The World Health Organization and the Centers birds and secondarily a pathogenic respiratory virus of mam- for Disease Control and Prevention continually plan for the mals. Moreover, the spectrum of its avian hosts immeasurably next worldwide pandemic and have stressed the importance of exceeds that of its mammalian ones; the virus is thought to be both disease and virus surveillance. Therefore, it is important fundamentally an archaic parasite of ancestral waterfowl. It is to identify all of the biotic as well as abiotic reservoirs for this a prominent waterborne virus, persisting chiefly through fecal- virus. Ice potentially constitutes an abiotic reservoir of prime oral circulation, and a common virus of holarctic migratory importance for influenza virus over short and long periods of aquatic birds. time, particularly in the Siberian region, which encompasses The Chinese-Siberian axis populations of migratory water- several migration routes of a variety of waterfowl. fowl play a role of paramount importance in the evolutionary Aquatic birds are the primary biotic reservoir for all influ- course and dynamics of influenza A virus, due to the high enza viruses (8). All influenza A virus subtypes (H1 to H16 and percentage of infected individuals, the wide exchangeability of N1 to N9) have been isolated from birds and are considered to viral RNAs, and the broad range of enduring variant viruses have descended from a primordial avian source (7, 30). Infec- (both at a given point of time and throughout long eras). Those tion in domestic and wild species is usually asymptomatic, with parameters are interrelated and yet independent of each other. occasional epizootic activity (29). Subclinically infected ducks One outcome of their coincidence is the periodical emergence have been shown to excrete enormous quantities of viral par- of pandemic and epizootic influenza A virus strains in the ticles (up to 108 particles/gram feces). Although the primary Chinese-Siberian domain. hosts are birds, a variety of influenza A virus subtypes have Most studies of influenza A viruses have focused on biotic been isolated from mammals, including swine, horses, seals, modes of transmission. Abiotic sources tested have almost exclusively been samples of water. In previous studies, we doc- umented the preservation of viruses, bacteria, and fungi in * Corresponding author. Mailing address: Department of Biological glacial ice up to 140,000 years old (2–4, 10–12, 24). In the Sciences, 217 Life Sciences Building, Bowling Green State University, present study, we report the results from testing for the pres- Bowling Green, OH 43403. Phone: (419) 372-2333. Fax: (419) 372- 2024. E-mail: [email protected]. ence of influenza A virus genes in ice and water samples from ᰔ Published ahead of print on 11 October 2006. three lakes in northeastern Siberia. The lakes are covered by

12229 12230 ZHANG ET AL. J. VIROL.

