1 Studies on Two Genomic Variants of Taura

Studies on Two Genomic Variants of Taura Syndrome Virus: Infection under Hyperthermic Conditions and Detection with a Novel Monoclonal Antibody

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Authors Cote, Isabelle

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STUDIES ON TWO GENOMIC VARIANTS OF TAURA SYNDROME VIRUS: INFECTION UNDER HYPERTHERMIC CONDITIONS AND DETECTION WITH A NOVEL MONOCLONAL ANTIBODY

by Isabelle Côté ______

A Dissertation Submitted to the Faculty of the DEPARTMENT OF VETERINARY SCIENCE AND MICROBIOLOGY In Partial Fulfilment of the Requirements For the Degree of

DOCTOR OF PHILOSPHY WITH A MAJOR IN MICROBIOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2008

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Isabelle Côté entitled: "Studies on Two Genomic Variants of Taura Syndrome Virus: Infection under Hyperthermic Conditions and Detection with a Novel Monoclonal Antibody” and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy.

______Date: __06/09/2008______Donald V. Lightner, Ph.D.

______Date: __06/09/2008______Bonnie T. Poulos, Ph.C.

______Date: __06/09/2008______Michael A. Cusanovich, Ph.D.

______Date: __06/09/2008______Carol L. Dieckmann, Ph.D.

______Date: __06/09/2008______Michael W. Riggs, DVM, Ph.D.

Final approval and acceptance of this dissertation is contingent upon de candidate's submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirements.

______Date: __06/09/2008______Dissertation Director: Donald V. Lightner

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfilment of requirements of an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library.

Brief quotations from this dissertation are allowable without special permission provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the Head of the Department of Veterinary Science and Microbiology or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interest of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED:______Isabelle Côté

ACKNOWLEDGEMENTS

Many thanks to Dr. Donald Lightner, who welcomed me in his laboratory and supported all the work presented here. Dr. Lightner's professionalism and outstanding qualities as a researcher and a pathologist have set an amazing example allowing me to grow as an aquatic animal health professional and a science person. Heartfelt acknowledgements are due to Bonnie Poulos, whose tireless work and constant joie de vivre have been tremendous blessings throughout the course of this project.

To my graduate committee members: Dr Michel Riggs, Dr. Michael Cusanovich and Dr. Carol Dieckmann. Your guidance and advices have been essential in shaping and completing this work.

I am very fortunate to have had the opportunity to work with a group of dedicated and amazing people. Present and past lab members have taught me many things about scientific thought, shrimp diseases, and even life in general. Specific thanks are extended to Allan Heres, Brenda White, Dr. Carlos Pantoja, Debbie Bradley-Dunlop, Dr. Kathy Tang, Linda Nunan, Leone Mohney, Paul Schofield, Rita Redman, Thales Andrade, Thiannarat Srisuvan and Wanda McCormack. All of whom, to one extent or another, contributed to this work. Michael Anderson and Dr. Helen Jost provided much appreciated help with experimental animals: Holly, Molly, Polly and Dolly (I and II). Dr. Janet Decker opened to me the doors of the wonderful world of teaching.

Financial support for this research was provided by the United States Marine Shrimp Farming Consortium under the grant: 2004-38808-02142 from the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture. The Department of Veterinary Sciences and Microbiology as well as the Department of Ecology and Evolutionary Biology have provided teaching and graduate research assistantships.

To Philip, for making it all worthwhile.

DEDICATION

To the shrimp and mice who gave their life for the advancement of science.

You will be remembered.

TABLE OF CONTENTS

Page

LIST OF FIGURES...... 8

LIST OF TABLES...... 9

ABSTRACT...... 10

CHAPTER 1. Literature review...... 12 Where it all began... the controversy...... …..12 Identification and description of the virus...... 14 Variants of the virus...... 17 Geographic distribution...... 19 Species of shrimp susceptible...... 20 Pathology and disease cycle...... 23 Cellular biology of the disease...... 26 Routes of transmission...... 27 Diagnostic methods...... 29 Methods of control...... 33

CHAPTER 2. Development and characterization of a Monoclonal antibody against Taura syndrome virus of penaeid shrimp...... 39 Abstract...... 39 Introduction...... 40 Material and Methods...... 42 Results...... 48 Discussion...... 50

CHAPTER 3. Taura Syndrome Virus from Venezuela is a New Genetic Variant...... 59 Abstract...... 59 Introduction...... 60 Material and Methods...... 62 Results...... 67 Discussion...... …...... 69

CHAPTER 4. Hyperthermia protects Kona stock Penaeus vannamei against infection by TSV-HI but not TSV-BZ...... 80 Abstract...... 80 Introduction...... 81 Material and Methods...... 83 Results...... 86 Discussion...... …..88

TABLE OF CONTENTS - Continued

REFERENCES...... ……98

LIST of FIGURES

Figure Page

1.1 Transmission electronic micrograph of TSV………………………...………………35

1.2 Schematic diagram of the genomic organization of TSV…………………….……...36

1.3 Acute phase histological lesions of TSV (H&E)…………………………………….37

1.4 Chronic phase histological lesions of TSV (H&E)……………………………..……38

2.1 Dob blot immunoassay of MAb 2C4………………………………………………...54

2.2 Representative ISH and IHC of TSV positive samples……………………………...55

2.3 SDS-PAGE of purified TSV-SI, TSV-HI and TSV-BZ……………………………..56

2.4 Western blot of purified TSV with Mab 2C4………………………………………..57

3.1 H&E, ISH, IHC (MAb 1A1) and IHC (PAbs) analysis of TSV-VE samples……….72

3.2 Phylogenetic tree of TSV isolates……………………………………………...…….75

3.3 Dot blot immunoassay of TSV-VE with MAb 1A1 and PAbs…………………...…77

4.1 Survival of shrimp injected with TSV-HI under hyperthermic conditions………..…92

4.2 Atypical LOS in shrimp infected with TSV-HI under hyperthermic conditions H&E and ISH compared with normal LOS……………………………………………………93

4.3 Survival of shrimp fed or injected TSV-BZ under hyperthermic conditions…..……95

4.4 Survival of shrimp fed TSV-BZ under conditions of low salinity and high temperature………………………………………………………………………………96

LIST OF TABLES

Table Page

2.1 Sensitivity of MAb 2C4 as determined by IHC…………………………...…………58

3.1 Year of collection and origin of the TSV isolates …………………………………...78

3.2 CP2 nucleotide and amino acid percentage identity among TSV-HI, TW, BZ and VE isolates……………………………………………………………………………………79

3.3 Survival of shrimp infected with TSV-VE versus shrimp infected with TSV-HI…..79

4.1 Viral copies number in shrimp injected with TSV-HI under hyperthermic conditions………………………………………………………………………………...97

ABSTRACT

Taura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against

TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).

The first manuscript describes the production of a monoclonal antibody reacting with the

Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and

TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.

Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus

vannamei . These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.

In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32 oC). Shrimp, infected with TSV-

HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susceptible to the infection by

TSV-BZ02. This susceptibility to TSV-BZ02 at higher temperatures was independent both of the route of infection and of the salinity of water. I conjecture that TSV-BZ02 might be a temperature permissible mutant of TSV.

CHAPTER 1: Literature Review

Where it all began... the controversy.

Taura syndrome (TS) was first described in Ecuador during the summer of 1992 (Jimenez

1992). In March 1993, it returned as a major epidemic and was the object of extensive media coverage (Rosenberry 1994b; Rosenberry 1995). Retrospective studies have suggested that a case of TS might have occurred on a shrimp farm in Columbia as early as 1990 (Brock 1995) and that the virus was already present in Ecuador in mid-1991

(Hasson et al. 1999a). Between 1992 and 1997 the disease spread to all major regions of the Americas where Penaeus vannamei was cultured (Lightner et al. 1997b; Hasson et al.

1999a). It has been estimated that the economic impact of TS in the Americas during that time might have exceeded 2 billion US dollars (Lightner et al. 1997b).

The 1992 Ecuadorian TS epidemic occurred concurrently with an outbreak of black leaf wilt disease in banana plantations (Brock 1997). The outbreak of black leaf disease led to an increase in fungicide usage within the Taura River basin district near the city of

Guayaquil (Colburn 1997). It was thought that the antifungicides Tilt

(propiconazole,Ciba-Geigy) and Calixin (tridemorph, BASF) used to control black leaf, ran off into nearby ponds and were responsible for TS (Brock 1997; Colburn 1997).

Analytical data demonstrated propiconazole in water, sediments and hepatopancreas

tissues of shrimp harvested from TS affected farms in Ecuador. No other pesticides were

discovered (Brock 1995).

An Ecuadorian shrimp farmer industry representative reported "According to the latest

research work, it seems to us that this delicate [environmental] balance was lost with the

growth of the banana industry and the measures taken in that sector to overcome sigatoka

black leaf spot disease. With the application of fungicides, a critical mass of toxic

substances was reached, producing Taura syndrome" (reported by Rosenberry 1994a).

Other articles in the popular press described the toxic etiology of the disease (Rosenberry

1994a). In his 1994 article, Wigglesworth mentions anomalies of the TS behavior with regard to a possible toxic etiology including higher resistance of wild post-larvae to the syndrome. The toxic etiology belief was strengthened by research performed by Intriago et al. (1997) and other groups (reported in Brock 1995). This was responsible for a controversy that lasted several years (Brock 1995; Brock et al. 1997; Lightner 1999) and might have contributed to the spread of the disease (Lightner 1999).

In January 1994, at the request of Ciba-Geigy, a TS workshop was held at the

Aquaculture Pathology of the University of Arizona. Experts from seven countries with expertise in shrimp and insect pathology, shrimp nutrition, toxicology, myocology, water quality and farm management participated in the workshop. Industry representatives from

Ecuador, Colombia, Switzerland, Belgium and the USA also participated (Colburn 1997).

The group developed recommendations as to the standardization of the research on TS

14 and suggested that studies be done to evaluate whether fungicides or an as-yet unrecognised agent were responsible for the syndrome.

James Brock first demonstrated the infectious etiology of the disease by feeding Taura victims to test shrimp (Chamberlain 1994). The dying test shrimp were then fed to a new set of shrimp who died at the same rate, precluding dilution of a toxic chemical and suggesting the active replication of an infectious agent within the shrimp (anon 1995).

Hasson et al. (1995) fulfilled Rivers' postulates and proved the viral etiology of the syndrome. The basis for histopathological diagnosis of the disease was described by

Lightner et al. (1995) and several diagnostic methods were later developed (discussed later in this review). The causative agent, Taura syndrome virus (TSV), of the disease has been designated as a notifiable disease by the Office international des Épizooties reflecting the serious and devastating impacts of the disease (Dhar et al. 2004; OIE 2006).

The causal agent is referred by the name of Infectious cuticular epithelial necrosis virus

(ICENV) by some authors (Jimenez et al. 2000).

Identification and description of the virus.

The causative agent of the disease, TSV, has been isolated and purified (Hasson et al.

1995; Bonami et al. 1997). Based on biological and physical characteristics, it was first classified as a possible member of the family Picornaviridae (Bonami et al. 1997;

Robles-Sikisaka et al. 2001). It was later reclassified in the Dicistroviridae family, genus

Cripavirus (Mari et al. 2002; Mayo 2002). It currently belongs to that same family, but it

is unassigned to any genus (Mayo and Ball 2006). Virus members of the Picorna-like

superfamily are prevalent in a variety of marine environments (Culley et al. 2003).

TSV is a 32nm non-enveloped particle with an icosahedral morphology (Fig. 1.1; Hasson

et al. 1995) and a buoyant density of 1.338g/ml (Bonami et al. 1997). Using light

microscopic in situ hybridization (ISH) it was found that the binding of a TSV-specific

gene probe was limited to the cytoplasm of infected cells, with no detectable signal

within the nucleus (Hasson et al. 1999a). Srisuvan et al. (2006b) used electron

microscopic in situ hybridisation (EM-ISH) to evaluate the replication of the virus within

the cell. They found that replicating TSV was associated with membranous structures

within the cytoplasm. They observed eccentricity of the nuclei of infected cells,

suggesting that TSV replication could take place in a defined region of the cell. In that

study the nuclei were relatively free of hybridization signals, but a positive reaction was evident between the inner and outer nuclear membranes.

TSV has a single-stranded positive-sense genome that consists of 10,205 nucleotides

(excluding the 3' poly-A tail). There is a 377 nucleotide untranslated region at the 5' end followed by two open reading frames (ORF) separated by an intergenic region of 226 nucleotides (Mari et al. 2002). ORF1 has motifs characteristic of a helicase, a protease, an

RNA-dependent-RNA-polymerase and a region of striking similarity with the

16 baculovirus IAP repeat (BIR) domain in the N-terminal region. The capsid proteins CP1

(40 kDa), CP2 (55 kDa) and CP3 (24 kDa) were mapped in ORF2 (Fig. 1.2). Protein analysis reveals a minor protein of 58 kDa. It has been hypothesized that the minor protein is produced by different proteolytic cleavage of the capsid polyprotein and could be a precursor for other capsid proteins (Mari et al. 2002).

