Nucelotide Substitution Rates in Hantavirus CD8 + T-Cell Epitopes

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12-2016 Nucelotide Substitution Rates in Hantavirus CD8+ T-Cell Epitopes Brian Gerald Sikes Winthrop University

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NUCLEOTIDE SUBSTITUTION RATES IN HANTAVIRUS CD8+ T-CELL EPITOPES

A Thesis Presented to the Faculty Of the College of Arts and Sciences In Partial Fulfillment Of the Requirements for the Degree Of Master of Science In the Department of Biology Winthrop University

December, 2016 By Brian Gerald Sikes

Abstract

Hantaviruses pathogenic to humans are exclusive to rodents. Typically each hantavirus is associated with only one host species. Due to their close association, the geographical distributions are the same. Old World species, such as Hantaan hantavirus and Puumala hantavirus, induce symptoms described as the Hemorrhagic Fever with

Renal Syndrome (HFRS). The New World hantaviruses, such as Andes hantavirus and

Sin Nombre hantavirus, share a suite of symptoms that have been described as Hantavirus

Pulmonary Syndrome (HPS). In this study, 16 well-characterized cytotoxic T lymphocytes (CTL) epitopes of the nucleocapsid (N) protein were identified from the literature. These 16 antigenic regions are reportedly CD8+ epitopes showing reactivity to one or more hantaviruses. A phylogenetic analysis was used to identify closely related sequences called sister pairs. The number of nonsynonymous differences per nonsynonymous site (pN) and the number of synonymous differences per synonymous site

(pS) were computed. Comparisons were then made between New World and Old World groups, as well as between pathogenic and non-pathogenic groups. It was expected that there would be evidence of viral escape, measured as elevated pN to pS means in the well characterized CTL epitopes of the N protein. However, evidence of CTL escape was not observed in this study. None of the epitopes exhibited positive selection. The mean pS value was greater than the mean pN value in all cases. The N protein appears to be have been highly conserved throughout most hantaviruses. Numerous epitopes did show evidence of possible negative, or purifying, selection. Because of genus specificity of the various epitopes, future studies comparing substitution rates within genera could be insightful.

Acknowledgements

I would like to thank my thesis committee members for their constructive and thoughtful guidance:

Dr. Kristi Westover, Thesis Advisor and Council Chair Dr. Matthew Stern Dr. Matthew Heard

I would like to express gratitude to my wife, Robin, and my daughters, Madeleine and Olivia, for their undaunted support and encouragement.

iii

TABLE OF CONTENTS

Abstract ...... ii Acknowledgements ...... iii List of Tables ...... v List of Figures ...... vi Introduction ...... 1 Human Pathogenic Hantaviruses ...... 1 Hantavirus Genome and Pathogenesis ...... 3 Host Immune Response ...... 4 Proposed Research ...... 7 Methods ...... 8 Data Collection ...... 8 Phylogenetic Analysis ...... 9 Substitution Rate Analysis ...... 9 Results and Discussion ...... 11 Phylogenetic Analysis ...... 11 Substitution Rate Analysis ...... 11 Conclusions ...... 16 References ...... 19

LIST OF TABLES

Table 1: The 520 complete nucleocapsid protein sequences used for phylogenetic analysis...... 32

Table 2: New World sister pair groupings of pathogenic and non-pathogenic hantaviruses...... 43

Table 3: Old World sister pair grouping of pathogenic and non-pathogenic hantaviruses...... 44

Table 4: Nucleocapsid amino acids of CD8+ epitopes found in Hantaan hantavirus (HTNV), Sin Nombre hantavirus (SNV), and Puumala hantavirus (PUUV) ...... 45

Table 5: Mean numbers of synonymous substitutions per synonymous site (pS) and mean numbers of nonsynonymous substitutions per nonsynonymous sites (pN) and standard errors are shown for each epitope for the New World group...... 46

Table 6: Mean numbers of synonymous substitutions per synonymous site (pS) and mean numbers of nonsynonymous substitutions per nonsynonymous sites (pN) and standard errors are shown for each epitope for the Old World group...... 47

LIST OF FIGURES

Figure 1: Phylogenetic tree of sister pairs of complete nucleocapsid protein sequences. Boot strap values shown were taken from the NJ Poisson phylogeny...... 48

Figure 2: Results from the Chi Square test of independence designed to test whether the distribution of the number of incidences where pS was greater than pN among each of the groups (ie, New World pathogenic, New World non-pathogenic, Old World pathogenic, and Old World non-pathogenic) differed...... 49

INTRODUCTION

Hantaviruses are enveloped, negative-sense, single-stranded RNA viruses belonging to the family Bunyaviridae. According to the International Committee on

Taxonomy of Viruses (ICTV 2015), the Hantavirus genus contains 24 species, though recent phylogenetic studies have reported numerous genetically-distinct genotypes as well (Bennett et al. 2014, Souza et al. 2014, Zhang 2014). The taxonomic status of many of these hantaviruses is unclear. For example, the Andes hantavirus was reported to be comprised of 12 genotypes in a study of the hantaviruses found in South America

(Figueiredo et al. 2014), calling into question whether this species should be sub-divided.

All hantaviruses are harbored by small mammal hosts and are the only viruses of the

Bunyaviridae that do not utilize an arthropod vector. In addition to its long recognized rodent hosts, hantaviruses have now been documented in bats and insectivores (Zhang

2014). However, hantaviruses that are pathogenic to humans are exclusive to rodents.

Human Pathogenic Hantaviruses

Currently there are 22 hantavirus genotypes pathogenic to humans described in the literature (Kruger et al. 2015 & Bi et al. 2008). Pathogenic hantaviruses can be divided into three groups according to the taxonomy of their rodent hosts: Cricertidae and

Murinae (rats and mice of Asia and Europe), Arvicolinae (voles and lemmings of North and South America, and Sigmodontinae (rats and mice of North America and South

America). Hantaviruses show a high degree of host specificity. The asymptomatic host is usually infected by a single hantavirus species, and each hantavirus in turn is associated usually with only one host species (or infrequently closely related species) (Plyusnin &

Sironen 2014). Therefore due to their close association, the geographical distributions are the same. The Murinae-associated hantaviruses, which are distributed in Europe and

Asia, are called Old World hantaviruses. Hantaviruses associated with the Arvicolinae and Sigmodontinae rodents are distributed in North and South American countries and are known as New World hantaviruses.

Old World species, such as Hantaan hantavirus and Puumala hantavirus, induce symptoms described as the Hemorrhagic Fever with Renal Syndrome (HFRS). According to the Ministry of Health of China, China reports 90% of the HFRS cases annually worldwide and has an annual mortality rate of approximately 15% (Fang et al. 2015). The earliest descriptions of an HFRS-like hemorrhagic fever was in China around 960 AD

(Lee et al. 2014). The disease was known as the Korean hemorrhagic fever when thousands of soldiers became infected with HFRS during the Korean War (Lee et al.

2014). More recent research supported the renaming of the Korean virus to Hantaan hantavirus when the antigen was discovered in the striped field mouse (Apodemus agrarius) (Lee et al. 1978). Soon after the description of Hantaan hantavirus, many pathogenic species of hantavirus were discovered in the Old World.

The New World hantaviruses share a suite of symptoms that have been described as Hantavirus Pulmonary Syndrome (HPS). Only several thousand cases have been reported in the twenty years since HPS was discovered (Lee et al. 2014). In 1993, the first New World species, Sin Nombre hantavirus, was described after an outbreak near the Four Corners region in southwestern US and was found to be associated with the deer mouse, Peromyscus maniculatus (Nichol et al. 1993). As of 2014, Sin Nombre hantavirus had the highest mortality rate of the hantaviruses at 40% (Lee et al. 2014). Numerous

2 non-pathogenic species have been discovered since 1993 in North America and South

America. The other significant New World pathogenic hantavirus species is the Andes hantavirus. In 1996, the Andes hantavirus was first recognized in the reservoir host

Oligoryzomys longicaudatus, the long-tailed pygmy rat (Lee et al. 2014). The Andes hantavirus is the first hantavirus to reportedly transmit from one human to another

(Padula et al. 1998).

Hantavirus Genome and Pathogenesis

The hantavirus genome consists of three segments designated as Small (S),

Medium (M), and Large (L). Each of the three segments contain an open reading frame

(ORF) flanked by non-coding regions at the 3′ and 5′ ends. The RNA’s ORF encodes for a spherical nucleocapsid protein (1,290 base pairs), a glycoprotein precursor (3,440 base pairs), and a RNA-dependent polymerase (6,470 base pairs) (Hepojoki et al. 2012). In addition to encoding the nucleocapsid protein, the S segment of some hantaviruses contains an overlapping reading frame that encodes the putative non-structural protein

(Jonsson et al. 2010). Each segment forms a circle enclosed in a nucleocapsid protein within a virion comprised of lipids (Hepojoki et al. 2012). The lipid envelope aids in transmission of the hantavirus by allowing it to remain infectious at room temperature for

15 days (Kallio et al. 2006).

Humans become infected with hantaviruses by inhaling excreta (e.g., urine, feces) from the asymptomatic hosts (Vaheri et al. 2013). The glycoprotein spikes of the virion bind to integrin receptors of the host cell which triggers the endocytosis of the virus via clethrin-coated pits (Jin et al. 2002). Once the virion has entered the cell, uncoating of the

3 vesicle occurs and the three viral genomes are released. Complimentary RNA is transcribed using primers from the host, followed by translation of viral proteins using host machinery. The viral RNA is replicated and assembled at the Golgi apparatus before finally being released through the plasma membrane.

Both hantavirus diseases are characterized by increased thrombocytopenia and capillary permeability causing vascular leakage (Hepojoki et al. 2014). At the time of patient death, viral antigens are detected in the endothelial cells of the lungs in the case of

HPS or the kidney in the case of HFRS. The virus also infects dendritic cells, macrophages and lymphocytes which spread the virus throughout the body to the capillaries of several organs, however mainly the lungs and kidneys (Jonsson et al. 2008).