MATERIALS AND METHODS

We sampled ice or water from three northeastern Siberian lakes in the Kolyma River region (Fig. 1) and tested the samples for the presence of influenza A virus using reverse transcription-PCR (RT-PCR) specific for the H1 version of the hemagglutinin gene. The lakes are approximately 100 km from the Arctic Ocean, in an area covered by hundreds of lakes. Ice forms on the lakes early in October and thaws early in June. The lakes sampled in our study do not contain perma- nent or semipermanent ice cover, as might probably be the case for Siberian lakes located north of 70°N. The significance of the lakes we sampled is for annual virus preservation and for demonstrating the feasibility of viral endurance in environmental ice. Maximum ice thickness ranged from 0.65 to 1.40 m. Lake water temperatures varied from 2.0 to 8.0°C at the bottom to 22.0 to 27°C at the surface during the summer, and from 0.3 to 2.5°C at the bottom to 0.0°C immediately below the ice during winter. Lake Edoma (also called Yedoma) and Lake Shchychie (also called Shuchi) are thermokarst lakes on a Late Pleistocene fluvial plain isolated from the Kolyma River, the primary river in the area. Lake Park also is a thermokarst lake but is within the floodplain of the Kolyma River between two residual outcrops of Late Pleistocene fluvial plain. Periodically, Lake Park is flooded by the Kolyma River. Due to its distance from human settlements, Lake Park is often visited by birds and has large avian nesting areas. Each of the lakes is frequented by migratory birds (although the visitation and nesting frequencies vary [Table 1]). The lakes are along the flight paths of migratory waterfowl, which fly into temperate and tropical Asia, North America, Europe, and Africa for wintering. Samples (300 to 500 ml each) were collected in September 2001 (water), from Downloaded from Lake Edoma, and March 2002 (ice), from Lake Park and Lake Shchychie. Water samples were collected in autumn during the beginning of mass migration of birds. The water was collected in sterile bottles at stations that were 1.5 to 2.0 m from the lake edge, very close to areas frequented by migratory waterfowl. Samples were kept at temperatures between 1 and 5°C during the transportation FIG. 1. Locations of lakes assayed. All of the lakes are in north- to the lab and then were frozen at Ϫ80°C. Ice was collected during the winter at eastern Siberia, near the Kolyma River (indicated by large arrow on 10 to 15 m toward the lake center from the edge (very close to areas frequented jvi.asm.org upper map). The lower satellite view shows the locations for each lake by waterfowl) to avoid the deep near-shore snow cover. Ice was removed with (indicated by arrows). Hundreds of lakes cover the Kolyma River delta sterilized instruments. The ice samples were placed into a double plastic pack region. Most, including Lake Park, are in the flood plains of the rivers and melted in the lab without contact with air. Then, the meltwater was placed and rivulets. Some of the lakes, including Lake Edoma and Lake into sterilized bottles and frozen at Ϫ80°C. by on October 22, 2007 Shchychie, are on an elevated area above the flood plain. In the lab, rigorous attention to avoidance of contamination was maintained throughout the procedures (10, 11, 12, 17, 19). The sterile culture room and ice for more than 6 months annually and are frequented by sterile biosafety laminar flow hood were bathed with germicidal UV radiation for at least 30 min prior to each work session (as well as 30 min after each session). large populations of migratory waterfowl, some of which travel All laboratory benches were cleaned with undiluted Clorox (5.25% sodium to North America and others that travel as far as southern hypochlorite) and 70% ethanol prior to, and following, a work session. The ice Asia, Europe, and Africa. and frozen water samples were melted at room temperature in the sterile laminar

TABLE 1. List of lakes assayed or to be assayed for the presence of influenza A virus

Elevation Width ϫ length; Influenza A virus H1 gene Lake Waterfowl observed (m) depth range (m) (isolation date, source) Edoma 25 200 ϫ 610; 1.0–14.0 Ducks (Anas spp. and Aythya spp.), gulls (Larus spp.), loons 1 sequence (September (Gavia arctica and G. stellata, some of which nested near the 2001, water) lake), sandpipers (Calidris spp., Limosa spp., and Tringa spp.), and terns (Sterna spp.); all fewer than for Lake Park and similar to Lake Shchychie Park 5 750 ϫ 1250; 3.0–3.5 Cranes (Grus canadensis), ducks (broad range of species, 83 unique sequences including Anas acuta, Anas clypeata, Anas formosa, Anas (March 2002, ice) penelope, Aythya fuligula, Aythya marila, Clangula hyemalis, Melanitta fusca, and M. nigra), geese (Anser spp.; A. fabalis is the dominant species, with some A. erythropus), gulls (Larus spp.; L. argentatus and L. canus are the dominant species, with some L. ridibundus and rarely Rhodostethia rosea), loons (Gavia arctica and G. stellata, some of which nested near the lake), sandpipers (Actitis spp., Alidris spp., Calidris spp., Limosa spp., Phalaropus spp., Philomachus pugnax, and Tringa spp.), swans (Cygnus bewickii and C. cygnus), and terns (usually Sterna paradisaea and S. hirundo) Shchychie 25 220 ϫ 450; 11.0–14.0 Ducks (Anas spp.), gulls (Larus spp.), sandpipers (Tringa spp., None detected (March Calidris spp., and Xenus spp.), and terns (Sterna spp.); all 2002, ice) fewer than for Lake Park and similar to Lake Edoma VOL. 80, 2006 INFLUENZA VIRUS IN ENVIRONMENTAL ICE 12231