The region separating the two non-overlaping ORFs contains an internal ribosome entry site (IRES) that directs the synthesis of the capsid proteins (Cevallos and Sarnow 2005).

Using Northern blot analysis Robles-Sikisaka et al. (2001) suggested that although the genome of TSV contained more than 1 ORF the entire genome was transcribed as a single transcript, the capsid protein gene not being transcribed as subgenomic RNA.

Despite reports by Audelo-del-Valle (2003) that certain primate cell lines could be used to culture TSV, Pantoja et al. (2004) and Luo et al. (2004) demonstrated that their report was based on misinterpretated data. Hence, at the present time, there is no continuous cell line that supports the growth of TSV. Therefore, all virus amplifications require the use of live shrimp.

Variants of the virus

RNA viruses such as TSV have rates of spontaneous mutation in the order of 10 -3 to 10 -4

per incorporated nucleotide (de Graindorge 2003). These very high rates might be due to

the lack of proofreading function of the RNA-dependent RNA polymerase (Tang and

Lightner 2005) and have resulted in the emergence of new genetic variants of the virus.

Direct analysis of genetic variations of CP2 has revealed initially two, and later three,

clusters of geographic isolates (Robles Sikisaka et al. 2002; Tang and Lightner 2005).

Four genetic clusters are currently recognised: Belize, America, South-east Asia and

Venezuela (Tang and Ligthner. 2005; Cote et al. this thesis). According to their mortality

rate and reaction with available antibodies, five distinct variants of the virus are

recognized. Isolates representing what are essentially clusters of TSV genotypes and the

year of collection are: TSV-HI94, TSV-SI98, TSV-BZ02, TSV-VZ05 and TSV-TH05 1

(Robles-Sikisaka et al. 2002; Erickson et al. 2002; Erickson et al. 2005; Tang and

Lightner 2005; Wyban 2004; Phalitakul et al. 2006; Cote et al. this thesis). Hereafter

referred to in this review as TSV-HI, TSV-SI, TSV-BZ, TSV-VE and TSV-TH.

The emergence of new strains could be caused by the rapid adaptation of the virus to new

local environmental conditions following transfer. This produces adapted local substrains

that facilitate infection of new hosts, resulting in the propagation of the new strain

(Chang et al. 2004). It has been suggested that new strains such as TSV-SI could have

1 Where HI= Hawaii, SI= Sinaloa, BZ=Belize, VZ= Venezuela and TH= Thailand

18 had evolved as a result of its infection of Penaeus stylirostris , a new penaeid host for

TSV (Erickson et al. 2002; Robles-Sikisaka et al. 2002). Species currently considered to be TSV tolerant might be at risk following the introduction of TSV into new areas

(Robles-Sikisaka et al. 2002).

Point mutations in TSV capsid proteins might provide a specific isolate with selective advantages such as host adaptability, increased virulence or increased replication ability

(Robles-Sikisaka et al 2002; Chang et al. 2004). Tang and Lightner (2005) noted that even small variations in the TSV genome can result in substantial differences in virulence. P. monodon infected with one isolate of TSV, isolated from brooders captured from Southern Taiwan coastal waters, showed no signs of active replication while another TSV isolate from Taiwan, isolated from a single Metapenaeus ensis specimen, was found to replicate freely within the shrimp (Chang et al. 2004).

Three variants were tentatively attributed to serotype according to their reaction with the then sole available Monoclonal antibody (MAb) 1A1. Serotype A: Hawaii, serotype B:

Sinaloa (Mexico) and serotype C: Belize (Erickson et al. 2003). Out of these, the Belize strain has consistently given higher mortality in bioassays and it is considered the most virulent (Erickson et al. 2005; Srisuvan et al. 2006a). In a bioassay using TSV-BZ infected tissue, mortalities began to occur as early as 1 day post-infection and occurred, in general, 1 day earlier than in control shrimp infected with TSV-HI (Erickson et al.

2005).

All TSV variants are similar in shape and size with slight variations. The average size of

TSV-BZ virus particles is 32.693+/- 1.834 nm compared to TSV-HI with a size of 31.485

+/- 1.187 nm (Erickson et al. 2005). A region of high genetic difference is within CP2

with pairwise comparison of nucleotides showing a 0 to 3.5% difference amongst isolates

while the difference for CP1 is 0 to 2.4% (Robles-Sikisaka et al. 2002). Tang and

Lightner (2005) reported up to 5.6% difference in CP2 using a larger group of isolates

collected over a longer time span (11 vs 1 year for Robles-Sikisaka et al. 2002). Most

variations in CP2 occur in the 3'-terminal sequence. This may be because it is less

constrained by structural requirements and more exposed than other regions of the protein

(Tang and Lightner 2005).

Geographic distribution

TSV has been reported from virtually all shrimp-growing regions of the Americas including Ecuador, Columbia, Peru, Brazil, El Salvador, Guatemala, Honduras, Belize,

Mexico, Nicaragua, Panama, Costa Rica, Venezuela as well as from the States of Hawaii,

Texas, Florida and South Carolina (Hasson et al. 1999a; Zarain-Herzberg and Ascencio-

Valle 2001; Conroy 2005). In 1997 the virus had spread to virtually all shrimp growing regions in the Americas, but had not yet been described in Asia and other parts of the world (Brock 1997). Until 1998, it was considered to be a Western Hemisphere virus

(Lightner and Redman 1998). The first Asian outbreak occurred in Taiwan (Tu et al.

1999; Yu and Song 2000). It has more recently been identified in Thailand, Myanmar,

China, Korea and Indonesia where it has been associated with significant epizootics in

farmed Penaeus vannamei and P. monodon (Tang and Lightner 2005; Nielsen et al. 2005;

Srisuvan et al. 2005; Do et al. 2006).

The wide distribution of the disease has been attributed to the movement of host stocks for aquaculture purposes (Lightner 1995). This might have been helped by the highly stable nature of the virus (Hasson et al. 1995). It is thought that importation of TSV- infected P. vannamei from the Western Hemisphere was at the origin of the outbreak in

Taiwan (Tu et al. 1999). This was further suggested by the genomic similarity of the

Taiwan and Western Hemisphere isolates (Lien et al. 2002). TSV appeared in Thailand in

2003. Due to the similarities in deduced CP2 protein sequence and the chronology of the disease outbreaks in relation to imported stocks it is likely that at least some of the Thai outbreaks originated from Chinese stocks (Nielsen et al. 2005).

Species of shrimp susceptible

TSV is known to affect many penaeid shrimp species. It causes serious diseases in the postlarval (PL), juvenile and adult stages of Penaeus vannamei , the most susceptible target host with up to 50-90% mortality in non-selected populations (Bonami et al. 1997;

Brock et al. 1997). It also severely affects P. setiferus , P. stylirostris, P. Schmitt and,

Metapenaeus ensis (Rosenberry 1995; Brock et al. 1997; Overstreet et al. 1997; Erickson

et al. 2005; Chang et al. 2004). Srisuvan et al. (2005) demonstrated the disease in P. monodon , a species previously thought to be resistant (Brock et al. 1997). Other species

such as P. chinensis (experimentally) present mortality (Overstreet et al. 1997). P.

aztecus, P. duorarum and P. japonicus have also been shown to be hosts for TSV in natural and experimental infections (Brock et al. 1997). P. aztecus and P. duorarum were

relatively tolerant to the virus and were not killed by TS (Overstreet et al. 1997).

TSV infection has no known impact on nauplii through the early postlarval stages.

Postlarval to adult sizes are susceptible to infection by TSV but seem to show increasing

TS resistance with increase in size (Brock 1997; Brock et al. 1997). The size of shrimp

affected with TS in Sinaloa, Mexico, has been reported as ranging from 0.063g to 15.0g

(Zarain-Herzberg and Ascencio-Valle 2001). Laboratory results have, however, failed to

find significant differences supporting the hypothesis that P. vannamei increases its

tolerance to TSV as it increases in size to up to 30g (Overstreet et al. 1997; Lotz 1997).

Lotz (1997) noted a consistent trend for larger shrimp to be more likely to succumb to

infection. The discrepancy between these findings and what is commonly observed in the

field might be due to the larger shrimp being more likely to have previously been

exposed and therefore having acquired resistance. The likelihood of infection and

mortality from TSV is also dependent on the initial dose. For both per os and water route of transmission the dose of TSV per g of shrimp will decrease as the shrimp increase in size (Lotz 1997).

There is variation within species and TSV-resistant strains of shrimp have been

developed (Argue et al. 2002). Wild stocks are showing increased resistance, perhaps

through intense natural selection (Laramore 1997; Lightner et al. 1997b; Lightner 1999).

Reports of TS in the wild are limited, but in February 1995, the Mexican Fisheries

Ministry reported the presence of TSV in wild-type shrimp captured on the border of

Mexico and Guatemala (reported in Zarain-Herzberg and Ascencio-Valle 2001).

Investigators have also reported the occurrence of TS in wild post-larvae from the Gulf of

Guayaquil, Ecuador during mid-1993 and in adult P. vannamei collected from the Pacific

Coast of Honduras and El Salvador (reported in Lightner et al. 1997b). There are no

confirmed reports indicating that TSV is infectious to other groups of decapod or non-

decapod crustaceans (Brock 1997).

As an RNA virus, TSV has shown rapid sequence evolution. This could be related to

adaptation to new hosts. According to Phalitakul et al. (2006) the sequence divergence of

an isolate of TSV from Thailand suggested adaptation to P. monodon as a new host species. Adaptation of the virus to a new host has also been suggested in the case of P. stylirostris and TSV-SI (Erickson et al. 2002; Robles-Sikisaka et al. 2002).

Pathology and disease cycle

In farm situations, TS often causes high mortality during the first 40 days of stocking into shrimp ponds (Brock 1997). The course of infection may be acute (5-20 days) to chronic

(more than 120 days) at the pond level (Brock et al. 1997).

The disease has three distinct but overlapping phases: acute, transition and chronic. The disease cycle has been characterized in detail in Penaeus vannamei (Hasson et al. 1999b).

Lotz et al. (2003) have developed a mathematical model for epidemics of TSV in a closed population. This model included the three phases described previously in five compartments: uninfected susceptible, prepatently infected, acutely infected, chronically infected and dead infected shrimp.

After the initial infection, the acute phase develops with clinical signs occurring as early as 7 hours post-infection in some individuals (Poulos et al. 1999) and last for about 4-7 days (Hasson et al. 1999b). Infected shrimp display anorexia, lethargy and erratic swimming behavior. They also present opacification of the tail musculature, soft cuticle and, in naturally occurring infections, a red tail due to the expansion of chromatophores

(Hasson et al. 1999a). Focal necrosis of the tail cuticular epithelium can be seen with a

10X hand lens (Lightner 1996; Flegel 2006). Mortality during this phase can be as high as 95% (Brock 1997).

The acute phase is characterized histologically by multifocal areas of nuclear pyknosis/karyorrhexis and numerous cytoplasmic inclusion bodies in the cuticular epithelium and the subcutis of the general body surface, all appendages, gills, hindgut, esophagus and stomach (Fig. 1.3; Lightner et al. 1995; Hasson et al. 1999b). The pyknosis and karyorrhexis give a “buckshot” appearance to the tissue and are considered pathognomonic for the disease (Lightner et al. 1995). In severe infections the antennal gland tubule epithelium, the hematopoietic tissues and the testis are also affected (Hasson et al. 1995; Lightner et al. 1995). This occurs mainly in severe infection following injection of viral particles and has not been reported from naturally infected P. vannamei

(Hasson et al. 1999b).

Transmission electron microscopic (TEM) evaluation of affected cells has allowed the observation of the inclusion bodies occupying a large area within the cytoplasm of infected cells (Srisuvan et al. 2006). The inclusion bodies are composed of an amorphous, granular, electron-dense matrix (Lightner et al. 1995) reacting with TSV by ISH using

TSV specific cDNA probes (Srisuvan et al. 2006). In early TEM studies, it was found that the inclusion bodies often contained needle-like crystals presumed to be made of calcium phosphate and associated to a disruption of the calcium metabolism and the calcification process of the exoskeleton (Lightner et al. 1995). It has later been stated that these crystalline deposits were a variable finding that could reflect an artefact from the use of phosphate buffered glutaraldehyde fixation (reported in Brock et al. 1997).

Shrimp that survive the acute stage enter a transitional stage and show randomly distributed melanized (brownish/black) lesions within the cuticle of the cepahlothorax and tail region (Hasson et al. 1999b) These foci are the sites of acute lesions which have progressed onto subsequent stages of hemocytic inflammation, cuticular epithelium regeneration and healing (Brock 1997), and which might be secondarily infected with bacteria (Hasson et al. 1999b). These foci are negative for TSV by ISH (Hasson et al.