Based on in vitro studies, hantavirus infection of the endothelial cells does not induce direct cytopathic effects (Mackow & Gavrilovskaya 2009). A number of studies have reported immune-response pathology rather than virus-induced cell death. It has been suggested, for example, that cytotoxic CD8+ T lymphocyte cells trigger capillary leakage and that cytokines further contribute to the increased capillary permeability (Terajima &

Ennis 2011).

Host Immune Response

Recognition of hantavirus epitopes by host cell receptors signals the activation of adaptive immune cells. In both HPS and HFRS, cytotoxic T lymphocytes (CTL) responses are essential for elimination of virus-infected cells and viral clearance

(Terajiima & Ennis 2011). At the onset of HFRS and HPS, increased amounts of circulating CTLs are observed. CTLs are the predominant cell type in the infiltrate of the

4 kidneys during the acute phase of HFRS, as well as in the lungs in lethal HPS cases

(Vaheri et al. 2013). Lung biopsies from patients with HFRS have revealed increased numbers of CD8+ T cells (Rasmuson et al. 2011). Higher frequencies of CD8+ T cells were detected in patients with severe HPS than in patients with less severe symptoms

(Kilpatrick et al. 2004).

The adaptive immune response by the host can also facilitate rapid viral evolution thus causing the pathogen to evade recognition (Capron & Dessaint 1989). For example, cell mediated clearance of the virus by host CD8+ T cells is a particular source of selective pressure. CD8+ T cells detect viral particles presented by Class I MHC proteins.

Because they lack proofreading polymerases, RNA viruses are prone to higher mutation rates. As a result, when there are changes in the viral epitopes presented by the MHC I complex, the viral pathogens may evade host immune recognition. This particular example of response to host natural selection pressure is known as viral escape.

Numerous studies have reported the positive selection of viral escape mutants against CTLs in several systems. Allen et al. (2000) and O’Connor et al. (2004) demonstrated that the response of CD8+ T cells in macaques selected for mutants of the

Simian Immunodeficiency Virus. They demonstrated that mutations within the viral CTL epitope were favored by positive Darwinian selection. CTL-driven selection in Human

Immunodeficiency Virus is well supported as well (Piontkivska & Hughes, 2004;

Piontkivska & Hughes, 2006). Data also indicate that Hepatitis C Virus undergoes positive selection from CD8+ T cell responses (Von Hahn et al. 2007). Westover &

Hughes (2007) provided support that CTL epitopes undergo CTL-driven selection in

Hepatitis B Virus.

Relatively little is known about escape mechanisms in hantaviruses. Levine et al.

(2010) report evidence that several hantaviruses develop species-specific mechanisms to escape innate immunity by evading type I IFN response. Only one analysis of CTL- driven selection in hantaviruses was found in the literature. Using two CTL epitopes,

Hughes & Friedman (2000) surveyed numerous Old World and New World hantaviruses for evidence of natural selection. Positive selection was not detected in the CTL epitopes of the nucleocapsid (N) protein, nor in antibody epitope regions of the glycoprotein.

However, they report a high level of amino acid residue charge changes in the N protein.

Hughes & Friedman (2000) conclude that these changes may indicate positive Darwinian selection from immune responses that occurred during the divergence of the three host subfamilies (i.e., Murinae, Arvicolinae, and Sigmodontinae). Though the data was non- significant, Lin et al. (2012) recently detected positive selection in the clade-defining sites of hantaviruses found in the S segment of Murinae subfamily.

The nucleocapsid protein is a major antigenic region in hantaviruses and frequently is used in serological diagnosis. A review by Yoshimatsu & Arikawa (2014) reports genus-common, group-common, and species-specific epitopes on the N gene.

CTLs are mostly directed against immunodominant epitopes of the N protein (Wang et al.

2015). This may be due to the fact that the N protein represents the most conserved and abundant hantavirus protein produced during infection (Kaukinen et al. 2005). These characteristics are specifically desirable for research on vaccines that induce cross- reactive immunity.

CTL escape is an important mechanism of evolution and is well documented in many of the major RNA viral systems. Review of the primary literature clearly showed

6 very little research on cell-mediated selection in hantaviruses. The N gene of hantaviruses is a good candidate for such an analysis because (1) Numerous reports indicate CTL- mediated clearance of the virus by the host immune system, (2) Many well-characterized

CTL epitopes of the N gene for several pathogenic hantaviruses are now available, and

(3) evidence suggests positive selection as a result of viral evasion. There are also complete N genes available for most pathogenic hantaviruses from a variety of hosts. A phylogenetic analysis based on the complete N gene would be a beneficial tool for a novel statistical analysis that uses a powerful pairwise sister-comparison method.

According to Hughes & Friedman (2000), this method is useful for detecting small evolutionary changes specific to host-pathogen interactions. Though epitopes are only described for a few hantaviruses and may not be antigenic in all available species, identifying cell-mediated host selection on the N gene by using all available sequences can be beneficial for vaccine research and understanding hantavirus evolution.

Proposed Research

In this study, well-characterized CTL epitopes of the nucleocapsid protein were identified from the literature. These antigenic regions are reportedly CD8+ epitopes showing reactivity to one or more hantaviruses (Table 4). A phylogenetic analysis was used to identify closely related sequences called sister pairs. The number of nonsynonymous differences per nonsynonymous site (pN) and the number of synonymous differences per synonymous site (pS) were computed using Nei & Gojobori’s (1986) method. Comparisons were then made between New World and Old World groups, as well as between pathogenic and non-pathogenic groups:

 It was expected that the nonsynonymous rates of nucleotide substitution in

antigenic regions of the N gene would be higher in the Old World (HFRS-causing

viruses) group because of the longer presence in Europe and Asia than the New

World (HPS-causing viruses) group has been in the Americas.

 It was expected that the nonsynonymous rates of nucleotide substitution in

antigenic regions of the N gene would be higher in pathogenic species than non-

pathogenic species because of CTL escape.

METHODS

Data Collection

All available hantavirus S segment nucleotide sequences were downloaded from the Virus Pathogen Resource (ViPR) website (https://www.viprbrc.org/). According to the website, the data were released on July 21, 2016. The dataset contained 952 complete

S segments comprised of all 24 species recognized by the International Committee on

Taxonomy of Viruses (2015) as well as numerous unclassified strains. The complete nucleocapsid protein sequences were extracted and genetically identical sequences were removed. The remaining 520 nucleotide sequences were translated to amino acid sequences and checked for errors (e.g., misplaced stop codons, gaps). Accession numbers and references for the sequences are provided in Table 1. Through an extensive literature review, source origination was confirmed and all clone and vaccine stock were removed.

The final dataset for this study contained 506 complete nucleocapsid protein sequences.

Amino acid sequence alignments were generated using ClustalW implemented in MEGA v6.0 (Tamura et al. 2013). Gap adjustments were performed as recommended by Hall

(2013). Nucleotide p-distances were calculated and thus confirmed no genetically identical sequences were present.

Phylogenetic Analysis

Phylogenetic trees of the 506 complete nucleocapsid protein sequences were constructed using MEGA v6.0 (Tamura et al. 2013). The neighbor-joining (NJ) method

(Saitou & Nei 1987) was utilized to construct two trees using the uncorrected proportion of amino acid differences (p-distance) and the Poisson-corrected amino acid distances.

The NJ method is a distance-based method that is based on the number of changes between the sequences to infer relatedness. Distance-based methods are computationally fast and are ideal for large datasets. Bootstrap analysis was performed using 1000 replications to determine reliability. Bootstrapping measures the robustness of a tree’s topology. Each clade receives a percentage which represents the percent of instances the bootstrap replicate supports the original tree. The phylogenetic analysis was used to identify closely related sequences called sister pairs. Each sister pair is phylogenetically and statistically independent (Felsenstein 1985). Only the pairs found in both NJ trees were used for comparison (Tables 2 & 3).

Substitution Rate Analysis

Natural selection by the host immune system was assessed by comparing nucleotide substitution rates within antigenic regions of the nucleocapsid protein sequence. Sixteen well-characterized CTL epitopes of the nucleocapsid protein were identified from the literature. These 16 antigenic regions are reportedly CD8+ epitopes showing reactivity to one or more hantaviruses (Table 4). For this study, the patterns of

9 synonymous and non-synonymous substitutions in the CTL epitopes were compared to demonstrate positive selection or negative selection. A synonymous substitution of a nucleotide occurs when one base is changed to another but the coded protein does not change. For example some protein-coding nucleotides can be substituted with any of the three other bases and the same amino acid is still made. A nonsynonymous substitution is a change of a nucleotide within a codon that does result in an amino acid change. The number of nonsynonymous differences per nonsynonymous site (pN) and the number of synonymous differences per synonymous site (pS) were computed using Nei &

Gojobori’s (1986) method implemented in Mega v6.0. When pN > pS, positive selection is occurring. This means, for example, a given gene is under selective pressures as a result of natural selection. When pN < pS, natural selection suppresses protein changes, therefore the sequence is under negative selection. Negative selection commonly occurs when a given protein has important properties that cannot tolerate any changes. Nonsynonymous changes can result in rapid evolution, whereas purifying selection from synonymous substitutions amounts to small changes over a long period of time (Hughes 1999). Mean substitution rates were calculated for New World hantaviruses (pathogenic versus non- pathogenic) and Old World hantaviruses (pathogenic versus non-pathogenic). To determine differences in New World and Old World hantaviruses, two-sample F-Tests were used to determine if the variances were equal or unequal and paired sample T-tests were used to test the hypothesis that pN = pS for each epitope. To determine whether the means were significantly different, two-sample F-Tests and Chi-Square tests of independence were used to test the hypothesis that there were differences in CTL mutation rates between the aforementioned groups.