flow hood. The meltwater was distributed into sterile 1.5-ml microfuge tubes. resulted from these 20 positive reactions. Lake Park has the Meltwater from five tubes for each sample was assayed immediately (as de- highest concentration of birds, including cranes, ducks, geese, Ϫ scribed below) while the other samples were frozen and stored at 80°C until gulls, loons, sandpipers, swans, and terns (Table 1). In 40 needed. Aliquots of 10 ␮l per sample were subjected to RT-PCR amplification. Primers attempts to amplify (by RT-PCR) H1 genes from Lake (forward, H1-1f [ATGCSAACAACTCAACCGACAC]) and reverse (an equimolar Shchychie ice, no amplification was evident. This lake had mixture of H1-5ra [GGGTTCCAGCAGAGTCCAGTAGTA] and H1-5rb [GGG the lowest bird visitation rate (including no observed visits ␮ TTCTAGCAAGGTCCAGTAATA]) were used at 25 pmol each, in 25- l reaction by geese) of the three lakes (Table 1). Only 1 of the 161 mixtures. Reverse transcription was performed with a GeneAmp EZ rTth RNA PCR kit (Applied Biosystems, Inc., Foster City, CA) using the reaction mix attempts to amplify the H1 gene from Lake Edoma water provided (10 ␮M Tris-HCl [pH 8.3]; 300 ␮M [each] dATP, dCTP, dGTP, and yielded an amplicon of the expected molecular weight. A dTTP; 2.5 mM manganese acetate; 50 mM bicine; 115 mM potassium acetate; single sequence resulted. This lake is only occasionally visited 8% [wt/vol] glycerol; and 2.5 U rTth DNA polymerase) at 60°C for 30 min. This by geese and other birds (Table 1). was followed by PCR (with the same reaction mix and enzyme) using the In BLAST searches of NCBI databases, the H1 gene ampli- following temperature regime: 94°C for 4 min and 35 cycles of 94°C for 1 min and 60°C for 90 s, followed by a final extension at 60°C for 8 min. Next, nested PCR cons were most similar to those previously isolated with neur- was performed using 0.5 ␮l of the RT-PCR mixtures described above. The nested aminidase gene subtypes N1, N2, and N5. Phylogenetic analy- primers consisted of a forward primer (an equimolar mixture of H1-2fa [TCAA ses indicated that while the viruses exhibit genetic diversity CCTACTTGAGGACAGTCACA] and H1-2fb [TTAACCTGCTCGAAGACA (Fig. 2), they form a monophyletic cluster (Fig. 3) in contrast to GCCACA]) and a reverse primer (H1-4r [CGGGTGATGAACACCCCATAG TA]) specific for the influenza A virus hemagglutinin H1 gene (25 pmol each). other H1 sequences. Comparison with a wide variety of H1 The following temperature regime was used: 94°C for 5 min; then 45 cycles of gene sequences indicates that the population in the Lake Park 94°C for 1 min, 54°C for 1 min, a 0.3°C-per-s increase to 72°C, and 72°C for 1 ice is most closely related to subtypes that were isolated from min; followed by 72°C for 8 min. both avian and porcine hosts in the 1930s and 1960s (Fig. 3). The PCR products were subjected to electrophoresis on 1.5% standard aga- They are distantly related to the H1 subtype from the 1918 Downloaded from rose (Bio-Rad Laboratories, Hercules, CA) gels in TBE (89 mM Tris base, 89 mM borate, 2 mM EDTA, pH 8.0, with 0.5 ␮g/ml ethidium bromide). Following pandemic. electrophoresis at 5 V/cm, the gels were viewed and photographed under 300 nm Our results indicate the following: (i) the highest frequencies UV light. In total, 373 amplification attempts were performed using meltwater of detection of influenza A virus RNAs are in the lakes with from Lake Park ice. An additional 40 amplification attempts were made using the highest concentrations of migratory waterfowl; (ii) influ- meltwater from Lake Shchychie ice. A total of 161 amplification attempts were performed with water from Lake Edoma. Multiple positive and negative controls enza A virus RNA is preserved in higher concentrations in lake were included for every set of RT-PCR experiments. When amplification bands ice than in lake water (also of note is that the fragment we jvi.asm.org of the expected molecular weight were observed, another aliquot of the reaction consistently were able to amplify was 610 nucleotides in length, mixture was subjected to electrophoresis on a 1.0% low-melting-point agarose indicating good preservation of the RNA, which implies good gel (NuSieve GTG; FMC, Rockville, ME) in