1999b). Shrimp in the transition phase are lethargic and anorexic, possibly because of the redirection of all their resources toward wound repair and recovery (Dhar et al. 2004).

Histologically these shrimp present focal active acute lesions and the onset of lymphoid organ spheroids (LOS) development. By ISH with TSV specific probes a diffuse positive signal can be observed within the walls of the LO of normal appearance with or without focal probe signals within developing LOS (Hasson et al. 1999b).

If the shrimp undergo another successful moult following the transitional phase they will cast off the melanized lesions (Hasson et al. 1995) and enter the chronic phase. The chronic phase is first seen 6 days post-infection and can persist for an undetermined period of time, at least 12 months under experimental conditions (Hasson et al. 1999b;

Poulos et al. 2008). The chronic phase is characterized histologically by the absence of acute lesions and the presence of LOS of successive morphologies (Fig. 1.4; Hasson et al.

1999c). These LOS are positive by ISH for TSV (Poulos et al. 2008). A low prevalence of ectopic spheroids can also be observed in some cases (Hasson et al. 1999c). LOS are not by themselves characteristic of TSV infection and can be found in other viral diseases

of shrimp such as lymphoid organ vacuolization virus (LOVV), lymphoid parvo-like

virus (LPV), lymphoid organ virus (LOV), rhabdovirus of penaeid shrimp (RPS) and yellow head virus (YHV) (Hasson et al. 1999c).

Diagnosis of the disease during the chronic phase is problematic as shrimp do not display any outward signs of the disease and do not show mortality from the infection (Brock et al. 1997; Hasson et al. 1999c). It has been suggested that shrimp with chronic TSV infection were not as vigorous as uninfected shrimp, as demonstrated by their inability to tolerate a salinity drop as well as uninfected shrimp (Lotz et al. 2005).

Cellular biology of the disease

Little is known regarding the effect of the virus at the cellular level. Lamr/p40 type receptor has been proposed as a putative attachment site for the virus CP2 (Senapin and

Phongdara 2006). Cevallos and Sarnow (2005) have hypothesized that the virus might counteract phosphorylation of eIF2 by kinase of the innate immune system by initiating translation without requirement for initiator-tRNA met . They also suggested the alternative hypothesis that the replication of the virus on cellular membranes may cause stress resulting in the activation of eIF2 kinase and an overall inhibition of initiator-tRNA met - dependent translation.

Viral particles have been detected in the hemolymph of infected shrimp (Bonami et al.

1997). These had the same morphology as virus purified from tissue. This indicates the

systemic nature of the acute phase infection and that the whole virion is transported by

the hemolymph. The expression of protein in the hemocytes of acutely infected shrimp

has been evaluated using two-dimensional gel electrophoresis. It showed that 11 forms of

eight proteins were significantly up regulated whereas nine forms of five proteins were

down regulated. The up regulated proteins were 14-3-3 Zeta protein, tubulin beta-2 chain,

beta-actin, acetylglucosamine, hemocyanin, protein disulfide isomerase precursors,

catalase, carboxylesterase and one unknown protein. The down regulated proteins were

TM, transglutaminase, the light chain of myosin, glutathione transferase and hemocyanin.

Only hemocyanin fragments (either C or N terminal) were altered, not the full-length

protein. These fragments could have a functional significance during the molecular

response of shrimp hemocytes to TS (Chongsatja et al. 2007).

Routes of transmission

The most likely route for transmission of TSV is cannibalism of dead infected shrimp.

The virus can be spread from one farm to another by seagulls and aquatic insects, such as

the water boatman, Trichocorixa reticulata (Lightner and Redman 1998). Shrimp farmers from Ecuador, Honduras and Columbia have reported numbers of these insects in TSV- infected ponds (reported by Hasson et al. 1995). ISH assays run on histological sections

of water boatman collected from ponds in which severe TS outbreaks were on going

showed several individuals with TSV-positive gut contents. There was, however, no

indication that the virus was infecting or replicating in the insect (Lightner et al 1997b).

Infectious TSV has been found in the feces of laughing gulls, Larus atricilla , that fed on

infected shrimp during an epizootic in Texas (Garza et al. 1997). Controlled laboratory

studies have documented that TSV remains infectious for up to one day after passage

through the gut of white leghorns chicken, Gallus domesticus , and laughing gulls

(Vanpatten et al. 2004). Vertical transmission of TSV is thought to occur based on anecdotal information, but this has not been experimentally confirmed (Lightner and

Redman 1998; Dhar et al. 2004).

Shrimp surviving an outbreak of TSV seem to be refractory to reinfection (Ken Hasson reported by anon 1995) albeit remain carriers and are the sources for infection of susceptible animals (Vincent et al. 2004). It has been hypothesized that TSV was introduced to Southeast Asia with chronically infected shrimp imported from the Western

Hemisphere (Yu and Song 2000). The ability of TSV to remain at least partly infectious after one or several freeze-thaw cycles might be a contributing factor facilitating its spread in the international commerce of frozen commodity products (Lightner 1995).

TSV is however easily degraded by multiple freeze-thaw cycles when in purified or pre- purified form (Bonami et al. 1997). Mechanisms by which infected frozen shrimp could spread the virus include: reprocessing of shrimp at processing plants with release of infectious liquid wastes, disposal of solid wastes in landfills where seagulls could acquire

the virus and then spread it, the use of shrimp as bait by sport fishermen and the use of

imported shrimp as fresh food for other aquatic species (Lightner et al. 1997a). The latter

was practiced at the National Zoo in Washington when freshwater crayfish became

infected with another exotic virus of shrimp White spot syndrome virus (WSSV)

(reported in Lightner et al. 1997a). The importation of infected commodity shrimp might therefore present a risk of accidental contamination of wild and cultured stocks (Lightner et al 1997a). The disease has potential to become established in wild stock and its long- term effects on commercial penaeid fisheries or on the culture of native species are unknown (Lightner et al. 1997a; Flegel 2006).

TS can spread rapidly when introduced in new areas. A shrimp farmer described the 1995 outbreak in Texas as " This thing spread like a forest fire... There was no stopping it. I just sat there and watched it, and in a matter of three days, my shrimp were gone. Dead!"

(reported in Rosenberry 1995).

Diagnostic methods

A presumptive diagnosis of acute TSV infection can be established by the presence of dead or dying shrimp in cast nets used for routine evaluation. Predatory birds are attracted to diseased ponds and feed heavily on the dying shrimp, contributing to the dispersion of the disease throughout a farm or a shrimp farming region (Garza et al.

1997). The unique signs of infection caused by TS such as the cuticular melanized spots can provide a strong presumptive diagnosis, but care must be taken as these can be confused with other diseases such as bacterial shell disease (Chamberlain 1994; Lightner et al. 1995, Brock 1997). In general, pathognomonic histopathological lesions are the first step in confirmatory diagnosis. Discrete foci of pyknotic and karyorhectic nuclei and inflammation are seen within the cuticular tissues (Lightner et al. 1995). The lymphoid organ might display spheroids, but is otherwise unremarkable (Hasson et al. 1999c).

The genome of the virus has been cloned and specific cDNA probes are available for diagnosis (Mari et al. 1998). ISH analysis of acute phase lesions has demonstrated that gene probe positive signals are strongest within intact cuticular cells in the early stages of infection. It is believed that following cell lysis, the virus is rapidly dispersed by the hemolymph resulting in a weakly positive to negative probe signal to the remaining cellular (e.g. pyknotic or karyorhectic nuclear fragments) components (Hasson et al.

1997; Hasson et al. 1999b).

Reverse transcriptase polymerase chain reaction (RT-PCR) methods have been developed for detection of TSV and have a detection limit of 10 4 copies/µg of RNA (Nunan et al.

1998). Real-time RT-PCR using either a Taq Man assay or SYBR green has a lower detection limit of 10 2 copies/µg of RNA (Mouillesseaux et al. 2003; Tang et al. 2004).

Real-time techniques allow for quantification of the virus (Nunan et al. 2004). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed.

This method is performed under isothermal conditions, requiring only a simple water bath (Kiatpathmochai et al. 2007). Another method requiring only a simple heating device, is the isothermal nucleic acid-based amplification coupled with a colorimetric solid-phase assay (dot blot) described by Teng et al. (2006).

Multiplex RT-PCR for simultaneous detection of WSSV and TSV have been described

(Tsai et al. 2002). Xie et al. (2007) have developed a multiplex RT-PCR that allows for the detection of three viral pathogens: TSV, WSSV and infectious hypodermal and hemapoietic necrosis virus (IHHNV). A nested RT-PCR based technique is commercially available. The IQ2000 TM TSV detection and prevention system is said to have a detection limit of 10 copies per reaction (IntelliGene, Tapei, China).

RNA-based methods are specific and sensitive and have proven to be very helpful in research and as diagnostic reagents. However, they are relatively expensive to perform and require specific equipment and highly trained personnel. They are also limited by the relative fragility of the viral RNA. Prolonged fixation in Davidson's fixative might result in RNA degradation due to fixative-induced acid hydrolysis (Hasson et al. 1997; Lien et al. 2002). The complexity of these approaches has limited their practical “field” applications (Poulos et al. 1999). An alternative for virus detection is the utilization of specific monoclonal antibodies, MAbs, directed against the relatively stable proteins in the viral capsid. Because of their speed, versatility and reasonably good sensitivity

MAbs-based tests could be potentially very useful as a routine diagnostic test for the

32 screening of a large number of samples at a relatively low cost and with a minimal requirement for technical expertise (Poulos et al. 1999).

Such rapid diagnostic tests are now in common use for WSSV and are being marketed under the commercial name of Shrimple® (Enbio Tech, Tokyo. Japan). Similar tests for

TSV, Yellow head virus (YHV) and IHHNV are currently under development. These tests are based on the principle of the sandwich immunoassay, no special tissue preparation is required and results can be obtained readily (Takahashi et al. 2003).

Polyclonal antibodies against TSV have been made (Poulos et al. 1999;

Chaivisuthangkura et al. 2006) and rabbit anti-TSV PAbs are commercially available

(Abcam, Cambridge, MA). Polyclonal antibodies react with a wide range of epitopes and do not present the reactional stability of MAbs. PAbs also require a constant use of animals, whether mice, rabbits or chickens, with possible lot-to-lot variation. Monoclonal antibodies against TSV CP2 have been developed (Poulos et al. 1999). However they do not recognize three of the five recognized strains of the virus: TSV-SI, TSV-BZ and

TSV-VE (Erickson et al. 2002; Erickson et al. 2005; Cote et al. this dissertation). There is a definite need to produce monoclonal antibodies against these strains, which could be used in rapid diagnostic tests and other assays for virus characterization (Erickson et al.

2003). Monoclonal antibodies recognizing the more conserved CP3 have been developed

(Longyant et al. 2008). Targeting this protein could prove to be an interesting avenue for diagnosis.

Prior et al. (2003) have developed a controlled bioassay system for the determination of

the lethal infective dose of tissue homogenate containing TSV. Their bioassay system

uses individual flasks to house TSV challenged indicator shrimp to eliminate the

possibility of viral dose magnification during the challenge due to cannibalism or the

shedding of infectious particles in the water by infected shrimp.

Methods of control

Management strategies for the disease have included raising more resistant species such as Penaeus stylirostris and stocking of specific pathogen free (SPF) or specific pathogen

resistant (SPR) shrimp (Lightner and Redman 1998). Relatively simple laboratory

challenges can be used to predict the performance of selected stocks at farms where TSV

is enzootic (White et al. 2002; Wyban et al. 2004). TSV-resistant shrimp are susceptible

to infection but have high survival following challenge with at least four variants of TSV

(HI, BZ, VE, TH) (Srisuvan et al. 2006). In challenge assays there was no significant

difference between the survivals of TSV-resistant lines. The author stated that resistant

lines have reached nearly complete resistance to TSV-HI and TH and further

improvement due to breeding for TSV-resistance should be minor (Wyban et al. 2004).

Significant improvements in TSV survival were made through selective breeding despite

low to moderate heritability for this trait (Argue et al. 2002).

A management strategy used to reduce the impact of TS has been the practice of stocking

PLs at increased stocking density (Laramore 1997). Following this strategy, farms would experience mortality due to TS at an early stage of the production cycle, before substantial feeding had begun, and the surviving shrimp would be resistant to further

TSV challenges (Lightner and Redman 1998). Other techniques used with limited efficacy have been the polyculture of shrimp with tilapia and maintenance of near optimal water quality conditions in the grow-out ponds with reduction of organic loading

(Chamberlain 1994; Brock 1997). Transgenic shrimp expressing an antisense TSV coat protein (TSV-CP) exhibited increased survival in TSV challenges (Lu and Sun 2005).

The public perception of transgenic animals as well as current technical limitations, limit the use of transgenic animals as a mean of disease control (Einsiedel 2005).