RESULTS AND DISCUSSION

Phylogenetic Analysis

The phylogenetic analysis of 506 complete nucleocapsid protein sequences was used to identify 60 sister pairs (not shown). The pairs were found on both p-distance and

Poisson NJ trees with branch supporting clustering receiving at least 70% bootstrap support in each tree. The typology of both NJ consensus trees closely resembled each other. In each tree, there were two clusters containing sequences whose rodent hosts can be broadly categorized as New World and Old World. Pathogenic hantaviruses are exclusively found in rodent hosts in the wild; therefore, 11 pairs of the 60 pairs found in bats and insectivores were excluded. The remaining 49 pairs chosen for this study fell into the two aforementioned categories (Tables 2 &3). The phylogeny of the 49 pairs in

Figure 1 is based on the aforementioned Poisson NJ tree.

Substitution Rate Analysis

Initially to test whether there were differences in mutation rates between the Old

World and New World hantavirus groups, mean pN and pS values for each epitope were calculated for both groups (not shown). Because very few of the mean comparisons of pN and pS values between Old and New World viruses were significant, two additional subgroups of hantaviruses were created for each: pathogenic and non-pathogenic.

Pathogenicity was determined according to Kruger et al. (2015) and Bi et al. (2008).

Overall mean pN and pS values were computed for each of the 16 epitope regions in each subgroup (Tables 5 & 6; New World and Old World, respectively). The mean pS value was greater than the mean pN value in all cases. In summary, the New World pathogenic

11 group had 11 out of the 16 epitopes with significantly different mean pN and pS values

(Table 5). The New World non-pathogenic group had only one epitope with a significant difference in mean pN and pS values (Table 5). One epitope, Epitope 11, had a significant difference in mean values in both New World subgroups. The Old World pathogenic and non-pathogenic groups had three and five epitopes with significant differences in mean pN and pS values, respectively (Table 6). Both Old World groups had significant pN and pS values for Epitope 6 and Epitope 14. Significant results are discussed below. To test whether the distribution of the number of incidences where pS was greater than pN among each of the groups (ie, New World pathogenic, New World non-pathogenic, Old

World pathogenic, and Old World non-pathogenic) was significantly different, a Chi-

Square test of independence was performed. The New World group had 11 pathogenic and one non-pathogenic comparisons where pS was significantly greater than pN ; and, the

Old World group had three and five, respectively (Figure 2). In the New World, there were more comparisons where pS was significantly greater than pN in the pathogenic hantaviruses and in the Old World, the opposite occurred (Figure 2). There were more comparisons where pS was significantly greater than pN in the non-pathogenic group

(Figure 2). Because the distributions were not equal, the null hypothesis was rejected (x2

= 6.715; df = 1; p<0.01). While it was originally expected that the nonsynonymous rates of nucleotide substitution in antigenic regions of the N gene would be higher in pathogenic species than non-pathogenic species because of CTL escape and there was no indication of that in any group, it is interesting there was evidence of purifying selection in Old versus New World group. In the New World viruses, perhaps the elevated number

12 of comparisons where pS was greater than pN in the pathogenic group is an early indication of potential for future nonsynonymous mutations.

The N protein consists of approximately 430 amino acids with numerous epitopes and can be divided into three antigenic domains (I, II, and III). Approximately 25%

(amino acids 1 to 100) of the protein consists of an N-terminal region comprised of two helices within Domain I that turn at the 36th position. Epitope 1 is restricted by HLA B51; it is also Hantaan hantavirus specific (Van Epps et al. 1999). Epitope 2 is H-2Kb- restricted and shows reactivity in Sin Nombre hantavirus and Puumala hantavirus

(Maeda et al 2004). This antigenic region of the N protein shows considerable variability among hantaviruses, reportedly due to differences in residues at the HLA binding site

(Van Epps et al. 1999). The HLA B51 is present in 8 to 12% of Asia populations where

Hantaan hantavirus is endemic (Yoshimatsu et al. 2014). It is possible that significant differences between distinct genotypes in Epitopes 1 and 2 were obscured by combining multiple hantaviruses into subgroups.

The central part of the N protein (Domain II) has three distinct regions.

Yoshimatsu et al. (2014) reported the presence of a highly conserved Region I (amino acids 100-125), an RNA-binding region located in the central locale (amino acids 175-

218), and a highly variable region located from amino acid 230 to 302. Eight epitopes in this study were located in Domain II of the N protein and resulted in significant differences in substitution rates (Table 5 & 6). Two of the eight epitopes (i.e., Epitopes 4 and 5) were located between Region I and the RNA-binding region. Epitope 4

(131LPIILKALY139) fits the HLA-B 35 binding motif. Epitope 5 (167DVNGIRKPK175) is

HLA-A33 restricted. According to Ma et al. (2015), Epitopes 4 and 5 are well conserved

13 with 67%–100% agreement in all hantaviruses and are especially conserved among the

Old World pathogenic group. Epitope 4 had a low mean value for the Old World non- pathogenic group (pS=0.081 ±0.037). Epitope 5 had moderately high mean values for Old

World non-pathogenic and New World pathogenic groups at pS=0.210 ±0.090 and pS=0.306 ±0.077 respectively. These epitopes are probably conserved throughout the multiple genotypes; therefore, the subgroups could be appropriate for Epitopes 4 and 5.

Two epitopes that exhibited significant mean differences were located within the

RNA-binding region of Domain II. The major human CTL epitopes of Sin Nombre hantavirus and Puumala hantavirus can be found primarily in this region and are thought to play a key role in spurring CTL activity (Maeda et al., 2004). Epitope 6

(175SMPTAQSTM189) is H2-b Class I restricted and is considered a major murine CTL epitope of Sin Nombre hantavirus. Unfortunately, only one Sin Nombre hantavirus sister pair was available for analysis, therefore it was combined with the New World group.

Epitope 7 (197RYRTAVCGL205) is HLA-A11 restricted and is reportedly conserved in

Old World pathogenic hantaviruses (Wang et al. 2011). Epitope 6 could potentially show positive selection, but shows moderately strong negative selection (pS=0.253 ±0.079) for the pathogenic New World group containing Sin Nombre hantavirus. The reportedly conserved Epitope 7 had a significant pS value of 0.170 ±0.070 in the Old World non- pathogenic group, but was statistically insignificant in the Old World pathogenic group

(pS=0.123 ±0.060). Two epitopes in this study were partially located in both the RNA- binding region and the variable region of Domain II. Epitope 8 (211SPVMGVIGF225) is

HLA-B35 restricted. Epitope 9 (217PVMGVIGFS231) is H-2Kb-restricted. Both reportedly play a key role in human and mice CTL responses in Sin Nombre hantavirus and/or

Puumala hantavirus (Maeda et al 2004). A reasonable hypothesis would be that the pathogenic subgroups would exhibit positive selection; however both epitopes had moderately high pS values for the pathogenic New World group and no significant difference in substitution rates for the pathogenic Old World (Table 5).

Four epitopes were analyzed within the variable region of Domain II on the N protein. Epitope 10 (234ERIDDFLAA242) is HLA-C7 restricted and is conserved in Sin

Nombre hantavirus but not conserved in other hantavirus species (Ennis et al. 1997).

HLA-C7 is prevalent in North American populations including Native Americans (Hjelle et al. 1994). Epitope 11 (245KLLPDTAAV253) is HLA-A24 restricted and very conserved

(Wang et al. 2011). This allele is found throughout the Asia where HTNV is endemic.

Epitope 13 (258GPATNRDYL266) fits the HLA-B7 binding motif and is also highly conserved in HTNV (Wang et al. 2011). This region contained the epitopes with the highest mean pS values (Table 5). In addition, the mean pS of Epitope 13 was higher in the pathogenic groups than the non-pathogenic group in both New and Old World hantaviruses. (Tables 5 and 6, respectively). These findings illustrate that the antigenic region has a significant difference in negative selection compared to the non-pathogenic groups and the Old World groups. While high means were primarily located in the New

World pathogenic group, Epitope 11 had the highest pS value for the New World non- pathogenic group. Epitope 14 (277ETKESKAIR285) is HLA-A33 restricted and reportedly less well conserved (Ma et al. 2013). Old World non-pathogenic Epitope 14 was moderately high (pS=0.216 ±0.097).

One epitope tested was located within the C terminus of Domain III. The C- terminal region also contains an RNA-binding region as well as two helices. Epitope 15

(333ILQDMRNTI341) fits the HLA-A2.1 binding motif. Lee et al. (2002) reports that

Epitope 15 shows strong CTL activity in HTNV. The New World pathogenic group had a moderately high pS value (pS=0.256 ±0.080). Epitope 15 had a very low pN value

(pN=0.005 ±0.005).

Conclusions

Large amounts of the N protein are present during the early stage of hantavirus infection. Early immune responses have been shown to be a result of the presence of the

N protein (Vapalahti et al. 1995). This response consists of significant CTL activity.

CTLs are mostly directed against immunodominant epitopes of the N protein (Wang et al.

2015). This may be due to the fact that the N protein represents the most conserved and abundant hantavirus protein produced during infection (Kaukinen et al. 2005). It was expected that there would be evidence of viral escape, measured as elevated pN to pS means in the well characterized CTL epitopes of the N protein. However, evidence of

CTL escape was not observed in this study. None of the epitopes exhibited positive selection. The mean pS value was greater than the mean pN value in all cases.