TBE. The bands were then eluted preservation of the virus); (iii) the H1 gene population in the from the gels (26, 27) and rehydrated in water. The eluted amplicons were then by on October 22, 2007 ligated into plasmid vectors (pCR2.1-TOPO; Invitrogen Corp., Carlsbad, CA). lakes is genetically heterogeneous; (iv) the single H1 gene Following transformation of host bacteria, the recombinant clones were identi- found in Lake Edoma is similar to the H1 genes in Lake Park, fied by growth on selective medium. The recombinant plasmids were extracted indicating that this gene likely is from the same population of from the bacterial host cells and purified, and the inserts were amplified using virions; (v) the H1 sequences in this study are closest to those M13 primers (forward primer, CAGGAAACAGCTATGAC; reverse primer, GTAAAACGACGGCCAG). The amplicons were gel purified (as described found in Europe during the 1930s and in Asia during the 1960s; above) and then used in DNA sequencing reactions. For sequencing, approxi- and (vi) the H1 sequence from an H1N1 specimen (Brevig mately 30 ng of DNA was added to a reaction with the Terminator Ready Mission, Alaska, 1918) (15) is distantly related to all of the H1 Reaction kit, version 3.0 (ABI, Foster City, CA). The cycling program was as genes from Lake Park and Lake Edoma that were character- follows: 1 min at 94°C and then 30 cycles of 94°C for 10 s, 50°C for 30 s, and 60°C ized in this study. for 4 min. The amplified DNA was precipitated by adding ethanol to a final concentration of 65% and incubating at room temperature for 15 min. Then the DNA was subjected to centrifugation in a microfuge (13,200 rpm) for 20 min. DISCUSSION The pellets were washed with 80% ethanol and then dried under a vacuum. The dried pellets were rehydrated with template suppression reagent (ABI, Foster This is the first report of the persistence of influenza A virus City, CA), denatured at 94°C for 4 min, and chilled on ice. Then the solutions were loaded into an ABI (Foster City, CA) 310 automated DNA sequencer for in lake ice, as reflected by enduring genes. It indicates a po- determination of the influenza A virus H1 gene sequences. tential long-term survival mechanism for the virus. Ice may act The sequences were compared to those in NCBI databases by using BLAST as a reservoir for influenza A viruses, preserving them for later searches (http://www.ncbi.nlm.nih.gov/BLAST/). The sequences exhibiting the release and infection of animals, including migratory waterfowl highest similarity to influenza A virus H1 sequences (a total of 84) were aligned and humans. Surveillance of Arctic and subarctic lakes for using CLUSTALW (http://searchlauncher.bcm.tcm.edu/multi-align/multi-align .html). A broad selection of H1 sequences from the NCBI database also were influenza virus may aid health professionals to improve pre- included in the alignment. Following alignment, the sequences were examined, diction of influenza virus subtypes that are circulating at par- and small manual adjustments were made as needed. Phylogenetic analyses were ticular points in time, thus facilitating long-term vaccination performed with PAUP (phylogenetic analysis using parsimony [25]) using neigh- strategies. Furthermore, surveillance may shed some light on a bor-joining and maximum parsimony. Consistency, homoplasy, retention, and rescaled consistency indices were calculated in the parsimony analysis. fundamental apparatus allowing for abiotic long-term perpet- uation of multiform influenza A virus strains. Cold temperatures and freezing preserve most types of vi- RESULTS ruses, including influenza virus (14, 17, 18, 22). Experimentally, The highest frequencies of influenza A virus H1 genes were the feasibility of influenza A virus endurance in the frozen found in ice from Lake Park. Twenty of the 373 RT-PCRs state has been demonstrated, implying its survivability in fro- using Lake Park ice meltwater yielded amplification bands of zen lakes. Inactivation of 99% of a virus population occurs in the expected size. After cloning, a total of 83 unique sequences approximately 1 week when water temperatures are between 12232 ZHANG ET AL. J. VIROL. Downloaded from jvi.asm.org by on October 22, 2007