There is one instance where TSV was successfully eradicated from one shrimp farming country (Belize). This required strict disinfection and quarantine protocols and was helped by the relative isolation and the relatively small size of the Belize shrimp farming industry (Dixon and Dorado 1997). TSV has since reappeared in Belize and eradication is now not considered a viable option for control of the disease, rather control has been achieved by using TSV-resistant shrimp stocks (Erickson et al. 2005; Argue et al. 2002).

The virus has been eradicated from the Hawaiian islands and was not detected in Hawaii during the period from 1994 to 2006 (Lightner et al. 2007). A case of TSV occurred in one Hawaiian farm and was confirmed in 2007 (OIE, 2007). TSV has not been reported from the State of Hawaii since clean-up occurred.

Figure 1.1. Transmission electronic micrograph of TSV purified from cesium chloride gradient ultracentrifugation. Stain 2% phosphotungstic acid (PTA). Mag: 53,000K.

Scale bars = 500nm

Figure 1.2. Schematic diagram of the genome organization of Taura syndrome virus

(TSV). Numbers indicate nucleotide positions. Open reading frames (ORFs) are shown as open boxes and untranscribed regions as a single line. The approximate positions of the

BIR-like sequence (BIR), helicase (H), protease (P) and RNA-dependent RNA polymerase (RdRp) are indicated. Arrows represent the N termini of the capsid proteins

(From Mari et al. 2002).

Figure 1.3. Histopathology of Taura syndrome (TS) in hematoxylin and eosin stained sections. Photomicrographs of sections of juvenile P. vannamei in acute phase of the disease. A) stomach (Mag 200X); B) gills (Mag 200X).

Figure 1.4. Histopathology of chronic Taura syndrome showing severe lymphoid organ spheroids (Mag 200X)

Development and characterization of a monoclonal antibody against Taura syndrome

virus.

Isabelle Côté 1, Bonnie T. Poulos 2, Rita M. Redman 1, Donald V. Lightner 1

1Department of Veterinary Sciences and Microbiology, The University of Arizona, Tucson AZ 85721

2Department of Ecology and Evolutionary Biology, The University of Arizona, Tucson AZ 85721

______

ABSTRACT: We produced a panel of Monoclonal antibodies (MAbs) from the fusion of

TSV-BZ immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting

SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot blot immunoassay and immunohistochemistry (IHC) analysis.

The MAb reacted against native TSV-BZ, TSV-SI and TSV-HI in dot blot immunoassay.

By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled

TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants but not for the TSV-VE and TSV-TH variants. MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody improves currently available TSV diagnostic applications.

INTRODUCTION

In 1992, Jimenez described Taura syndrome (TS) in Ecuador. Since, TS has become a major threat to the shrimp farming industry worldwide causing outbreaks in most shrimp farming areas of the Americas (Hasson et al. 1999a) and South-East Asia (Tu et al. 1999;

Nielsen et al. 2005). The causative agent of the disease is a non-enveloped, single- stranded positive sense RNA virus member of the Discistroviridae family: Taura syndrome virus (TSV) (Mari et al. 2002; Mayo and Ball 2006). The fully sequenced genome is 10,205 nucleotides in length and encodes two open reading frames (ORF). The first ORF encodes the housekeeping genes: a helicase, a protease and a RNA-dependent

RNA polymerase. The capsid consists of three major polypeptides encoded in the second

ORF: CP1 (40 kDa), CP2 (55 kDa) and CP3 (24 kDa), and a minor protein of 58 kDa

(Mari et al. 2002).

The disease has three overlapping phases: acute, transition and chronic (Hasson et al.

1999b). Histologically, the acute and transitional phases are characterized by multifocal pyknosis/karyorrhexis associated with cytoplasmic inclusion bodies in the cuticular epithelium and the subcutis of the general body surface, appendages, gills, hindgut, oesophagus and stomach (Lightner et al. 1995; Hasson et al. 1999b). At histology, chronically infected animals have lymphoid organ spheroids (LOS) in the absence of other lesions (Hasson et al. 1999c).

Treatment measures against shrimp viruses are limited. Efforts to prevent TSV infection

have concentred on the raising of TSV-resistant animals. Resistant lines have been

successfully developed and their use has limited the impact of the disease. However, viral

variants have emerged to which resistant shrimp lines might be susceptible (Argue et al.

2002; Erickson et al. 2002).

Reverse transcriptase PCR and gene probes are available for diagnosis of TSV infection

(Mari et al. 1998; Nunan et al. 1998). These RNA-based detection methods require

preservation of the relatively fragile viral RNA (Hasson et al. 1997). Alternatively,

antibody-based methods recognizing the more stable capsid proteins are more

advantageous in field situations because they require less technical expertise and no toxic

chemicals (Sithigorngul et al. 2006).

Mutation rates are higher in most RNA viruses than in DNA viruses due to the lack of

fidelity and proofreading activity of RNA dependent RNA polymerase. The frequency of

errors during RNA genome replication can be as high as 1 misincorporation per 10 3-10 4 nucleotides polymerised (Holland et al. 1982). Several TSV isolate have been identified and are named with the place and date of first isolation: TSV-HI94, TSV-SI98, TSV-

BZ02, TSV-VE05 and TSV-TH05 (hereafter referred to as: TSV-HI, TSV-SI, TSV-BZ,

TSV-VE and TSV-TH) (Erickson et al. 2002; Erickson et al. 2005; Tang & Lightner

2005; Srisuvan et al. 2006).

The gene encoding CP2, where regions are sequenced, is a zone of high variability

amongst TSV isolates (Tang and Lightner 2005). TSV isolates have been tentatively

attributed to serotypes with regard to their reactions with MAb 1A1, an antibody that

reacts to the CP2 of TSV (Poulos et al. 1999; Erickson et al. 2003). MAb 1A1 recognizes

TSV-HI, TSV-TH and TSV-VE (native form only) and not other variants of TSV (Poulos

et al. 1999, unpublished observation). Here we report on the development of a

monoclonal antibody that recognizes TSV-BZ, TSV-HI and TSV-SI but not TSV-TH and

TSV-VE.

MATERIAL AND METHODS

Experimental shrimp. The Oceanic Institute's SPF breeding program in Hawaii provided specific pathogen free (SPF) Kona stock Penaeus vannamei (Holthuis, 1980;

Wyban et al. 1992). The shrimp were maintained at the Aquaculture Pathology

Laboratory of the University of Arizona as described in White et al. (2002).

Antigens. For the production of antigens, Kona Stock P. vannamei received a 50 µm intramuscular injection of a 1:10 homogenate of TSV-BZ infected tissue in 2% saline.

Shrimp were maintained in 90 L tanks equipped with a biofilter. Moribund and dead shrimp were removed from the tank and kept frozen at –70 oC until used for viral purification. Viral purification was performed as described by Bonami et al. (1997) with

minor modifications. Following the sucrose gradient the fractions were diluted in 10 mM

Tris- 400 mM NaCl (TN) 1:2 and centrifuged for 3 h at 286,000 g. The resulting pellet

was suspended in 1 ml of filter sterilized TN with 1 µl (0.1%) of Triton X-100 and

incubated for 1 h on ice on a rocking apparatus (modified from Tsukamoto et al. (1990).

The solution was thereafter layered onto a 15-45% cesium chloride gradient and

centrifuged for 13 h at 208,500 g. Fractions associated with the band were collected,

pooled and diluted 1:2 in TN and centrifuged at 286,000 g for 3 h. The supernatant was

discarded and the pellet was suspended in filter-sterilized TN. The virus preparation was

negatively stained with 2% phosphotungstic acid (PTA) and examined with a JEM-

100CXII transmission electron microscope for purity analysis. The same procedure was

used to purify TSV-HI, TSV-SI and TSV-VE using archived shrimp or tissue

homogenate. The triton wash was not performed on these later viruses. The genome

copies number in all four viral preparations was determined with a real-time RT-PCR

TaqMan assay as described by Tang et al. (2004).

The CP2 region of the purified viruses was amplified using the primers 55P1 and 55P2 as

described by Tang and Lightner (2005), using 1 µl of the purified virus solution in the

PCR reaction. A 10 µl aliquot of the amplified product was analysed in a 1.5% agarose

gel containing ethidium bromide. The amplified fragments were cleaned using a

QIAQuick PCR purification kit (Quiagen) according to the manufacturer's recommendations. The fragments were sequenced with an ABI Prism 377 automated

DNA sequencer by the sequencing facility of the University of Arizona. The sequence

obtained was blasted against TSV sequences in the NIH database

(www.ncbi.nlm.nih.gov) for homology.

Hemolymph was collected from shrimp infected with yellow head virus (YHV), White

spot syndrome virus (WSSV) as well as from specific pathogen free (SPF) Penaeus

monodon and P. vannamei . The hemolymph was frozen at -70 oC until used in dot blot

analysis.

Immunization. 5 weeks old Balb/c mice, Mus musculus (Linnaeus ), were obtained from

Jackson Laboratory (Bar Harbor, ME) and kept at the Central Animal Care facility of the

University of Arizona for at least 2 weeks prior to the immunization procedure. All mice

manipulations were done in accordance with the recommendations from the Institutional

Animal Care and Use Committee of the University of Arizona. For the immunization of

the mice, a mixed viral solution of native and denatured (boiled for 5 min) in a ratio of

1:1 was prepared using purified TSV-BZ. The viral antigen solution was diluted with

Titermax gold adjuvant (Sigma) prepared following the manufacturer's recommendations.

Mice were injected with 100 µl in two sites on the flank (50 µl each site), followed by a

booster injection 3 weeks later. An intraperitoneal injection booster without adjuvant was

given exactly 72 h prior to the fusion.

Fusion . The fusion was carried out using the standard PEG-mediated procedure described by Gefter et al. (1977). Spleen cells from the immunized mice were fused with

approximately 1E 7 SP2 myeloma cells pretreated with 8-azaguanine (Sigma). The resulting cell solution was plated in 24 wells Costar plates (Corning) with RPMI conditioned culture medium with 20% heat inactivated fetal calf serum and 5% Briclone

(QED Bioscience) for 24 h. After 24 h, culture medium with 2X hypoxanthine- aminopterin-thymidine (HAT) (Sigma) was added for a final concentration of 1X HAT.

The cells were fed as deemed necessary by their appearance and the pH of the media.

After 15 days, the cells were fed HT media without aminopterin. At all stages of culture,

o cells were maintained at 37 C in a 5% CO 2 moisturized atmosphere. The water bath in

the incubator was changed monthly and contained 0.5% Aquaclean (WAK-Chemie). The

cells were maintained under conditions of strict sterility and no antibiotics were added

during the culture process. The hybridoma cultures supernatants were tested for the

production of antibodies specific to TSV by dot blot immunoassay on purified TSV and

SPF shrimp hemolymph.

The dot blot was performed as described by Poulos et al. (1999) with minor

modifications. Briefly, MAHAN 45 plates (Millipore) were dotted with 1 µl of purified

virus and 1 µl of SPF hemolymph and let dry. They were blocked at 37 oC for 30 min with

150 µl of 10% normal goat serum (NGS), 10% fat-free powdered milk in PBS. The wells

were then reacted with 100 µl of hybridoma supernatant fluid for 30 min (sometimes

incubation times up to 4 h were used) at 37 oC; mice sera (PAbs) diluted 1:100 in PBS

was used as a positive control. The wells were washed three times with PBS and reacted

with 100 µl of a 1:1000 dilution of phosphatase-labelled goat-anti mouse F(ab') 2

fragments (Kierkegaard & Perry) for 30 min at 37 oC. Wells were washed three times with

PBS and the reactions were visualized by development with nitroblue tetrazolium and bromo-chloro-indoyl phosphate for 30-45 min in the dark. Wells specific to TSV were cloned four times by limiting dilution. Cell numbers were expanded in 25 cm 2 flasks and frozen at all stages of culture. The Experimental Mouse Shared Service of the University of Arizona tested the cell culture for mycoplasma contamination with the MycoAlert

Mycoplasma Detection Kit (Lonza).

Dot blot immunoassay. Dot blot immunoassay was performed as previously described on hemolymph infected with YHV, WSSV, SPF P. vannamei , SPF P. monodon as well

as purified TSV-BZ, TSV-HI, TSV-SI and TSV-VE.

Immunohistochemistry and In situ hybridisation. Immunohistochemistry (IHC) was

performed on fixed samples that were found positive for TSV by standard histology

(Poulos et al. 2001). Briefly, paraffin embedded samples were cut in 4 µm sections,

heated at 65 oC for 30 min and rehydrated. The slides were washed for 5 min in PBS and

blocked for 30 min in a humid chamber at 37 oC using blocking buffer (PBS + 2% dry

milk + 10% NGS). The slides were reacted with 500 µl of hybridoma supernatant fluid

from cell culture that was positive for TSV-BZ in immunodoblot. The slides were washed

three times (2 min each wash) with PBS and incubated with 500 µl of 1:1000

o phosphatase-labelled goat-anti mouse F(ab') 2 fragments at 37 C for 30 min. The slides

were washed three more times and incubated with developing solution for 1 h at RT in

47 the dark. The slides were counter-stained with Bismarck brown, dehydrated and coverslipped using permount. In situ hybridization (ISH) was performed on sections from the same tissue sample with the P15-Q1 probe using the procedure described by Mari et al. (1998).