It was also expected that the nonsynonymous rates of nucleotide substitution in antigenic regions of the N gene would be higher in the Old World (HFRS-causing viruses) group because of the longer presence in Europe and Asia than the New World

(HPS-causing viruses) group has been in the Americas. The means were not significantly different; the Old World group had a nonsynonymous rate of pN=0.00532 ±0.001 and the

New World Group a nonsynonymous rate of pN=0.00279 ±0.001. And finally, it was expected that the nonsynonymous rates of nucleotide substitution in antigenic regions of

16 the N gene would be higher in pathogenic species than non-pathogenic species because of

CTL escape. The means were not significantly different; the pathogenic group of hantaviruses had a nonsynonymous rate of of pN=0.0035 ±0.001 and the non-pathogenic group had a nonsynonymous substitution rate of pN=0.0035 ±0.001. Determining sister- pair taxa groups allowed for the use of conventional statistics (t-tests) when comparing synonymous and nonsynonymous mutation rates. However, in this study, the particular groupings of hantaviruses (i.e. New World pathogenic and non-pathogenic and Old

World pathogenic and non-pathogenic) may have limited ability to detect patterns of interest. There were relatively small numbers of pairs in the groups and for Old World pathogenic group, seven of the 21 pairs represented Puumala hantavirus (Table 3).

Future studies should, if possible, include more equal representation form all the genotypes in each group. Future studies could also be strengthened by better understanding what the term ‘pathogenic’ means. For example, using the aforementioned criteria, pathogenic strains elicited disease. However, it is possible that a pathogenic virus may not elicit a disease response if an individual can clear it and remain asymptomatic.

The N protein appears to be have been highly conserved throughout most hantaviruses. Numerous epitopes did show evidence of possible negative, or purifying, selection. The New World group had 11 pathogenic and one non-pathogenic epitopes with a significantly higher synonymous rate. The Old World group had three and five epitopes with a significantly higher synonymous rate. Because of genus specificity of the various epitopes, future studies comparing substitution rates within genera could be insightful, especially Domain I. This antigenic region of the N-terminal shows

17 considerable variability among hantaviruses, reportedly due to differences in residues at the HLA binding site (Van Epps et al. 1999).

Furthermore, identifying the variability within changing codons would be beneficial in targeting selective pressures related to binding sites. This could include an analysis of codon changes associated with biophysical properties as done by Hughes and

Friedman (2000). A better understanding of the evolutionary changes in the antigenic regions of the hantavirus may be important to developing methods to predict new outbreaks and/or the possibility of novel host infections. This level of characterization may also aid the diagnosis and treatment of the disease in humans.

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Table 1. The 520 complete nucleocapsid protein sequences used for phylogenetic analysis. The accession number, strain name, and published references are included where available.

Species Accession Strain Name Sequence Reference Adler KP013568 Mm/98-08 Tkachenko et al. 2015 Adler KP013569 Mm/172-11 Tkachenko et al. 2015 Adler KP013570 Mm/173-11 Tkachenko et al. 2015 Adler KP013571 Mm/296-08 Tkachenko et al. 2015 Adler KP013572 Mm/302-08 Tkachenko et al. 2015 Adler KP013573 Mm/340-08 Tkachenko et al. 2015 Adler KP013576 Mm/603-11 Tkachenko et al. 2015 Adler KP013577 Mm/5779-09 Tkachenko et al. 2015 Adler KP013578 Mm/5844-09 Tkachenko et al. 2015 Alto Paraguay DQ345762 AlPa Chu et el. 2006 Amga KF974360 AH301 Bennett et al.c2014 Amga KM201411 MSB148558 Arai et al. 2016 Amga KC136244 H8205 Zhang et al. 2013 Amga AB071184 Solovey/AP63/1999 Lokugamage et al. 2002 Amga AB127996 H5 Lokugamage et al. 2004 Amga AB620028 Khekhtsir/AP209/2005 Kariwa et al. 2012 Amga EF121324 JilinAP06 Jiang et al. 2007 Amga JQ061291 NA33 Chen & Chen 2012 Amga JX473004 ApJLCB2011-99 Yao et al. 2012 Anajatuba JX443690 H759113/BRA270 Firth et al. 2012 Andes AB689164 HV-97021050 Koma et al. 2012 Andes AF004660 AH-1 Lopez et al. 1997 Andes AF325966 AND Nort Gonzalez et al. 2002 Andes AF482713 Oc22531 Bohlman et al. 2002 Andes AF482714 22819 Bohlman et al. 2002 Andes AF482715 22996 Bohlman et al. 2002 Andes AF482716 13796 Bohlman et al. 2002 Andes AF482717 14403 Bohlman et al. 2002 Andes AY228237 CHI-7913 Tischler et al. 2003 Andes AY267347 HV-97021050 Fulhorst et al. 2004 Andes DQ345763 Neembucu Chu et al. 2006 Andes EF571895 P5/Cajuru de Sousa et al. 2008 Andes JF750418 FVB640 Cruz et al. 2012 Andes KP202360 LBCE 9480 Guiterres et al. 2015 Anjozorobe/Rr/MDG/2009/ Anjozorobe KC490914 ATD261 Reynes et al. 2014 Anjozorobe/Rr/MDG/2009/ Anjozorobe KC490916 ATD56 Reynes et al. 2014 Anjozorobe/Em/MDG/2009/ Anjozorobe KC490918 ATD49 Reynes et al. 2014 Araucaria AY740627 HPR/02-85 Raboni et al. 2005 Araucaria AY740628 HPR/03-95 Raboni et al. 2005 Asama EU929070 H4 Arai et al. 2008 Asikkala KC880341 CZ/Beskydy/412/2010/Sm Radosa et al. 2013 DE/Duerrbach/1912/2009/ Asikkala KC880343 Sm Radosa et al. 2013

Asikkala KJ136616 Asikkala Ling et al. 2014 Bayou GQ200820 HV F0260003 Richter et al. unpublished Bayou L36929 UNKNOWN-L36929 Morzunov et al. 1995 Black Creek Canal AB689163 UNKNOWN-AB689163 Koma et al. 2005 Bowe KC631782 VN1512 Gu et al. 2013 Cano Delgadito DQ285566 VHV-574 Milazzo et al. 2006 Cao Bang EF543524 3 Song et al. 2007 Cao Bang KJ162406 ROM117784 Gu et al. 2016 Carrizal AB620093 2/2006 Saasa et al. 2012 Carrizal AB620103 26/2006 Saasa et al.2012 Castelo dos Sonhos-2 JX443691 AN717307/BRA299 Firth et al. 2012 CASV JX443693 H745332/BRA261 Firth et al. 2012 CASV-2 JX443692 AN717313/BRA300 Firth et al. 2012 Catacamas DQ256126 HV C1280001 Milazzo et al. 2006 Choclo DQ285046 UNKNOWN-DQ285046 Nelson et al. 2010 Choclo KM597161 Uk (ex Panama) Atkinson et al. 2015 Dabieshan JF796018 Wencheng-Nc-469 Lin et al. 2012 Dabieshan JF796019 Wencheng-Nc-470 Lin et al. 2012 Dabieshan JF796020 Yongjia-Nc-15 Lin et al. 2012 Dabieshan JF796022 Yongjia-Nc-58 Lin et al. 2012 Dabieshan JF796023 Yongjia-Nc-95 Lin et al. 2012 Dobrava-Belgrade KP878311 10636/Ap Kruger et al. 2015 Dobrava-Belgrade KP878313 10752/hu Kruger et al. 2015 Dobrava-Belgrade AJ009773 DOB/Saaremaa/160V Nemirov et. al. 1999 Dobrava-Belgrade AJ009775 Saaremaa/90Aa/97 Nemirov et. al. 1999 Dobrava-Belgrade AJ131672 Kurkino/44Aa/98 Plyusnin et al. 1999 Dobrava-Belgrade AJ131673 Kurkino/53Aa/98 Plyusnin et al. 1999 Dobrava-Belgrade AJ269549 East Slovakia-856-Aa Sibold et al. 2001 Dobrava-Belgrade AJ269550 East Slovakia-862-Aa Sibold et al. 2001 Dobrava-Belgrade AJ410619 DOBV/Ano-Poroia/13Af/99 Nemirov et al. 2003 Saaremaa/Lolland/Aa1403/ Dobrava-Belgrade AJ616854 2000 Nemirov et al. 2004 Dobrava-Belgrade AY168576 East Slovakia/400Af/98 Klempa et al. 2003 Dobrava-Belgrade AY533118 Esl/29Aa/01 Klempa et al. 2004 Dobrava-Belgrade AY961615 SK/Aa Klempa et al. 2005 Dobrava-Belgrade EU188449 Ap1584/Sochi-01 Klempa et al. 2008 Dobrava-Belgrade EU188452 Aa1854/Lipetsk-02 Klempa et al. 2008 Dobrava-Belgrade GQ205401 GER/07/293/Aa Schlegel et al. 2009 Dobrava-Belgrade GQ205402 GER/07/607/Af Schlegel et al. 2009 Dobrava-Belgrade GQ205404 GER/07/1064/Aa Schlegel et al. 2009 Dobrava-Belgrade GQ205406 GER/05/477/Af Schlegel et al. 2009 Dobrava-Belgrade GQ205407 GER/08/118/Aa Schlegel et al. 2009 Dobrava-Belgrade GQ205408 GER/08/131/Af Schlegel et al. 2009 Dobrava-Belgrade GU904030 UNKNOWN-GU904030 Kirsanovs et al. 2010 Dobrava-Belgrade JF920150 Ap/Sochi/hu Dzagurova et al. 2012 Dobrava-Belgrade JF920152 Ap/Sochi/79 Dzagurova et al. 2013 Dobrava-Belgrade KC848495 DOB/Gola/235Af/07 Nemeth Unpublished Dobrava-Belgrade KC848498 DOB/Gola/343Af/07 Nemeth Unpublished Dobrava-Belgrade KC848499 DOB/Pecs/27Aa/06 Nemeth Unpublished Dobrava-Belgrade KC848500 DOB/Pecs/31Aa/06 Nemeth Unpublished