FIG. 2. Neighbor-joining phylogram of the influenza virus hemagglutinin H1 gene sequences isolated from Lake Park ice (collected in March 2002) and Lake Edoma water (collected in September 2001). Numbers indicate sequences from clones derived from nested RT-PCR mixtures. The number before the decimal point indicates the RT-PCR number, while the number after the decimal point indicates each unique clone from the reaction. Sequences from the control virus (A/WS/33, clone p1.9) and from the Brevig Mission, Alaska, subtype H1 (accession number AF116575), are also shown. The Brevig Mission sequence was used as the outgroup. LPI indicates cloned sequences from Lake Park ice, while LEW indicates the one clone from Lake Edoma water.

22 and 25°C. However, 10 weeks is required to inactivate the continues to decrease as the temperature is lowered. This same proportion of the virus population if the temperature is trend continues to below Ϫ80°C. We have found that viruses between 3 and 5°C. The rate of virus degradation slows down and bacteriophage (as prophage) frozen in glaciers can be to an even greater extent at and below freezing, and it preserved for well over 100,000 years (3, 4). We previously VOL. 80, 2006 INFLUENZA VIRUS IN ENVIRONMENTAL ICE 12233 Downloaded from jvi.asm.org by on October 22, 2007

FIG. 3. Maximum parsimony phylogram of a wide selection of hemagglutinin H1 gene sequences, including selected sequences from this study. Gaps were scored as a fifth base. There were more than 2,000 most-parsimonious trees, with placement of several of the sequences varying. Primarily, very closely related sequences shifted relative to one another. However, the relationships of the influenza A virus H1 sequences from this study with the other sequences were consistent in all of the trees. The tree shown has 1,128 steps, with consistency, homoplasy, retention, and rescaled consistency indices equal to 0.5417, 0.4583, 0.8562, and 0.4638, respectively. The H1 genes from Lake Park ice and Lake Edoma water are closest to those from avian strains isolated in Asia in 1933 and 1967. Also, they were related to strains from 1938 and 1939 isolated from swine in the United Kingdom. These are embedded within a large clade that includes a wide range of H1 sequences isolated from humans from the 1930s to the present. There is a more distant relationship with the H1 gene from the 1918 H1 influenza A virus (upper clade) and avian strains from 1976 through 1985 (lower ingroup clade). Sequences from H6 influenza virus strains were used as representatives of the outgroup. Abbreviations for sources: Av, avian; Hu, human; Sw, swine. 12234 ZHANG ET AL. J. VIROL. reported on viral RNA preserved in ice that was approximately to Europe and North Africa. As the birds visit lakes along their 140,000 years old (4) and have additional unpublished data paths they shed viruses into the lakes and onto the ice (when supporting preservation to many times this age. Therefore, present) and drink water containing viruses discharged by glaciers and ice-covered lakes may be an unrecognized major other birds or released from the ice by thawing. Therefore, reservoir of microbes. For pathogens, this is advantageous, these lakes become abiotic mixing pools for the viruses, while since upon reemergence specific genotypes may interact with the birds are the biotic vessels where mixing occurs (including host populations that may lack resistance or immunity. replication and recombination). Since there are susceptible Some influenza virus strains have appeared, disappeared, hosts along their migration path, they may pass the viruses to and then reemerged decades later virtually unchanged (13, 20). other birds as well as to swine, humans, or other animals. Our This may indicate the presence of an abiotic mode of preser- results support the hypothesis that ice acts as a long-term vation. For example, the Russian influenza virus subtype abiotic storage matrix for influenza