IHC was also performed on samples infected with hepatopancreatitis parvovirus (HPV), infectious hypodermal and hemapoietic necrosis (IHHNV), infectious myonecrosis virus

(IMNV), YHV, WSSV, Spiroplasma penaei and SPF P. vannamei .

SDS-PAGE and Western blot analysis. TSV preparation was denatured in Laemmli buffer (Laemmli 1970) with 10% urea and separated using a 12% resolving polyacrylamide gel (Bonami et al. 1997) at 25 mAmp for 100 min. A prestained kaleidoscope polypeptide standard (Biorad) and a pre-mixed molecular mass marker

(Roche) were run alongside the virus for reference. Gels were either stained with 0.1%

Coomassie blue (Wilson 1983) or transferred to nitrocellulose membrane for Western blot analysis as described by Poulos et al. (1999). For the Western blot, the membranes were dried overnight at RT and reacted with undiluted supernatant or mouse PAb diluted

1:1000 in PBST + 10% NGS. Antibody incubations were performed in bags for 1h on a rocking apparatus. The reactions were visualized as in the dot blot immunoassay.

Isotype determination. The isotype of the antibody was determined using the

ImmunoPure Monoclonal Antibody Isotyping kit II (Pierce) following the antigen-

independent ELISA procedure as described by the manufacturer.

RESULTS

Antigens. With TEM the purified virus preparations of TSV-VE, TSV-BZ and TSV-SI were found to contain only one type of virus particles that were consistent with TSV as previously illustrated in Hasson et al. (1995). The viral load of the TSV-BZ antigens used for the mouse immunization was 6.84E 9 viral RNA copies per µl as determined by real- time RT-PCR. Viral titers of all viruses were obtained and these were diluted in PBS to obtain identical concentrations for use in the dot blot immunoassay.

Monoclonal antibody production. The selected clone, 2C4, was stable in culture and produced antibodies even after being subcloned 25 times over a period of 2 months. This clone could be frozen and retrieved without loss of antibody production and did not present bacterial or mycoplasma contamination. No other clone proved to be specific to

TSV and highly stable. Some clones showed faint positive reactions to TSV but these disappeared with subsequent clonings.

Determination of isotype. Mabs 2C4 was determined to be of the IgG 1κ isotype using the ImmunoPure Monoclonal Antibody Isotyping kit II.

Antigen specificity of Mabs. We tested MAb 2C4 by dot blot immunoassay (four variants) and IHC (five variants). By dot blot immunoassay the antibody reacted against the following variants: TSV-BZ, TSV-HI and TSV-SI, but not against TSV-VE (Fig.

2.1). By IHC the antibody reacted with shrimp infected with TSV-BZ, TSV-HI and TSV-

SI but not in shrimp infected with TSV-VE and TSV-TH (Table 2.1). For IHC, samples from both the acute and chronic phases were included in the analysis. All TSV-infected slides examined were positive by ISH. It was observed that, in general, IHC with 2C4 did not give as strong results as ISH (See Fig. 2.2 for representative ISH and IHC images).

The antibody was, however, sensitive as all shrimp infected with TSV-BZ, TSV-HI or

TSV-SI that were positive by ISH were also detected by IHC.

The antibody was highly specific for TSV. It did not react by dot blot assay with YHV,

WSSV or SPF hemolymph. It was negative by IHC against all other shrimp pathogens tested (HPV, IHHNV, IMNV, YHV, WSSV and S. penaei ) (data not shown).

SDS-PAGE. By SDS-PAGE, the protein profile of the purified virus (TSV-BZ, TSV-HI and TSV-SI) contained three major proteins of 55 kDa, 40 kDa and 24 kDa. This is consistent with what was described by Bonami et al. (1997). The TSV-HI preparation was more concentrated and showed stronger bands (Fig. 2.3).

Western blot analysis. Western blot was performed with the BZ, HI and SI variants of

TSV. MAb 2C4 recognized CP2 of all three variants (Fig. 2.4). A small faint band was

observed under the TSV-HI variant CP2 (Fig. 2.4 arrowhead). PAbs reacted strongly with

CP2 of all variants tested (data not shown). The absence of strong reactions with the other

bands CP1 and CP3 is consistent with what was observed in Poulos et al. (1999) who

observed a very weak reaction with CP1 and no reaction with CP3 using PAbs against

TSV-HI.

DISCUSSION

The purpose of this study was to develop a panel of highly specific monoclonal

antibodies that could be used in a cocktail recognizing all strains of TSV and individually

recognize specific TSV serotype. Only one stable mouse hybridoma cell line producing

monoclonal antibodies against TSV was obtained. The antibody produced, designated

MAb 2C4, was of the IgG 1κ isotype. It consistently demonstrated highly specific

reactions in all formats (IHC, dotblot and Western blot) tested.

Using IHC, no positive reaction was found in healthy shrimp tissue, tissue infected with

other pathogens or non-target TSV tissue. There was no reaction with any of the TSV-TH

and TSV-VE slides examined by IHC. These slides were, however, positive for TSV by

ISH. Two samples labelled as TSV-HI did not react by ISH and IHC despite presenting

51 good lesions upon histopathological examination (data not shown). Protein (Ramos-Vara

2005) and RNA (Hasson et al. 1997) can be denatured upon the point of non-recognition following excessive fixation, this could explain this unexpected result in these archived

TSV specimens.

The antibody reacted strongly with the band associated with CP2 in Western blot as well as with a band of slightly lower molecular weight for TSV-HI. The significance of this band is not known. It could represent a cleavage product of CP2 that conserves some of the same protein determinants. Some viruses, such as Acyrthosiphon pisum virus , present proteolytic breakdown of some of their polypeptides resulting in capsid proteins of varying lengths having identical series of amino acids (van der Wilk et al. 1997). It is unknown why this band was present in TSV-HI but not in TSV-BZ nor TSV-SI. The quantity of virus particles used in the different preparation may account for the bands visualized.

CP2 is a region of high genetic variability amongst TSV variants. MAbs recognize epitopes sometime as small as four amino-acids (Sominskaya et al. 1992). The amino acids responsible for antibody reactivity might be conserved for BZ, HI and SI but not for

VE and TH. Similar to MAb 1A1, MAb 2C4 reacts against CP2 opening interesting venues for classifying strains of TSV into serotypes. The detecting ability of the currently available cocktail of MAbs (1A1 and 2C4) includes TSV-TH, TSV-HI, TSV-SI, TSV-BZ and the native form of TSV-VE. This captures most variants currently encountered in the

Americas and Asia and might provide a good first line diagnostic test for TS. Other

authors have reported the production of MAbs against another capsid protein of TSV:

CP3 however these MAbs have not been tested against all the variants examined in this

study (Longyant et al. 2008).

Despite numerous fusions and the production of several healthy clones, only one

monoclonal antibody was produced. Poulos et al. (1999) reported similar problems during the development of MAb 1A1. They developed three clones that reacted with

TSV, out of which only 1 (MAb 1A1) presented consistently strong reactions. This success rate is lower compared to other aquatic pathogens whether bacterial such as

Piscirickettsia salmonis (Jamett et al. 2001) or viral such as White spot syndrome virus

(Shih et al. 2001). In these cases, panels of 28 and 20, respectively, monoclonal antibodies or positive hybridoma clones were produced.

Many of the first clones produced showed reactivity with SPF shrimp hemolymph. As stated by Berry (2005), whole pathogen immunogen preparations are inherently contaminated unless they can be grown in pure form. Since TSV can not be cultivated in vitro , we tried to address this issue by washing the virus with Triton-X. Shih et al. (2001) have described a MAb produced following injection of purified WSSV that bounded specifically to two shrimp proteins and reacted to healthy and non-target infected tissues.

These results strongly suggest that MAb 2C4 is useful for the diagnosis of TSV-HI, TSV-

SI and TSV-BZ infection and provides useful information for the classification of TSV variants. It presents a step towards improved rapid detection methods for TSV.

Acknowledgments. The principal author is most thankful to D. Bradley-Dunlop

(Department of Immunobiology, University of Arizona) for technical advices. Grant support for this study was from the United States Marine Shrimp Farming Consortium under Grant No. 2004-38808-02142 from the Cooperative State Research, Education and

Extension Service, U.S. Department of Agriculture.

Figure 2.1. Dot blot assay using MAb 2C4, From left to right dotted TSV-BZ, TSV-HI,

TSV-SI, TSV-VE reacted with MAb 2C4 followed by the viruses in the same order reacted with PAbs. A positive reaction is indicated by a blue spot on the membrane.

Figure 2.2. IHC and ISH reaction in TSV-HI in the stomach of a P. vannamei infected with TSV-HI 7 post-infection a) ISH with e labelled cDNA-TSV probe. b) IHC with 2C4

(Mag 100X).

Figure 2.3. SDS-PAGE of proteins from purified TSV isolates stained with Coomassie

Blue. Lane 1, TSV-BZ. Lane 2, TSV-HI. Lane 3, TSV-SI. Lane 4, Molecular weight marker. The molecular masses of the proteins are indicated in kDa.

Figure 2.4. Western blot of TSV preparations reacted with MAB 2C4: Molecular weight marker (lane 1), TSV-BZ (lane 2), TSV-HI (lane 3) and TSV-SI (lane 4). Arrow indicates faint band below 55 kDa protein in TSV-HI preparation.

Table 2.1. Sensitivity of the anti-TSV MAb 2C4 as determined by IHC on fixed tissue sections and compared with ISH and histological findings of the same sample. MAb 2C4 failed to detect two variants of TSV by this method.

Sample # variant Days pi ISH IHC Histological findings 1 BZ 15 Pos Pos LOS 24 2 BZ 1 Pos Pos Buckshot 3 G1-44 3 BZ 2 Pos Pos LOS3, buckshot G2-3 4 BZ 3 Pos Pos LOS2, buckshot G2-4 5 BZ 3 Pos Pos Buckshot G2-4, no LO 6 HI 15 Pos Pos LOS3-4 7 HI 5 Pos Pos Buckshot G1-2 8 HI 4 Pos Pos LOS2, buckshot G2-3 9 HI 4 Pos Pos LOS2, buckshot G1 10 HI 9 Pos Pos LOS3, buckshot G2 11 HI 7 Pos Pos LOS2, buckshot G2-4 12 SI 2 Pos Pos LOS2, buckshot G3-4 13 SI 15 Pos Pos LOS3 14 SI 15 Pos Pos LOS4 15 SI 2 Pos Pos Buckshot G1-2 16 SI 3 Pos Pos Buckshot G1-2, Granuloma G4 LO 17 TH 3 Pos Neg Buckshot G2 18 TH 3 Pos Neg Buckshot G2-3 19 TH 4 Pos Neg LOS1-2 20 TH 4 Pos Neg LOS2, buckshot G2-3 21 TH 15 Pos Neg LOS3 22 VE 6 Pos Neg LOS2, Buckshot G2-3 23 VE 4 Pos Neg LOS2, buckshot G2-3 24 VE 2 Pos Neg LOS1, buckshot G1 25 VE 4 Pos Neg LOS1, buckshot G1-2 26 VE 14 Pos Neg LOS3

2 LOS: lymphoid organ sheroid 3 Buckshot: acute TSV lesions 4 G1 to G4: severity grade of infection/lesion according to the scale described in Hasson et al. 1995

CHAPTER 3

Taura Syndrome Virus from Venezuela is a New Genetic Variant.

I. Cote, S. Navarro, K.F.J. Tang, B. Noble, D.V. Lightner

Aquaculture Pathology Laboratory, Department of Veterinary Sciences and Microbiology, The University of Arizona, Tucson, Arizona 85721, USA

______

ABSTRACT: In early 2005, the Aquaculture Pathology Laboratory of the University of

Arizona received samples of diseased shrimp, Penaeus vannamei , from a Taura syndrome (TS) outbreak in the Lake Maracaibo region of Venezuela. Histopathology and in situ hybridization (ISH) were performed and the results confirmed the presence of

Taura syndrome virus (TSV). The viral isolate was sequenced and presented a 94% similarity with the TSV reference strain from Hawaii (TSV-HI94).

Immunohistochemistry (IHC), dot blot immunoassay and bioassays were also performed.

While processed samples reacted only faintly with the TSV monoclonal antibody MAb

1A1, the virus in its native state reacted strongly with the antibody. In bioassay, the

Venezuelan isolate of TSV (TSV-VE05) presented mortality comparable to TSV-HI94 in

P. vannamei SPF Kona stock. These data indicate that a new variant of the virus was responsible for the outbreak of TS in Venezuela.