Dobrava-Belgrade KC848501 DOB/Sarmellek/77Aa/06 Nemeth Unpublished Dobrava-Belgrade L41916 Dobrava Avsic-Zupanc et al. 1995 El Moro Canyon U11427 RM-97 Hjelle et al. 1994 Eothenomys miletus LX309 HM756286 LX309 Zhang et al. 2011 Gou JQ912775 LongquanRn-08-712 Wang et al. 2013 Gou JQ912779 LongquanRn-09-641 Wang et al. 2013 Gou JQ912789 LongquanRn-11-148 Wang et al. 2013 Gou JQ912795 LongquanRn-11-220 Wang et al. 2013 Hantaan KP970578 JS7 Wang Unpublished Hantaan KT935024 Aa03-161 Kim et al. 2016 Hantaan KT935034 Aa09-948 Kim et al. 2016 Hantaan KT935036 Aa10-123 Kim et al. 2016 Hantaan KT935038 Aa10-434 Kim et al. 2016 Hantaan KT935045 Aa14-204 Kim et al. 2016 Hantaan KT935051 Aa14-386 Kim et al. 2016 Hantaan KT935057 Aa14-438 Kim et al. 2016 Hantaan AB027097 Q32 Wang et al. 2000 Hantaan AB027101 Chen4 Wang et al. 2000 Hantaan AB027523 NC167 Wang et al. 2000 Hantaan AB127998 Bao14 Lokugamage et al. 2004 Hantaan AF288296 RG9 Yao et al. Unpublished Hantaan AF288644 E142 Yao et al. Unpublished Hantaan AF288646 A16 Yao et al. Unpublished Hantaan AF288657 SN7 Yao et al. Unpublished Hantaan AF288659 S85-46 Yao et al. Unpublished Hantaan AF321094 Maaji-1 Choi et al. Unpublished Hantaan AF321095 Maaji-2 Choi et al. Unpublished Hantaan AF366568 84FLi Liu et al. Unpublished Dekonenko & Ivanov Hantaan AF427318 AA1028 Unpublished Dekonenko & Ivanov Hantaan AF427319 AA1719 Unpublished Hantaan AF427322 AP708 Kariwa et al. 2012 Hantaan AF427323 AP1168 Kariwa et al. 2012 Hantaan AY017064 84FLi Li et al. Unpublished Hantaan AY748308 YU61 Zhang et al. Unpublished Hantaan AY839871 TJJ16 Li et al. Unpublished Hantaan D25530 cl-1 Isegawa et al. 1994 Hantaan DQ658415 N8 Lu et al. Unpublished Hantaan EF208929 CJAp93 Zhang et al. 2007 Hantaan EF595840 Z251 Xu et al. Unpublished Hantaan EF990904 CGRn93P8 Zou et al. 2008 Hantaan EF990905 CGRn93MP8 Zou et al. 2008 Hantaan EF990906 CGRn5310 Zou et al. 2008 Hantaan EF990907 CGRn53 Zou et al. 2008 Hantaan EF990909 CGHu3612 Zou et al. 2008 Hantaan EF990910 CGAa31P9 Zou et al. 2008 Hantaan EF990912 CGAa1015 Zou et al. 2008 Hantaan EF990913 CGAa1011 Zou et al. 2008 Hantaan EF990915 CGAa4MP9 Zou et al. 2008

Hantaan EU092218 CGHu1 Zou et al. 2008 Hantaan EU092219 CGAa2 Zou et al. 2008 Hantaan EU092220 CGAa75 Zou et al. 2008 Hantaan EU092221 CGRn45 Zou et al. 2008 Hantaan EU363810 CGRn15 Zou et al. 2008 Hantaan EU363811 CGRn2616 Zou et al. 2008 Hantaan EU363812 CGRni1 Zou et al. 2008 Hantaan EU363813 CGHu2 Zou et al. 2008 Hantaan FJ753396 ZLS6-11 Yao et al. Unpublished Hantaan GU329991 YN509 Cao et al. 2010 Hantaan HQ611981 YaluRiver13 Yao et al. Unpublished Hantaan HQ834499 CA09082007 Ma et al. 2012 Hantaan HQ834500 CA10081109 Ma et al. 2012 Hantaan HQ834501 CA10081113 Ma et al. 2012 Hantaan HQ834502 CA10081203 Ma et al. 2012 Hantaan HQ834503 CA10081206 Ma et al. 2012 Hantaan HQ834504 CA10081708 Ma et al. 2012 Hantaan HQ834505 CA10081905 Ma et al. 2012 Hantaan HQ834506 H10150 Ma et al. 2012 Hantaan JQ665905 HubeiHu02 Kang et al. 2012 Hantaan JQ665906 WuhanAaJ10 Kang et al. 2012 Hantaan JQ912697 LongquanAa-08-150 Wang et al. 2013 Hantaan JQ912709 LongquanAa-08-314 Wang et al. 2013 Hantaan JQ912714 LongquanAa-08-573 Wang et al. 2013 Hantaan JQ912716 LongquanAa-09-113 Wang et al. 2013 Hantaan JQ912719 LongquanAa-09-191 Wang et al. 2013 Hantaan JQ912748 LongquanAa-09-698 Wang et al. 2013 Hantaan JQ912754 LongquanAa-10-46 Wang et al. 2013 Hantaan KC344246 LongquanAa-11-754 Wang et al. 2013 Hantaan KC570386 DandongHu-32 Chen et al. 2014 Hantaan KC570387 DandongHu-34 Chen et al. 2014 Hantaan KC570388 DandongHu-44 Chen et al. 2014 Hantaan KC570389 DandongHu-89 Chen et al. 2014 Hantaan KC570390 DandongHu-91 Chen et al. 2014 Hantaan KC844228 SXRn20120013 Ma et al. 2014 Hantaan KJ857347 Fuyuan-Aa-26 Wang et al. 2014 Hantaan KJ857349 Fuyuan-Aa-145 Wang et al. 2014 Hantaan KM355414 WCL Chen Unpublished Hantaan NC_005218 Schmaljohn Schmaljohn et al. 1986 Hantaan U37768 CUMC-B11 Kim & Jung Unpublished Hantaan X95077 CFC94-2 Lee et al. 1997 Hantavirus AF329390 an A9 Shi et al. Unpublished Hantavirus AF285264 AH09 Zhihui et al. Unpublished Hantavirus AF288647 AH211 Yao et al. Unpublished Hantavirus EF990903 CGRn8316 Zou & Hu 2008 Hantavirus EF990902 CGRn9415 Zou et al. 2008 Hantavirus EU072480 Fusong-Mf-682 Zou & Wang 2008 Hantavirus EU072481 Fusong-Mf-731 Zou & Wang 2008 Hantavirus FJ816031 HMT 08-02 Raboni et al. 2009 Chenqgun et al. Hantavirus AF252259 261 Unpublished

Hantavirus GQ274940 Jurong TJK/06(RT49) Johansson et al. 2010 Hantavirus GU140098 UNKNOWN-GU140098 Zhang et al. 2010 Hantavirus AF288649 Liu Lokugamage et. al. 2004 Hantavirus U32591 Pm Song et al. 1996 Hantavirus JN196141 NFG289 Chu et al. Unpublished Hantavirus JN196142 NFG354 Chu et al. Unpublished Hantavirus JN196140 NFG357 Chu et al. Unpublished Hantavirus KM853162 NMAp117 Zuo Unpublished Hantavirus FJ170797 Shenyang-Mf-135 Zou_Unpub_2008 Hantavirus FJ170796 Shenyang-Mf-136 Zou Unpublished Hantavirus AF482711 Hu39694 Bohlman et al. 2002 Hantavirus AF482712 Andes 9718133 Bohlman et al. 2002 Hantavirus EF576661 patient 2 Melo-Silva 2009 Hantavirus AB186420 741 Pattamadilok et al. 2006 Hantavirus JF421283 XAHu09047 Ma et al. 2012 Hantavirus JF421284 XAHu09066 Ma et al. 2012 Hantavirus EU072484 Yakeshi-Mm-182 Zou & Wang 2008 Hantavirus EU072483 Yakeshi-Mm-59 Zou & Wang 2008 Hantavirus HQ589247 YN06-862 Ge et al. 2016 Hantavirus FJ170794 Yuanjiang-Mf-15 Zou & Xiao 2008 Hantavirus FJ170792 Yuanjiang-Mf-78 Zou & Xiao 2008 Hantavirus AF184987 Z10 Zhihui et al. Unpublished Hantavirus EF533944 Z10 Vaccine Zhu et al. Unpublished Hantavirus EF103195 Z5 Xie et al. Unpublished Hokkaido AB675450 Tobetsu35S/2010 Kariwa et al. Unpublished Hokkaido AB675479 ShariG2-1S-As/2010 Kariwa et al. Unpublished Huitzilac AB620106 200/2006 HUIV_Saasa_2012 Imjin EF641804 Cl 05-11 MJNV_Song_2009 Imjin EF641805 Cl 04-55 MJNV_Song_2009 Imjin KJ420559 UNKNOWN-KJ420559 MJNV_Lin_2014 Isla Vista U19302 MC-SB-47 Song et al. 1995 Isla Vista U31534 MC-SB-1 Song et al. 1995 Isla Vista U31535 PC-SB-77 Song et al. 1995 Itapua DQ345766 Itapua 38 Itapua_Chu_2006 Jabora JN232078 Akm9635 JABV_Oliviera_2012 Jabora JN232079 Aks9826 JABV_Oliviera_2012 Jabora JN232080 Akp8048 JABV_Oliviera_2012 Jeju HQ663933 SH42 JJUV_Arai_2012 Jeju HQ834695 10-11 JJUV_Arai_2012 Jemez Springs FJ593499 MSB144475 JMSV_Kang_2009 Jemez Springs FJ686859 MSB90752 JMSV_Kang_2009 Jemez Springs FJ686860 MSB90111 JMSV_Kang_2009 Jemez Springs FJ686862 MSB147533 JMSV_Kang_2009 Jemez Springs FJ686864 MSB147745 JMSV_Kang_2009 Jemez Springs KF963284 FXC_MSB144181 JMSV_Kang_2009 Jemez Springs KF963286 PWB_Sv742 JMSV_Kang_2009 Jemez Springs KF963288 TLN_DGR7087 JMSV_Kang_2009 Juquitiba KC422347 Oln6470 Guterres et al. 2013 Juquitiba KC422344 olnSVS220 Guterres et al. 2013 Juquitiba KC422345 olnSVS221 Guterres et al. 2013