virus and other microbes, (H1N1) that caused an epidemic in 1977 was nearly identical to including pathogens (18, 21–23). Furthermore, the cold lake the subtype (H1N1) that caused an epidemic in 1950. Other water is also capable of preserving the virus, although presum- strains, most notably specific genotypes of H2N2 and H3N2 ably for shorter time periods. and several H1 variants, have made similar returns. Since in- Although the findings of this study are limited to the testing fluenza virus is an RNA virus, the rates of mutation should of three lakes, they point to a principal mechanism that may have been rapid if the viruses had been reproducing in biotic underlie a wider natural apparatus of abiotic long-term preser- hosts during those years. A possible explanation for the slow vation of avian influenza viruses. The prevalence and extent of rates of mutation is that the strains may have been preserved in such a mechanism, which may bear a wealth of implications, some way during the decades between the epidemics. Preser- should be further demonstrated through additional studies, vation in ice is a possible explanation (2, 14, 18, 22, 23). Ice and including the exploration of more geographical sites, assays for Downloaded from ice-covered lakes (as well as glaciers) may act as huge reser- related genes, and examination of viral endurance. At present, voirs of preserved viruses. Therefore, annually and perennially we describe the feasibility of the mechanism and supportive frozen lakes (some are frozen continuously for decades or evidence at the level of gene recovery and analysis. longer) along the paths of waterfowl migration routes have the potential for being major sources of viruses that cause pan- ACKNOWLEDGMENT demics and epizootics in birds and other animals. Virological This work was supported by the National Institutes of Health, Na- jvi.asm.org surveillance of these lakes is needed in order to assess the tional Institute of Allergy and Infectious Diseases (grant number relationships between the prevalence of current as well as 5R03AI063144-02, awarded to S.O.R.). earlier influenza A virus subtypes (including sequence charac- REFERENCES by on October 22, 2007 teristics) and endemic and epidemic occurrence of disease. 1. Abyzov, S. S., M. N. Poglazova, J. N. Mitskevich, and M. V. Ivanov. 2005. This probably relates to other diseases as well, but this awaits Common features of microorganisms in ancient layers of the Antarctic ice sheet, p. 240–250. In J. D. Castello and S. O. Rogers (ed.), Life in ancient ice. thorough examination. One expectation in relation to this phe- Princeton University Press, Princeton, N.J. nomenon would be an increased rate of release of these mi- 2. Castello, J. D., and S. O. Rogers (ed.). 2005. 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Gang Zhang, Dany Shoham, David Gilichinsky, Sergei Davydov, jvi.asm.org John D. Castello, and Scott O. Rogers Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403; Begin-Sadat Center for Strategic Studies, Bar-Ilan University, Ramat-Gan, ; Soil Cryology Laboratory, Institute for Physicochemical and Biological Problems in by on October 22, 2007 Soil Science, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, ; Pacific Institute of Geography, Russian Academy of Sciences, 678830 Cherskii, Republic of Sakha (Yakutia), Russia; and Environmental and Forest Biology, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210

Volume 80, no. 24, p. 12229–12235, 2006. Page 12231, column 2, line 20: “avian” should read “human.” Page 12233, Fig. 3. Sequences U38242 (Tokyo/3/67) and U08904 (A/WS/33) were isolated from humans; therefore, they should have the Hu prefix rather than the Av prefix. Page 12233, legend to Fig. 3, line 6. “avian strains isolated in Asia in 1933 and 1967” should read “human and swine strains isolated in the UK during the 1930s and from a human in Asia in 1967.”

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