INTRODUCTION

Since its first appearance in 1992, Taura syndrome (TS) has caused significant economic

losses to the penaeid shrimp culture industry worldwide. The first TS epizootic was

reported in Ecuador in 1992 (Jimenez 1992). Outbreaks have since been reported from

most shrimp farming regions of the America and Asia (Hasson et al. 1999a; Yu & Song

2000; Nielsen et al. 2005; Do et al. 2006). This disease is known to affect many penaeid

shrimp species. It has no known impact on the naupliar through mysis stages. However, it

seriously affects the post-larvae, juvenile and adult stages of Penaeus vannamei , the most

susceptible host (Brock 1997).

The clinical signs of acute TS include anorexia, lethargy, erratic swimming behavior and

expansion of red chromatophores (Lightner et al. 1995; Hasson et al. 1999b). This phase

is followed by a transition phase, characterized by the presence of multiple melanized

lesions on the cuticle of the tail and the cephalothorax, and a definitive chronic phase

during which animals do not display any outward signs of infection. The chronic phase is

characterized histologically by the presence of numerous lymphoid organ spheroids

(LOS) in the absence of other pathology (Hasson et al. 1999c).

TS is caused by Taura syndrome virus (TSV) a member of the Dicistroviridae family

(Mari et al. 2002; Mayo and Ball 2006). TSV is a non-enveloped, single-stranded positive sense RNA virus (Hasson et al. 1995; Bonami et al. 1997). The viral genome has been

61 completely sequenced and consists of 10,205 nt encoding two open reading frames

(ORF). The 5' ORF (ORF1) encodes the non-structural proteins: a helicase, a protease and a RNA-dependent RNA polymerase. ORF2 encodes three major and one minor capsid proteins (CP) of 40 kDa (CP1), 55 kDa (CP2), 24 kDa (CP3), and 58 kDa

(Bonami et al. 1997; Mari et al. 2002).

Comparison of genome sequences has allowed the categorization of emerging TSV isolates into different genotypic clades. Phylogenetic analysis of TSV isolates from 1993 to 2004 revealed three major clusters designated as the "Americas", "Belize" and "SE

Asia" (Tang & Lightner 2005). TSV variants have also been identified according to the reaction to the TSV monoclonal antibody: MAb 1A1 (Erickson et al. 2003). MAb 1A1 is the only monoclonal antibody currently available for the antibody-based classification of

TSV isolates. It has been shown to react to the 55 kDa CP2 (Poulos et al. 1999). The variants TSV-A (HI94 reference strain), TSV-B (MX98) and TSV-C (BZ02) are currently recognized (Erickson et al. 2003). Of these, TSV-A is the only variant reacting with MAb 1A1.

In 2005, the Aquaculture Pathology Laboratory (APL) received samples of diseased

Penaeus vannamei from an apparent TS outbreak in Venezuela. This represented the first recognized outbreak of TSV infection in farmed P. vannamei in Venezuela (Conroy

2005). This isolate (hereafter referred to as TSV-VE05) did not show positive reaction by

IHC with MAb 1A1. The genome was sequenced and compared to the Hawaiian

62 reference strain (TSV-HI). The CP2 sequence was compared against other isolates collected during 1993-2005 (Tang and Lightner 2005). The virulence of this isolate was evaluated in a bioassay using specific pathogen free (SPF) P. vannamei Kona stock

(White et al. 2002). The findings suggest that TSV Venezuela is a new variant of the virus.

MATERIALS AND METHODS

Shrimp and TSV isolates samples . The taxonomy used for the penaeids in this paper is according to Holthuis (1980). Shrimp samples ( Penaeus vannamei) from several farms in northwest Venezuela displaying gross signs characteristic of a TSV infection were received at the APL for diagnosis. The samples of P. vannamei juveniles, subadults and adults were fixed in Davidson’s alcohol-formalin-acetic acid (AFA) for histological examination (Bell and Lightner 1988). A separate set of samples was fixed in 95% ethanol for RT-PCR analysis. Additional frozen material was received and used in infectivity and virulence studies with SPF P. vannamei Kona stock (White et al. 2002).

In situ hybridization (ISH) and Immunohistochemistry (IHC) . Shrimp samples from the original case and the experimental bioassay were fixed by injection of Davidson's

AFA followed by immersion in 10 volumes of fixative. The samples were transferred to

70% ethanol after 24 h, followed by a second transfer after 48 h. They were processed for

63 routine paraffin embedding with Paraplast X-tra (McCormick Scientific), sectioning and staining with conventional Mayer-Bennett’s hematoxylin/eosin-phloxin as described by

Lightner (1996). Selected samples were evaluated by ISH with digoxigenin-labeled gene probes P15/Q1 as described by Mari et al. (1998). IHC was performed using either MAb

1A1 specific for the detection of the original strain of TSV (TSV-HI94) (Poulos et al.

1999) or a polyclonal antibody (PAb) as described by Poulos et al. (2001). The Mab 1A1 consisted of hybridoma supernatant fluid purified by anionic exchange and diluted 1:5 in blocking buffer (PBS with 2% powdered skim milk and 10% normal goat serum). The

PAb 1:100 in blocking buffer consisted of serum from mice immunized with TSV-BZ02 purified viral particles diluted. The PAb is known to react with TSV-HI94 and TSV-

BZ02 in a dotblot immunoassay and in IHC.

TSV RT-PCR and genomic sequencing . Total RNA was extracted from the ethanol fixed tissue samples using a High Pure RNA extraction kit (Roche Biochemical) according to the manufacturer's recommendations. Primers were designed using Primer3

(http://biotools.umas smed.edu/bioapps/primer3_www.cgi) and used to amplify the

Venezuela TSV genome. SuperScript II one-step RT-PCR with Platinum Taq

(Invitrogen) was used for the amplification. The cycling parameters consisted of one cycle at 50ºC for 30 min, 94ºC for 2 min and 39 cycles at 94ºC for 30 s, 55ºC for 30 s,

68ºC for 1.5 min with a final extension cycle of 68ºC for 5 min. The RT-PCR products were visualized by electrophoresis of ethidium bromide stained agarose gels under UV light. The resulting bands were excised with a scalpel blade, cleaned with a QIAQuick

PCR purification kit (Quiagen) and sequenced with a DNA sequencer ABI Prism 377

(Applied Biosystems) by the sequencing facility at the University of Arizona. The

10,095 nt sequence is available in Genbank under the accession number: DQ212790.

Phylogenetic analysis . The nt 7952-9151 sequence corresponding to the region encoding

CP2 was compared against the TSV isolates from the UAZAPL collection (Table 3.1).

The amino acid sequence was deduced from the nucleic acid sequence and the phylogenetic analysis was performed using the MEGA software package

(http://www.megasoftware.net). The phylogenetic tree was constructed with the neighbor-joining (NJ) method (Saitou and Nei 1987). The deduced amino acid sequences from the protein CP2 from Venezuela TSV and Hawaii TSV were further analysed using the ProtParam open source software (http://ca.expasy.org/tools/protparam.html).

Virus purification . Virus purification followed the technique described by Bonami et al.

(1997) with minor modifications. Frozen shrimp heads were homogenized 1:3 in TN buffer (20 mM Tris, 0.4 M NaCl) using an Ultra-turrax tissue blender and clarified three times (10 min at 1000 g using an IEC HN-SII centrifuge followed by 15 min at 4300 g and 30 min at 30,600 g using an SS34 rotor in a Sorvall RC5-B Superspeed centrifuge).

The pooled supernatant fluid was centrifuged at 205,100 g for 3 h (T647.5 rotor in a

Sorvall Ultracentrifuge). The pellet was resuspended, homogenized and extracted 3 times with freon. Charcoal was added for 5 min and the supernatant fluid was filtered through

Celite535 (Fluka) under vacuum and pelleted at 205,100 g for 3 h (T647.5 rotor). The

65 pellet was homogenized and layered onto a 15-40% sucrose gradient and centrifuged at

233,000 g for 2 h (TH641 rotor). The gradient fractions were removed with an

Autodensiflow IIC (Buchler) and collected using an ISCO Retriever II. The absorbance readings of the fraction were plotted upon collection by an ISCO UA5 monitor at a wavelength of 254 nm. The fractions associated with the peak were washed in TN and pelleted (233,000 g, 3 h, TH641 rotor). The pellet was homogenized in TN buffer and layered onto a 15-45% cesium chloride gradient (12 h, 208,500 g, TH641 rotor). The fractions were recovered as described above. Fractions associated with the peak were pooled, diluted with TN and pelleted at 233,000 g for 3 h (TH641 rotor). The final virus preparation was negatively stained with 2% phosphotungstic acid (PTA) at pH 7 on 200 mesh collodion carbon-coated grids and examined using a JEM-100CXII microscope

(JEOL).

Dot blot immunoassay . The dot blot immunoassay was performed using MANAH45 multiscreen plates (Millipore). Purified TSV-VE05 virus preparations, native or boiled for 5 min, were dotted on the plate. The wells were blocked using 150 µl of blocking buffer for 30 min. The buffer was poured off and 100 µl of MAb 1A1 (1:5 in blocking buffer) or 100 µl of a PAb dilution (1:100 in PBS) was added to the wells and incubated for 30 min. The antibodies were poured off and washed 3 times with PBS before adding alkaline phosphatase labelled goat anti-mouse IgG F(ab') 2 heavy and light chains

(Kierkegaar and Perry) as secondary antibody (30 min incubation with 100 µl of a 1:500 dilution). The supernatant fluid was poured off and the plate washed 3 times with PBS.

The wells were developed using 100 µl of developing solution: Buffer III (100 mM Tris

HCl pH 8.0, 100 mM NaCl and 50 mM MgCl 2) containing nitroblue tetrazolium (NBT)

(Roche), 0.3375 mg/ml and 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate), 0.175 mg/ml for 30 min. A positive reaction was indicated by the presence of a dark blue precipitate on the membrane. All incubations were done in the dark at 37 oC.

Bioassay . The bioassays followed the method described by White et al. (2002) and were performed on five separate occasions. Briefly, 20 Kona stock Penaeus vannamei (mean weight: 1-2.5 g) were housed in 90 L aquaria fitted with a biological filter. Salinity was adjusted to 25 ppt by the use of Crystal Sea Salt Marine Mix (Marine Enterprise

International, Baltimore, MD). Shrimp were fed minced shrimp tissue generated with tissue from the original outbreak in Venezuela or tissue infected with TSV-HI at 10% body weight (BW) on day 1, 10% BW on days 2 and 10% BW on day 3. All animals were fed ad libidum with pelleted shrimp feed (Rangen, Buhl, ID) for the duration of the experiment. The experiments were conducted over 15 days. Mortality was recorded daily.

Surviving shrimp were fixed in Davidson's AFA and examined by routine histopathology.

RESULTS

Histopathological analysis of the initial samples from Venezuela showed severe Taura syndrome infection characterized by "buckshot lesions" in the cuticular epithelium of the gills, appendages, cuticle and/or cuticular epithelium of the foregut. Some shrimp also had lymphoid organ spheroids (Hasson et al. 1999c). No signs of infection by other shrimp viruses including White spot syndrome virus (WSSV), Baculovirus Penaei (BP) , hepatopancreatitis parvovirus (HPV), infectious myonecrosis virus (IMNV) or yellow head virus (YHV) were observed upon histopathological examination of the samples. The samples were also examined by RT-PCR for the presence of infectious myonecrosis virus

(IMNV) and were found to be negative.

By ISH for TSV, a strong blue-black precipitate was observed in focal lesions in cuticular tissue (data not shown). TSV specific probes were found to react strongly with the cytoplasm of the cuticular epithelial cells of the gills, appendages, cuticle and foregut.

However, no positive reaction was observed on the consecutive histology sections analysed by IHC using the MAb 1A1 (data not shown). ISH was also performed in chronically infected shrimp form the bioassays and these showed strong positive reactions (Fig. 3.1b). On IHC these slides were strongly positive using PAb (Fig. 3.1c), however only a very faint positive reaction could be observed with the MAb 1A1 (Fig.

3.1d)

The full TSV-VE05 genome was sequenced (GenBank DQ212790) and the 10,095 nucleotide sequence was compared to TSV-HI94. An overall 94% identity was obtained from the comparison (Table 3.2). It has been shown that the CP2 region of TSV has variations that may account for the serological classifications of the TSV isolates

(Erickson et al. 2002). CP2 also has the highest genetic variation of the three major capsid proteins (Tang & Lightner 2005). The CP2 sequence (1200 nts) of TSV-VE05 was pair-wise compared to TSV variants representing the three major clusters: "Americas",

"SE Asia" and "Belize". The nucleotide sequence identity of TSV-VE05 was 93% with

TSV-HI94 and TSV-TW99 strains and 91% with TSV-BZ02. The amino acid sequence identity was the highest with TSV-HI94 (96%) followed by TSV-TW99 (95%) and finally by TSV-BZ02 (94%). An unrooted phylogenetic tree was generated by the neighbor-joining method using the deduced amino acids (aa) sequence from TSV-VE05 and 36 isolates collected from 1993 to 2006 by the APL. The resulting tree showed TSV-

VE05 as a branch separate from the three major clusters suggesting that it is a new variant (Fig. 3.2). Samples analysed in 2006 suggest that TSV-VE05 is still circulating and might be spreading. The TSV-VE05 detected in Aruba (AW05 and AW06) was detected in a hatchery with operational connections to a Venezuelan farm.