Juquitiba KF913850 LBCE 12071 Guterres et al. 2014 Kenkeme GQ306148 MSB148794 Kang et al. 2010 Kenkeme KJ857341 Fuyuan-Sr-326 Wang et al. 2014 Khabarovsk KJ857342 Fuyuan-Mm-217 Wang et al. 2014 Khabarovsk KJ857343 Fuyuan-Mm-228 Wang et al. 2014 Khabarovsk KJ857344 Fuyuan-Mm-250 Wang et al. 2014 Khabarovsk KJ857345 Fuyuan-Mm-254 Wang et al. 2014 Khabarovsk KJ857346 Fuyuan-Mm-312 Wang et al. 2014 Khabarovsk U35255 MF-43 Horing et al. 1996 Kilimanjaro JX193698 FMNH174124 Kang et al. 2014 Laguna Negra AF005727 510B Johnson et al. 1997 Laguna Negra KP202359 LBCE 12234 Guterres et al. 2013 Laibin KM102247 BT20 Xu et al. 2015 LANV-2 JX443684 H713174/BRA255 Firth et al. 2012 LANV-2 JX443687 H711879/BRA250 Firth et al. 2012 Lianghe JX465404 Lianghe-As-1 Guo et al. 2013 Lianghe JX465406 Lianghe-As-217 Guo et al. 2013 Lianghe JX465408 Lianghe-As-221 Guo et al. 2013 Lianghe JX465409 Lianghe-As-222 Guo et al. 2013 Lianghe JX465410 Lianghe-As-238 Guo et al. 2013 Longquan JX465414 Longquan-Ra-14 Guo et al. 2013 Longquan JX465415 Longquan-Ra-25 Guo et al. 2013 Longquan JX465418 Longquan-Ra-90 Guo et al. 2013 Maripa JQ611712 BOR Matheus et al. 2012 Maripa KC876041 GOU Thoisy et al. 2014 Montano AB620077 3/2006 Saasa et al. 2012 Montano AB620079 42/2006 Saasa et al. 2012 Montano AB620084 107/2006 Saasa et al. 2012 Montano AB620085 111/2006 Saasa et al. 2012 Montano AB620086 113/2006 Saasa et al. 2012 Montano AB620088 119/2006 Saasa et al. 2012 Montano AB620089 121/2006 Saasa et al. 2012 Montano AB703012 95/2007 Saasa et al. 2012 Montano AB703016 122/2007 Saasa et al. 2012 Montano AB703019 141/2007 Saasa et al. 2012 Muleshoe U54575 SH-Tx-339 Rawlings et al. 1996 Necocli KF481954 HV O0020002 Montoya et al. 2015 Nova FJ539168 MSB95703 Kang et al. 2009 Nova JX990924 1135 Gu et al. 2014 Nova JX990929 2095 Gu et al. 2014 Nova KF515970 2086 Gu et al. 2014 Nova KT004445 BE/Namur/TE/2013/1 Laenen et al. 2015 Oxbow FJ539166 Ng1453 Kang et al. 2009 Playa de Oro EF534079 Mexico Chu et al. 2008 Prairie vole U19303 MO46 Song et al. Unpublished Prospect Hill M34011 Par Parrington & Kang 1990 Prospect Hill U47136 PH-NY1 Huang et al. 1996 Puumala AF367069 CRF161 Dekokenko et al. 2003 Puumala DQ138128 00-18 Song et al. 2007 Puumala DQ138142 99-28 Song et al. 2007

PUUV/Ardennes/Mg156/20 Puumala KT247592 11 PUUV_Castel et al. 2015 Puumala KT247594 PUUV/Orleans/Mg23/2010 PUUV_Castel et al. 2015 Puumala KT247596 PUUV/Jura/Mg2/2010 PUUV_Castel et al. 2015 Puumala KT247597 PUUV/Jura/Mg214/2010 PUUV_Castel et al. 2015 Puumala AB010730 Kamiiso-8Cr-95 Kariwa et al. 1999 Puumala AB010731 Tobetsu-60Cr-93 Kariwa et al. 1999 Puumala AB433843 Samara_49/CG/2005 Kariwa et al. 2009 Puumala AF294652 Opina916 Leitmeyer et al. 2001 Puumala AF367064 CG144 Dekokenko et al. 2003 Puumala AF367065 CG168 Dekokenko et al. 2003 Puumala AF367067 CG222 Dekokenko et al. 2003 Puumala AF367068 CG315 Dekokenko et al. 2003 Puumala AF367070 CRF308 Dekokenko et al. 2003 Puumala AF367071 CRF366 Dekokenko et al. 2003 Puumala AF442613 CG17/Baskiria-2001 Dekokenko et al. 2003 Puumala AJ238779 PUU/Cg-Erft Heiske et al. 1999 Puumala AJ238788 Karhumaki Asikainen et al. 2000 Puumala AJ238789 Kolodozero Asikainen et al. 2000 Puumala AJ238790 Gomselga Asikainen et al. 2000 Puumala AJ238791 Fyn Asikainen et al. 2000 Puumala AJ277030 Thuin/33Cg/96 Escutenaire et al. 2001 Puumala AJ277031 Montbliart/23Cg/96 Escutenaire et al. 2001 Puumala AJ277033 Momignies/55Cg/96 Escutenaire et al. 2001 Puumala AJ277034 Couvin/59Cg/97 Escutenaire et al. 2001 Puumala AJ277075 CG14444 Escutenaire et al. 2001 Puumala AJ277076 CG14445 Escutenaire et al. 2001 Puumala AJ314597 Pallasjarvi/63Cg/98 Sironen et al. 2001 Puumala AJ314598 Baltic/49Cg/00 Sironen et al. 2001 Puumala AJ314599 Baltic/205Cg/00 Sironen et al. 2001 Puumala AJ314600 Balkan-1 Sironen et al. 2001 Puumala AJ314601 Balkan-2 Sironen et al. 2001 Puumala AJ888751 PUU/Klippitztoerl/Cg9/1995 Plyusnina et al. 2006 PUU/Ernstbrunn/Cg641/19 Puumala AJ888752 95 Plyusnina et al. 2006 PUU/Mignovillard/CgY02/2 Puumala AM695638 005 Plyusnina et al. 2007 Puumala AY526219 Umea/hu Johansson et al. 2004 Puumala DQ016430 Bavaria CG 33/04 Essbauer et al. 2006 Puumala DQ016432 Bavaria CG 41/04 Essbauer et al. 2006 Puumala DQ138133 96-1 Song et al. 2007 Puumala EF211819 Fusong 84-05 Tang & Li Unpublished Puumala EF442087 Fusong-Cr-247 Zhang et al. 2007 Puumala EF488803 Fusong 199-05 Wu et al. Unpublished Puumala EF488804 Fusong 114-05 Wu et al. Unpublished Puumala EF488806 Fusong 900-06 Wu et al. Unpublished Puumala EU439968 Bavaria 151/05 Mertens et al. 2011 PUUV/Mg9/HungaryTR17/ Puumala FN377821 00 Plyusnina et al. 2009 PUUV/Mg23/HungaryTR17 Puumala FN377822 /00 Plyusnina et al. 2009

Puumala GQ339475 Kiviniemi/Mg6/05 Nemirov et al. 2010 Puumala GQ339480 Gyttjea/Mg19/05 Nemirov et al. 2010 Puumala GQ339481 Ljustrask/Mg20/05 Nemirov et al. 2010 Puumala GQ339483 Bergsjobo/Mg25/05 Nemirov et al. 2010 Puumala GQ339484 Faboviken/Mg26/05 Nemirov et al. 2010 Puumala GQ339485 Mangelbo/Mg1/05 Nemirov et al. 2010 Puumala GQ339486 Munga/Mg2/05 Nemirov et al. 2010 Puumala GQ339487 Munga/Mg16/05 Nemirov et al. 2010 Sironen et al. Puumala GU808824 Kuhmo/X5 Unpublished Sironen et al. Puumala GU808825 Kuhmo/X11 Unpublished PUUV/Jelgava/Mg149/200 Puumala JN657228 8 Razzauti et al. 2012 Puumala JN657229 PUUV/Madona/Mg99/2008 Razzauti et al. 2012 PUUV/Jelgava/Mg136/200 Puumala JN657230 8 Razzauti et al. 2012 PUUV/Jelgava/Mg140/200 Puumala JN657231 8 Razzauti et al. 2012 Puumala JN696358 Mu362Osnabrueck/05 Ettinger et al. 2012 Puumala JN696373 MuEb10Karlstadt/10 Ettinger et al. 2012 Puumala JN696374 MuEb12Lackenberg/10 Ettinger et al. 2012 Puumala JN696376 MuEb51Elsenthal/10 Ettinger et al. 2012 PUUV/Pieksamaki/Mg7/20 Puumala JN831943 08 Plyusnina et al. 2012 PUUV/Konnevesi/Mg_O57 Puumala JQ319161 A/2005 Razzauti et al. 2013 PUUV/Konnevesi/Mg_O78 Puumala JQ319162 A/2005 Razzauti et al. 2013 PUUV/Konnevesi/Mg_M94 Puumala JQ319163 A/2005 Razzauti et al. 2013 PUUV/Konnevesi/Mg_O6B/ Puumala JQ319164 2005 Razzauti et al. 2013 PUUV/Konnevesi/Mg_O22 Puumala JQ319168 B/2005 Razzauti et al. 2013 PUUV/Konnevesi/Mg_O74 Puumala JQ319170 B/2005 Razzauti et al. 2013 PUUV/Konnevesi/Mg_M11 Puumala JQ319171 4B/2005 Razzauti et al. 2013 Puumala JX028273 11-1 Lee et al. 2014 Puumala JX046487 11-5 Lee et al. 2014 Puumala KJ994776 Mu/07/1219 Ali et al. 2015 Puumala L08804 K27 Xiao et al. 1993 Puumala L11347 P360 Xiao et al. 1993 Puumala M32750 CG1820 Stohwasser et al. 1990 Puumala U14137 Vranica Reip et al. 1995 Puumala U22423 CG 13891 Bowen et al. Puumala X61035 Sotkamo Vapalahti et al. 1992 Puumala Z21497 Udmurtia/894Cg/91 Plyusnin et al. 1994 Puumala Z30703 Evo/13Cg/93 Plyusnin et al. 1995 Puumala Z30704 Evo/14Cg/93 Plyusnin et al. 1995 Puumala Z30705 Evo/15Cg/93 Plyusnin et al. 1995 Puumala Z30707 Udmurtia/458Cg/88 Plyusnin et al. 1995