According to the ProtParam software TSV-VE05 CP2 is 492 aa in length, with a total molecular weigh of 53 882.5 Da and a theoretical pI of 5.44. This protein was slightly heavier than the deduced molecular weight of CP2 for TSV-HI94 (53 862.5 Da).

Homogenous icosahedral particles were observed by EM. These were similar in size and

shape to the viral particles described by Hasson et al. (1995) (data not shown). In dot blot

immunoassay, purified native TSV-VE05 reacted strongly to MAb 1A1 and PAb.

Denatured TSV-VE05 reacted against PAb but not against MAb 1A1 (Fig. 3.3).

In challenge bioassays with Penaeus vannamei , shrimp fed the TSV-HI94 infected tissue had a 30% survival while shrimp fed the TSV-VE05 infected tissue had a 28% survival

(Table 3.3). All surviving shrimp challenged with either virus type examined had mild to severe lymphoid organ spheroids indicative of chronic TSV infection upon histopathological examination (Fig. 3.1a).

DISCUSSION

The TSV-VE05 reacted only faintly with MAb 1A1 by IHC. This excludes the isolate from the serotype A group which includes the Hawaiian TSV isolate (Erickson et al.

2002). According to Poulos et al. (1999) Mab 1A1 reacts with the 58 kDa TSV structural proteins and the 55 kDa CP2 which is an area of high genetic distance when compared amongst TSV isolates from Hawaii, Mexico, Taiwan, Thailand and Belize (Tang &

Lightner 2005).

MAbs are developed to recognize a specific epitope and they represent a pure antibody preparation of pre-defined specificity (Köhler & Milstein 1976). The affinities of MAbs are often lower than those observed for PAbs (Chappey et al. 1992). Antigen-antibody

(Ag-Ab) binding is the result of both the binding of the complementary side groups of the proteins associated with the conformational fitting of two very delimited areas: the epitope and the paratope (Montero 2003). The Davidson's fixative commonly used in shrimp pathology contains 33% of ethyl alcohol, 33.5% acetic acid and 22% formaldehyde. Formaldehyde is a cross-linking fixative which in solution is capable of binding the following amino acids: lysine, arginine, cysteine and glutamine. The final result of formaldehyde fixation is a profound and significant change in the tertiary structure of the protein, making recognition of Ag by Ab difficult (Ramos-Vara 2005).

Most samples of tissues infected with TSV-VE05 examined by IHC with MAb 1A1 showed no reactivity. A very faint positive cell-associated reaction was observed in the lymphoid organ of two chronically infected shrimp, suggesting a partial and limited recognition of the antigen by the antibody. This recognition appears to be too limited to be of diagnostic use.

The native form of TSV-VE05 virus presented a strong positive reaction with the MAb by dot blot immunoassay. MAb 1A1 was produced using a mixture of purified TSV isolated from three geographic regions, Ecuador, Hawaii and Texas, in their native state

(Poulos et al. 1999). It has been shown to react to denatured TSV-HI94 in IHC assay. It appears that while denatured TSV-VE05 does not react with MAb 1A1, purified TSV-

VE05 in the native state does react with the MAb. This is useful from a diagnostic standpoint since rapid immuno-assay tests using fresh shrimp tissue are more likely to be used as diagnostic samples (Takahashi et al. 2003; Sithigorngul et al. 2006).

The Venezuela isolate caused similar mortalities compared to TSV-HI94 as shown by the bioassay results. TSV-BZ02 is still the most virulent strain observed to date (Erickson et al. 2005).

The results from these experiments lead to the conclusion that the virus isolated from

Venezuela in 2005 was indeed a genetic variant of TSV. The isolate shows enough variation to designate it as a new strain of TSV.

Acknowledgement. The funding for this work was provided by the USDA, CSREES grant No. 2004-388808-02142 to the US Marine Shrimp Farming Consortium. We are grateful to the Venezuelan farmers who submitted the samples used in this study. B.

Poulos, P. Schofield and R. Redman provided much appreciated technical assistance.

Figure 3.1. H & E histopathology, ISH and IHC in TSV-VE05 infection in P. vannamei

a) LOS stained with hematoxylin and eosin in shrimp chronically infected with TSV-

VE05 (Mag 200X). b) In situ hybridization of the sample shown in 3.1a) with DIG- labeled TSV specific gene probe illustrating TSV lesions in animal chronically infected with TSV-VE05. TSV positive cells are marked by a blue precipitate indicating reaction of the probe to cell-associated TSV (Mag 200X). c) Immunohistochemistry of the sample shown in 3.1 a) using TSV PAbs. The TSV PAbs reacted strongly with several TSV-

VE05 infected cells (Mag 200X). d) Immunohistochemistry of the sample shown in 3.1a using MAb 1A1. The MAb 1A reacted faintly with a few TSV-VE05 infected cells

(arrows) (Mag 200X)

Figure 3.2. Phylogenetic relationships among 32 TSV isolates based on CP2 amino acid sequence. Tree constructed using the neighbor-joining with Clustal X program. Numbers indicate the percentages of bootstrap support from 1000 replicates, bootstrap values

(>50%) are indicated. The year and origin of isolation are reflected in the isolate's name

(see Table 3.1).

05VE/06AW 05AW 99 Venezuela 06-1 53 06-2 05MX3 04MX

88 05MX1 05MX2/06MX 50 93 04ER 89 98MX 96MX Americas 2KMX 99MX1 85 79 99MX2 94US-HI 80 93EC 94EC 99TW 68 53 05ID 06ID 04CH SE Asia 05CH 04TH2 04TH1 84 81 06TH 66 04BZ2 63 05BZ1 63 02BZ 01BZ Belize 05BZ2 99 04BZ1 04BZ3

Figure 3.3. Dot blot immunoassay. From left to right TSV-VE05 native and denatured reacted with PAbs, followed by native and denatured virus reacted with MAb 1A1.

Table 3.1. Year of collection and origin of the TSV isolates collected from Penaeus spp.

TSV isolate Year Origin TSV isolate Year Origin 93EC 1993 Ecuador 04ER b,9 2004 Eritrea 94US-HI 1 1994 US-Hawaii 04CH 2004 China 94EC 1994 Ecuador 05MX1 2005 Mexico 98MX 2 1998 Mexico 05MX2 2005 Mexico 99MX1 a,3 1999 Mexico 05MX3 2005 Mexico 99MX2 a,4 1999 Mexico 05CH 2005 China 99TW 5 1999 Taiwan 05VE 2005 Venezuela 2KMX a,6 2000 Mexico 05AW 2005 Aruba 01BZ 7 2001 Belize 05ID 2005 Indonesia 02BZ 2002 Belize 05BZ1 2005 Belize 04TH1 2004 Thailand 05BZ2 2005 Belize 04TH2 b,8 2004 Thailand 06ID 2006 Indonesia 04US-TX 2004 US Texas 06AW 2006 Aruba 04MX 2004 Mexico 06-1 2006 Not specified 04BZ1 2004 Belize 06-2 2006 Not specified 04BZ2 2004 Belize 06MX 2006 Mexico 04BZ3 2004 Belize 06TH 2006 Thailand a P. stylirostris . b P. monodon . Not labeled: P. vannamei . GenBank no. 1AF277675,

2AF510515, 3AF510516, 4AF277378, 5AF406789, 6AF510517, 7AY590471, 8DQ000306,

9DQ000302.

Table 3.2. CP2 nucleotide and amino acid percentage identity among Hawaii, Taiwan,

Belize and Venezuela TSV isolates.

TSV Isolates Nucleotide Amino acid sequence % sequence % identity identity TSV-VE05 vs. TSV-HI94 (AF277675) 93 96

TSV-VE05 vs. TSV-TW99 93 95 (AF406789) TSV-VE05 vs. TSV-BZ02 91 94 (AY590471)

Table 3.3. Survival of TSV-VE05 infected shrimp versus TSV-HI94 infected shrimp.

TSV Number Number of Percent Isolate Stocked Survivors Survival HI 20 6 30 HI 20 3 15 HI 20 6 30 HI 20 9 45 Average HI 30 VE 20 4 20 VE 20 2 10 VE 20 9 45 VE 20 3 15 VE 20 7 35 VE 20 9 45 Average VE 28

CHAPTER 4:

Hyperthermia protects Kona stock Penaeus vannamei against infection by TSV-HI but

not TSV-BZ.

Isabelle Côté, Donald V. Lightner

Aquaculture Pathology Laboratory, Department of Veterinary Science and Microbiology, University of

Arizona, 1117 East Lowell Street, Tucson, AZ 85721, USA

______

ABSTRACT: We compared the susceptibility of Kona stock Penaeus vannamei to infection by two variants of Taura syndrome virus (TSV) under hyperthermic conditions

(32 oC). Shrimp infected with the Hawaii variant of TSV (TSV-HI) were resistant to infection as demonstrated by the absence of mortality, viral load by Real-time PCR and histopathological lesions. However, shrimp were fully susceptible to the disease caused by the Belize variant of TSV (TSV-BZ) under the same conditions, with high mortality, high viral load and histopathological lesions. This susceptibility to TSV-BZ at the higher temperature was independent of the route of infection (injection vs per os ) and the salinity of the water (low 11 ppt vs high 27 ppt). This behavior of TSV-BZ seems unique amongst known shrimp viruses. We hypothesize that TSV-BZ might be a temperature permissible mutant of TSV.

INTRODUCTION

Taura Syndrome (TS) first appeared in Ecuador in 1992 (Jimenez 1992). Since, the disease has been reported from most shrimp farming regions of the Americas and South-

East Asia (Hasson et al. 1999, Yu and Song 2000, Nielsen et al. 2005). TS is a major problem in Penaeus vannamei culture and has been responsible for massive economic losses (Lightner 1999). The viral agent responsible for the disease is Taura syndrome virus (TSV), a member of the Discistroviridae family (Hasson et al. 1995, Mari et al.

2002, Mayo and Ball 2006).

TSV has a single stranded positive sense RNA genome, which has been fully sequenced

(Mari et al. 2002). The genome is 10,205 nucleotides in length and encodes 2 open reading frames (ORF). The first ORF encodes the housekeeping genes: a helicase, a protease and a RNA-dependent RNA polymerase. The capsid consists of three major polypeptides encoded in the second ORF: CP1 (40 kDa), CP2 (55 kDa) and CP3 (24 kDa), and a minor protein of 58 kDa.

There is no known treatment for TSV infections. Control of TS is done through husbandry and management practices. Species differ in their susceptibility and there is a variation in disease resistance within populations. Stocking of resistant animals has therefore been advocated as a means of control. Manipulation of the environment could

provide an interesting opportunity for disease management if environmental conditions

conductive to the expression of the disease were known (Guan et al. 2003).

Jimenez et al. (2000) evaluated the correlation between climatic variations and the

frequency of outbreaks in the gulf of Guayaquil, Ecuador. A significant negative

correlation was found between TS outbreaks and water temperature. Low water

temperatures were associated with increased frequency of TS outbreaks. Laboratory

studies have demonstrated that increased water temperature is associated with improved

survival of shrimp exposed to the Hawaiian reference strain for TSV (TSV-HI94) per os

(Montgomery-Brock and Shimojo 2003). These shrimp also appeared to be free of viral nucleic acid following the bioassay, as demonstrated by RT-PCR analysis (Dee

Montgomery-Brock personal communication).

In an unrelated study performed in our laboratory, it was unexpectedly found that Kona stock P. vannamei exposed to the Belize-02 variant of TSV (referred to as TSV-BZ in the remainder of this paper) (Erickson et al. 2005) presented high mortalities under hyperthermic conditions. We conducted assays under hyperthermic conditions (32 oC) to assess the following factors: route of exposure, salinity and variant of the virus.

MATERIALS AND METHODS

Shrimp stock. Specific pathogen free (SPF) Kona Stock Penaeus vannamei were obtained from the Oceanographic Institute in Hawaii and maintained in our laboratory.

These shrimp are known to be particularly susceptible to TSV infection and are the reference stock used for most TSV-related research performed in our laboratory (White et al. 2002). Juveniles weighing between 1-2 g were used for most bioassays. For the route of exposure assay with TSV-BZ the shrimp weighed approximately 3-4 g.

Bioassay. Variant of the virus.

TSV-HI. Shrimp were housed in 90 L aquaria at a density of 9-10 shrimp per aquarium.

Each aquarium was fitted with a biological filter. Salinity was adjusted to 28 ppt by the use of artificial crystal sea salt marine mix (Marine Enterprise International, Baltimore

MD). Water temperature in the heated tank was kept constant by the use of an electric heater and increased from 28 oC to 32 oC over the course of 4 days. Shrimp were

acclimated to their definitive assay conditions for 3 days prior to the start of the

experimental challenge. The temperature of the water was monitored twice daily with the

use of a hand-held thermometer. Shrimp were fed ad lib with pelleted Rangen feed (Buhl,

ID; 35% protein) for the duration of the experiment.

Shrimp held under hyperthermic conditions were injected with 50µl of TSV-HI infected tissue homogenate in the second abdominal segment of the tail muscle. The homogenate

was diluted 1:10 in 2% saline. This treatment was performed in duplicate. A positive

control group was kept at room temperature (27-28 oC) and injected with the same infected homogenate. Negative control shrimp were kept at 32 oC and injected with the same volume of 2% saline. On the 6 th day, two survivors per tank were fixed with

Davidson's AFA and processed for histopathological analysis according to the procedure described by Bell and Lightner (1988). ISH was also performed on these samples according to the procedure described by Mari et al. (1998).

A second assay was performed to evaluate the viral load of the shrimp infected with

TSV-HI at different time-points. The bioassay was set up as described previously with a density of 25 shrimp per tank. There were duplicate tanks for both the high and low temperature treatments. Two negative control tanks containing five shrimp injected with

2% saline were kept at low and high temperature. Each tank, except the negative control, was sampled every 12 h (12, 24, 36 and 48 h post-infection). At the selected time-points, shrimp were randomly selected and their pleopods clipped and preserved in 100% ethanol at 4 oC for 1 week. They were then dried and frozen at -80 oC until used for RNA extraction. Extractions were performed using the High Pure RNA Tissue kit (Roche) according to the manufacturer recommendation for standard reaction. Real-time PCR was performed using a Taq Man assay as described by Tang et al. (2004). Histopathology was also performed on 5 animals per infected tank collected at the 48 h time point and fixed with Davidson's AFA.

TSV-BZ. Two bioassays were run separately to evaluate the role of the route of exposure and salinity on TSV-BZ infection under hyperthermic (32 oC) conditions.

Route of exposure . The bioassay was run as described previously for TSV-HI, using 10 to 11 shrimp in each tank. The injected shrimp were treated as previously described except that the negative control received a dilution of SPF tissue homogenate diluted 1:10 in 2% sterile saline. The animals infected by the oral route, received 10% of their body weight (BW) of TSV-BZ infected minced tissue on day 1, followed by a second 10% feeding on day 2. Each treatment was performed in duplicate. Moribund animals throughout the assay were fixed for examination by histopathology. The pleopods were fixed in 95% ethanol for examination by real-time PCR (negative control animals were not tested by real-time PCR).

Salinity . The bioassay was performed as described previously. For the shrimp held at the lower salinity, salinity was adjusted to 11 ppt (from 28 ppt) over the course of 4 days by the addition of aerated fresh water. The salinity of the water was measured twice daily by the use of a hand-held refractometer. The low salinity treatment was done in duplicate alongside one, untreated, low salinity control and one positive control at high salinity. All tanks were maintained at 32 oC by the use of submersible heater. Animals were fed 10%

BW of TSV-BZ infected minced tissue on days 1 and 2. Tanks were monitored several times daily and moribund animals were fixed for examination by histopathology.

RESULTS

Variant of the virus.

TSV-HI.

Shrimp infected with TSV-HI under hyperthermic conditions presented higher survival compared with the positive control kept at room temperature (Fig. 4.1). Two mortalities at the beginning of the TSV-HI challenge bioassay were attributed to stress. Shrimp are known to be cannibalistic (Wu et al. 2001) and the dead animals were probably quickly cannibalized by the remaining healthy animals in the tank. Hence, they were not available for further analysis.

Upon histological examination, lesions typical of acute TSV infection were found in one shrimp from the positive control group fixed 3 days after the start of the challenge. The other moribund animals from the positive control tank were found dead and could not be processed for histopathological analysis. Lymphoid organ spheroids (LOS), which are diagnostic of chronic phase TSV infections, were observed in the 2 survivors from the positive control and at a low to moderate grade in 2 out of 4 examined animals from the hyperthermic tanks (see Hasson et al. 1995 for detail on the grading system). The LOS from infected animals kept at high temperature were poorly organized (Fig. 4.2a) and negative for TSV by ISH (Fig. 4.2b). They did not have the typical circumscribed organization normally found in chronic TSV infection (Fig. 4.2c).

There was a large variation in viral copy number/ng RNA amongst animals within each

treatment (Table 4.1). Viral titers in the low temperature treatments were consistently

higher compared to the high temperature treatments. Shrimp under hyperthermic

conditions presented low titers that decreased following the challenge. Histopathology

results showed low levels of early TSV lesions in 6/10 animals under low temperature

conditions and 0/10 animals under hyperthermic conditions. Atypical LOS were not

observed in those animals.

TSV-BZ

Route of exposure. Shrimp presented high mortality when challenged with TSV-BZ at

high temperature (32 oC) independently of the route of exposure, whether per os (Fig.

4.3a) or by injection (Fig. 4.3b). Negative control animals kept at the higher temperature

did not present any mortality.

All moribund animals collected throughout the study regardless of treatment presented

lesions characteristic of the acute or transitional phase of TSV with "buckshot" lesions in

the cuticular epithelium of the gills, cuticle and stomach of various degree of severity G1-

G4 and/or mild to moderate LOS. They all also had significant viral titers as determined

by real-time PCR, varying from 1.87E10 6 to 2.78E10 8. Negative control animals had no lesions indicative of TSV upon histological examination.

Salinity. All moribund animals sampled throughout the bioassay from the low salinity tanks (9/9) had lesions suggestive of acute or transitional TS. All survivors (3 out of 3) sampled at the end of the bioassay presented severe LOS indicative of a chronic infection. Negative control animals (2 of 2 examined) were normal upon histological examination and had a 100% survival (Fig. 4.4).

DISCUSSION

Shrimp infected with TSV-HI under hyperthermic conditions (32 o) presented lower mortalities compared to the shrimp kept ambient temperature (28 oC). The only mortalities that occurred in the hyperthermic tank could be associated with stress following the manipulation and injection of the infectious material. The remainder of the hyperthermic animals remained healthy throughout the challenge. These shrimp were also free of typical TSV lesions by histopathology and presented very low viral titers. Atypical LOS were found at low level in 2 of 4 shrimp infected with TSV-HI at high temperature after 6 days. The significance of these LOS is unclear. They could be associated to the clearing of residual viral particles. The low level of TSV lesions in animals kept at low temperature in the second bioassay could be explained by their low viral titers (Table

4.1). For example, Tang et al. (2004) reported average levels of 5.88 E10 7 TSV copies/µg of RNA in the pleopods of acutely infected shrimp which is much higher than the maximum value of 1.06E10 6 in this bioassay.

Shrimp infected with TSV-BZ presented high mortality. This was independent of the

route of infection ( per os vs injection) and the salinity of the water (11 vs 28 ppt).

Moribund animals were found to have severe typical TSV lesions associated with high

viral titers. This indicates the replication of the virus at the higher temperature.

This data confirms that shrimp are protected against TSV-HI infection under

hyperthermic conditions, but not against TSV-BZ. For TSV-HI, this is consistent, with

the behavior of other shrimp viruses. Hyperthermia has been shown to reduce shrimp

susceptibility to viral infections such as White spot syndrome virus (WSSV) (Vidal et al.

2001; Granja et al. 2003; Guan et al. 2003) and Infectious hypodermal and hematopoietic

virus (IHHNV) (Dee Montgomery-Brock personal communication).

Two hypotheses could account for the unresponsiveness of shrimp to TSV-HI infection

and their susceptibility to TSV-BZ under hyperthermic conditions. The first relates to the

immune response of the shrimp and the second to the temperature permissibility of the

viruses.

Du et al. (2006) demonstrated that hyperthermia reduced viral replication in

Procambarus clarkii infected with WSSV associated with a sharp reduction in mortality.

Healthy crayfish at high temperature (32 oC) suffered from severe white spot syndrome if transferred from high to low temperature (24 oC) suggesting that viral inactivation was not

responsible for the reduction in mortality. Using subtractive suppressive hybridisation

Reyes et al. (2007) showed that the principal effect of hyperthermia on subticular

epithelial cells in WSSC infected shrimp was a reduced expression of WSSV. Their study

did not demonstrate induction of specific host genes that could contribute to the non-

specific immune response of the shrimp and the control of infection.

De la Vega et al. (2006) proposed that short-term hyperthermic treatment in Penaeus

monodon increased the expression of heat shock protein 70 that could be linked to a

lower level of gill-associated virus in chronically infected shrimp. Heat shock has also

been associated with increased survival of Artemia franciscana larvae against challenge with virulent vibrios (Yik Sung et al. 2007).

It unknown at this point why TSV-BZ, but not TSV-HI, causes mortality in shrimp held under hyperthermic conditions. TSV-BZ causes higher mortalities than TSV-HI in laboratory challenges and in the field (Tang and Lightner 2005; Erickson et al. 2005).

The TSV-BZ variant belongs to its own genetic clade and has an overall difference of

2.4% in its genomic sequence with the TSV-HI reference strain (Tang and Lightner

2005). This difference is principally located within the capsid protein 2 (CP2) where regions are sequenced.

Temperature sensitive mutants, sometime varying in as little as one nucleotide, are known to have different permissible temperatures. A single nucleotide change in a capsid protein of poliovirus was sufficient to prevent the maturation of the virus at the non-

91 permissive higher temperature (Compton et al. 1990). Temperature sensitive RNA polymerase mutants of Foot and mouth disease virus (FMDV, a picornaviridae) have been described (Lowe et al. 1981). The mutants were the result of a single base pair change in the gene encoding the RNA polymerase. The partial blockage of the FMDV at higher temperature as long been recognized (Sharpe 1958). It is hypothesized that TSV-

BZ is a temperature permissible mutant of TSV. However, further studies will be needed to demonstrate this possibility

In view of these results, we propose that infection with TSV-BZ causes mortality under hyperthermic (32 oC) conditions in Kona stock P. vannamei , independently of the route of exposure and the salinity (in a range of 11 ppt-28 ppt). According to the currently available data, this is unique amongst shrimp viruses and does not apply to another strain of TSV, Hawaii-94 (TSV-HI).

Acknowledgements . Grant support for this study was from the United States Marine

Shrimp Farming Consortium under Grant No. 2004-38808-02142 from the Cooperative

State Research, Education and Extension Service, U.S. Department of Agriculture. The senior author is thankful to Brenda White and Rita Redman for technical assistance and to Bonnie T. Poulos who reviewed this manuscript.

Figure 4.1. Survival of shrimp injected with TSV-HI under hyperthermic conditions (32 oC).

100

80 neg. control 60 pos. control hyperthermia 1

% survival % 40 hyperthermia 2

0 1 2 3 4 5 6 days

Figure 4.2. Microscopic examination of tissues from shrimp infected with TSV-HI and held at 32 oC. Poorly organized LOS noted by histopathology (a) were negative for TSV when tested by ISH (b). This contrast with well defined LOS seen in TSV-HI infection at ambient temperature (28 oC) (c). Mag 200X

Figure 4.3a. Survival of P. vannamei fed TSV-BZ infected tissues under conditions of high salinity (25 ppt) and high temperature (32 oC).

100

80 neg. control 60 pos. control hyperthermia fed 1

% survival % 40 hyperthermia fed 2 20

0 1 2 3 4 5 6 7 8 910 Days

Figure 4.3b. Survival of P. vannamei injected TSV-BZ infected tissues under conditions of high salinity (25 ppt) and high temperature (32 oC).

100

80 neg. control 60 pos.control hyperthermia inj. 1

% % survival 40 hyperthermia inj. 2

0 1 2 3 4 5 6 7 8 910 Days

Figure 4.4. Survival of P. vannamei fed TSV-BZ infected tissues under conditions of low salinity (11 ppt) and high temperature (32 oC).

100

80 neg. control 60 pos. control low salinity 1

% survival % 40 low salinity 2

0 1 2 3 4 5 6 7 8 910 Days

Table 4.1 Viral copies number/ng of RNA in shrimp injected with TSV-HI

RT (28 oC) 32 oC hours p.i. Min. Median Max. Min. Median Max. 12 13.23 577.45 69,180.05 0.43 5.46 140.45 24 1383.73 6861.00 335,028.03 0.97 7.24 272.65 36 528.53 29,098.56 286,733.47 0 0.23 2.00 48 1011.77 34,101.89 1,060,984.67 0 0.44 8.06

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