Puumala Z30708 Udmurtia/338Cg/92 Plyusnin et al. 1995 Puumala Z84204 Puu/Kazan Lundkvist et al. 1997 Qian Hu Shan GU566023 YN05-284 Zuo et al. Unpublished Rio Mamore FJ532244 HTN-007 Richter et al. 2010 Rio Mamore KF584259 LH 060/2011 de Oliveira et al. 2014 Rio Mamore U52136 OM-556 Bharadwaj et al 1997 Rio Segundo U18100 RMx-Costa-1 Hjelle et al. 1995 RIOMV-3 JX443667 AN683313/BRA271 Firth et al. 2012 RIOMV-4 JX443670 AN693231/BRA278 Firth et al. 2012 RIOMV-4 JX443671 AN693239/BRA279 Firth et al. 2012 RIOMV-4 JX443678 AN693288/BRA292 Firth et al. 2012 Rockport HM015218 MSB57411 Kang et al. 2011 Rockport HM015224 MSB57413 Kang et al. 2011 Sangassou JQ082300 SA14 Klempa et al. 2012 Sangassou JQ082303 SA22 Klempa et al. 2012 Seewis EF636024 mp70 Song et al. 2007 Seewis JQ425265 Horst092292_N_Sa Schelegel et al. 2012 CeskeBudejovice145_N_S Seewis JQ425267 a Schelegel et al. 2012 Seewis JQ425272 Kosice260_N_Sa Schelegel et al. 2012 Seewis JQ425276 Drahany327_N_Sa Schelegel et al. 2012 Seewis JQ425281 Drahany344_N_Sa Schelegel et al. 2012 Seewis JQ425286 Drahany360_N_Sm Schelegel et al. 2012 Seewis JQ425293 Beskydy389_N_Sa Schelegel et al. 2012 Seewis JQ425297 Beskydy395_N_Sa Schelegel et al. 2012 Seewis JQ425299 Beskydy399_N_Sa Schelegel et al. 2012 Seewis JQ425304 Beskydy407_N_Sa Schelegel et al. 2012 Seewis JQ425306 Beskydy415_N_Sa Schelegel et al. 2012 Seewis KJ136609 48/Lappeenranta Ling et al. 2014 Seewis KJ136610 21/Muonio Ling et al. 2014 Seewis KJ136614 EWS25/Tammela Ling et al. 2014 Seoul KU204960 Tchoupitoulas/POR Miles e al. 2016 Seoul AB027522 Gou3 Wang et al. 2000 Seoul AF184988 Gou3 Zhihui et al. Unpublished Seoul AF187082 Z37 Zhihui et al Unpublished Seoul AF288295 R22 Yao et al. Unpublished Seoul AF288299 L99 Zhihui et al. Unpublished Seoul AF288643 Hb8610 Yao et al. Unpublished Seoul AF288651 Gou3-v9 Yao et al. Unpublished Seoul AF288655 K24-v2 Yao et al. Unpublished Seoul AF329388 IR461 Shi et al. 2003 Seoul AF329389 Tchoupitoulas (TCH) Shi et al. 2003 Seoul AF406965 zy27 Sun et al. Unpublished Seoul AF488707 R22 Shi Shi et al. 2003 Seoul AF488708 L99 Shi Shi et al. 2003 Seoul AY006465 Pf26 Sun et al. Unpublished Seoul AY273791 80-39 Song et al. Unpublished Seoul AY627049 BjHD01 Zuo et al. Unpublished Seoul AY750171 ZT71 Xie et al. Unpublished Seoul AY766368 ZT10 Xie et al. Unpublished Seoul DQ217791 JUN5-14 Tao et al. 2007

Seoul EF192308 93HBX12 Li et al. Unpublished Seoul FJ753400 ZJ5 Yao et al. 2008 Seoul FJ803201 CixiRf23 Zhang et al. 2010 Seoul FJ803202 CixiRf56 Zhang et al. 2010 Seoul FJ803205 CixiRn21 Zhang et al. 2010 Seoul FJ803206 CixiRn76 Zhang et al. 2010 Seoul FJ803208 OuhaiRf35 Zhang et al. 2010 Seoul FJ803210 OuhaiRn146 Zhang et al. 2010 Seoul FJ803213 RuianRf74 Zhang et al. 2010 Seoul FJ803214 RuianRn23 Zhang et al. 2010 Seoul FJ803215 RuianRn33 Zhang et al. 2010 Seoul GQ274945 Singapore/06(RN46) Johansson et al. 2010 Seoul GQ279379 HuBJ22 Zuo et al. Unpublished Seoul GQ279380 HuBJ16 Zuo et al. Unpublished Seoul GQ279382 Rn-CP7 Zuo et al. Unpublished Seoul GQ279383 Rn-M11 Zuo et al. Unpublished Seoul GQ279386 Rn-DC8 Zuo et al. Unpublished Seoul GQ279389 HuBJ19 Zuo et al. Unpublished Seoul GQ279391 HuBJ3 Zuo et al. Unpublished Seoul GQ279395 CUI Zuo et al. Unpublished Seoul GU361893 SC106 Chen et al. Unpublished Seoul GU592932 GanyuRn66 Guo et al. Unpublished Seoul GU592942 FeixianRn1 Guo et al. Unpublished Seoul HQ611980 YaluRiver12 Yao et al. Unpublished Seoul JN377553 HeB38 Qi Unpublished Seoul JQ665911 WuhanMm24 Kang et al. 2012 Seoul JQ665912 WuhanRf02 Kang et al. 2012 Seoul JQ665915 WuhanRf11 Kang et al. 2012 Seoul JQ898106 SEO/Belgium/Rn895/2005 Plyusnina et al. 2012 Seoul KC626089 Cherwell Jameson et al. 2013 Seoul KF745942 Gongzhuling42 Fan et al. Unpublished Seoul KF745943 Gongzhuling45 Fan et al. Unpublished Seoul KF745944 Gongzhuling58 Fan et al. Unpublished Seoul KF745945 Gongzhuling85 Fan et al. Unpublished Seoul KF745946 Gongzhuling97 Fan et al. Unpublished Seoul KF745947 Gongzhuling108 Fan et al. Unpublished Seoul KF745948 Gongzhuling147 Fan et al. Unpublished Seoul KF745949 Gongzhuling415 Fan et al. Unpublished Seoul KF745950 Taonan52 Fan et al. Unpublished Seoul KF745951 Taonan420 Fan et al. Unpublished Seoul KP645198 Fj372/2013 Wang et al. 2015 Seoul KP859512 JiangxiXinjianRn-09-2011 Liu et al. Unpublished Seoul M34881 Sapporo Rat 11 Arikawa et al. 1990 Serang AM998808 Serang/Rt60/2000 Plyusnina et al. 2009 Sin Nombre JQ690278 3 Bagamian et al. 2012 Sin Nombre JQ690282 2 Bagamian Unpublished Miles & Lewandowski Sin Nombre KT885046 CC107/POR Unpublished Sin Nombre L25784 NM H10 Spiropoulou et. al. 1994 Sin Nombre L33683 Convict Creek 107 Scmaljohn et al. 1995 Sin Nombre L33816 Convict Creek 74 Scmaljohn et al. 1995

Sin Nombre U47135 H-NY1 Huang et al. 1996 Soochong AY675351 SC-3 Baek et al. 2006 Soochong AY675349 SC-1 Baek et al. 2006 Soochong AY675350 SC-2 Baek et al. 2006 Soochong AY675352 SC-4 Baek et al. 2006 Nakhon Thailand AM397664 Ratchasima/Bi0017/2004 Hugot et al. 2006 Thottapalayam AY526097 UNKNOWN-AY526097 Song et al. Unpublished Thottapalayam JF784172 LongwanSm53 Guo et al. 2011 Thottapalayam JF784173 LongwanSm450 Guo et al. 2011 Thottapalayam JF784175 LongwanSm505 Guo et al. 2011 Thottapalayam JF784176 LongwanSm512 Guo et al. 2011 Thottapalayam KJ420560 UNKNOWN-KJ420560 Lin et al. 2014 Thottapalayam KJ420561 UNKNOWN-KJ420561 Lin et al. 2014 Thottapalayam KJ420562 UNKNOWN-KJ420562 Lin et al. 2014 Thottapalayam KJ420563 UNKNOWN-KJ420563 Lin et al. 2014 Thottapalayam KJ420564 UNKNOWN-KJ420564 Lin et al. 2014 Thottapalayam KJ420565 UNKNOWN-KJ420565 Lin et al. 2014 Thottapalayam KJ420566 UNKNOWN-KJ420566 Lin et al. 2014 Topografov AJ011646 Ls136V Vapalahti et al. 1999 Tula AF063897 Lodz-2 Song et al. 2004 Dekonenko & Yakimenko Tula AF442621 MG23/Omsk Unpublished CHEVRU/Hu/FRA/2015/15. Tula KT946591 00453 Reynes et al. 2015 Tula AF017659 Song Song et al. 2002 Tula AF063892 Lodz-1 Song et al. 2004 Schmidt-Chanasit et al. Tula AF164093 g20-s 2010 Scharninghausen et al. Tula AF164094 c109-s 2002 Tula AF289819 D5-98 Klempa et al 2003 Tula AF289820 D17-98 Klempa et al 2003 Tula AF289821 D63-98 Klempa et al 2003 Tula AJ223600 Tula/Koziky/5247Ma/94 Sibold et al. 1999 Tula AJ223601 Tula/Koziky/5276Ma/94 Sibold et al. 1999 Tula AM945877 TUL/Karatal/Ma322/2003 Plyusnina et al. 2008 Schmidt-Chanasit et al. Tula EU439949 Sennickerode Sen05/175 2010 Schmidt-Chanasit et al. Tula EU439950 Sennickerode Sen05/204 2010 Tula Y13979 (Tula/Kosice144/Ma/95) Sibold et al. 1999 Tula Y13980 (Tula/Kosice667/Ma/95) Sibold et al. 1999 Tula Z69991 (Tula/Moravia/5302v/95) Vapalahti et al. 1996 Ussuri AB677476 Khekhtsir3S/1998 Kariwa et al. Unpublished Ussuri AB677477 Khekhtsir17S/2002 Kariwa et al. Unpublished Xinyi KT901298 MVZ180982 Gu et al. 2016 Xuan son KF704709 F42682 Gu et al. 2014 Xuan son KF704710 F44580 Gu et al. 2014 Xuan son KF704711 F44583 Gu et al. 2014 Xuan son KF704712 F44601 Gu et al. 2014 Yakeshi JX465423 Yakeshi-Si-210 Guo et al. 2013

Table 2. New World sister pair groupings of pathogenic and non-pathogenic hantaviruses. There were nine pairs of taxa for each group. Pairs were identified in both NJ uncorrected p-distance and Poisson distance phylogenies and supported by a bootstrap greater than or equal to 70.

Pathogenic Non-Pathogenic

Sister Pair Name Sequences Sister Pair Name Sequences

Andes hantairus AF325966 AF482715 Jabora hantavirus JN232079 JN232080 Juquitiba hantavirus KC422344 KC422347 Maporal hantavirus AB689164 AY267347 Rio Mamore-4 JX443671 JX443678 Maripa hantavirus JQ611712 KC876041 hantavirus Rio Mamore3 JX443667 KF584259 Unknown JN196140 JN196141 hantavirus hantavirus Choclo hantavirus DQ285046 KM597161 Carrizal hantavirus AB620093 AB620103 Bayou hantavirus GQ200820 L36929 Rockport HM015218 HM015224 hantavirus Andes hantavirus AF482712 AY228237 Prospect Hill M34011 U47136 hantavirus Araraquara EF576661 KP202360 EL Moro Canyon AB620106 U11427 hantavirus hantavirus Sin Nombre JQ690278 L25784 Convict Creek-107 L33683 L33816 hantavirus hantavirus

Table 3. Old World sister pair grouping of pathogenic and non-pathogenic hantaviruses. There were 21 pairs of taxa for the pathogenic group and 10 for the non-pathogenic group. Pairs were identified in both NJ uncorrected p-distance and Poisson distance phylogenies and supported by a bootstrap greater than or equal to 70.

Pathogenic Non-Pathogenic

Sister Pair Name Sequences Sister Pair Name Sequences

Tula hantavirus Y13979 Y13980 Adler hantavirus KP013570 KP013577 Tula hantavirus EU439949 EU439950 Adler hantavirus KP013571 KP013572 Tula hantavirus AF063892 AF063897 Yuanjiang FJ170792 FJ170794 hantavirus Puumala hantavirus AJ888751 AJ888752 Ussuri hantavirus AB677476 AB677477 Puumala hantavirus AF367064 AF367071 Muju hantavirus JX028273 JX046487 Puumala hantavirus L08804 L11347 Muju hantavirus DQ138128 DQ138142 Puumala hantavirus JN657230 JN657231 Serang hantavirus AM998808 GQ274940 Puumala hantavirus JQ319161 JQ319163 Anjozorobe KC490916 KC490918 hantavirus Dobrava-Belgrade AJ009773 AJ009775 Seoul hantavirus AY750171 AY766368 hantavirus Dobrava-Belgrade GQ205404 GQ205406 Unknown HM756286 HQ589247 hantavirus hantavirus Dobrava-Belgrade AJ131672 AJ131673 hantavirus Sangassou JQ082300 JQ082303 hantavirus Thailand hantavirus AB186420 AM397664

Hantaan hantavirus HQ834500 HQ834504

Hantaan hantavirus EF990912 EF990913

Amur hantavirus AB127996 AF288649

Hantaan hantavirus EF990902 EF990903

Hantaan hantavirus AF288659 D25530

Puumala hantavirus AJ314598 AJ314599

Puumala hantavirus AY526219 U14137

Tula hantavirus AF164093 KT946591

Table 4. Nucleocapsid amino acids of CD8+ epitopes found in Hantaan hantavirus (HTNV), Sin Nombre hantavirus (SNV), and Puumala hantavirus (PUUV). The amino acid sequence and position if the N gene, the MHC allele, the virus species, and the published references are included.

Epitope No. Amino Acid Sequence MHC Allele Species Reference

1 12NAHEGQLVI20 HLA-B51 HTNV Van Epps et al. 1999

2 94SSLRYGNV101 H2-b class I SNV & PUUV Maeda et. al. 2004

3 129FVVPILLKA137 HLA-A2 HTNV Ma et. al. 2013

4 131LPIILKALY139 HLA-B35 HTNV Ennis et al. 1997

5 167DVNGIRKPK175 HLA-A33 HTNV Ma et. al. 2013

6 175SMPTAQSTM189 H2-b class I SNV & PUUV Maeda et. al. 2004

7 197RYRTAVCGL205 HLA-A11 HTNV Wang et. al. 2011

8 211SPVMGVIGF225 HLA-B35 PUUV Maeda et. al. 2004

9 217PVMGVIGFS231 H2-Kb SNV & PUUV Maeda et. al. 2004

10 234ERIDDFLAA242 HLA-C7 SNV Ennis et al., 1997

11 245KLLPDTAAV253 HLA-A2.3 HTNV Wang et. al. 2011

12 247LPDTAAVSL255 HLA-B35 HTNV Ma et. al. 2013

13 258GPATNRDYL266 HLA-B7 HTNV Wang et. al. 2011

14 277ETKESKAIR285 HLA-A33 HTNV Ma et. al. 2013

15 333ILQDMRNTI341 HLA-A2.1 HTNV Lee et. al. 2002

16 421ISNQEPLKL429 HLA-A1 HTNV Van Epps et al. 1999

Table 5. Mean numbers of synonymous substitutions per synonymous site (pS) and mean numbers of nonsynonymous substitutions per nonsynonymous sites (pN) and standard errors are shown for each epitope for the New World group.

New World Pathogenic Non-Pathogenic

pN pS pN pS Epitope 1 0.000 ±0.000 0.078 ±0.031a 0.000 ±0.000 0.020 ±0.020 Epitope 2 0.000 ±0.000 0.121 ±0.052 a 0.008 ±0.008 0.270 ±0.223 Epitope 3 0.000 ±0.000 0.272 ±0.133 0.000 ±0.000 0.207 ±0.119 Epitope 4 0.000 ±0.000 0.495 ±0.251 0.007 ±0.007 0.327 ±0.187 Epitope 5 0.005 ±0.005 0.306 ±0.077b 0.000 ±0.000 0.275 ±0.162 Epitope 6 0.000 ±0.000 0.253 ±0.079 b 0.000 ±0.000 0.164 ±0.098 Epitope 7 0.000 ±0.000 0.289 ±0.127a 0.000 ±0.000 0.198 ±0.133 Epitope 8 0.000 ±0.000 0.279 ±0.080 b 0.000 ±0.000 0.189 ±0.095 Epitope 9 0.000 ±0.000 0.270 ±0.090 b 0.000 ±0.000 0.141 ±0.076 Epitope 10 0.000 ±0.000 0.353 ±0.093 b 0.000 ±0.000 0.235 ±0.144 Epitope 11 0.012 ±0.012 0.673 ±0.295 a 0.000 ±0.000 0.426 ±0.191 a Epitope 12 0.031 ±0.031 0.259 ±0.119 0.000 ±0.000 0.364 ±0.212 Epitope 13 0.000 ±0.000 0.306 ±0.099b 0.000 ±0.000 0.044 ±0.044 Epitope 14 0.007 ±0.007 0.240 ±0.150 0.006 ±0.006 0.239 ±0.143 Epitope 15 0.005 ±0.005 0.256 ±0.080 b 0.000 ±0.000 0.123 ±0.081 Epitope 16 0.000 ±0.000 0.328 ±0.161 0.009 ±0.009 0.281 ±0.177 Remainder 0.001 ±0.000 0.216 ±0.064 b 0.004 ±0.002 0.302 ±0.155 a b p<0.05, p<0.01 test of hypothesis pS = pN

Table 6. Mean numbers of synonymous substitutions per synonymous site (pS) and mean numbers of nonsynonymous substitutions per nonsynonymous sites (pN) and standard errors are shown for each epitope for the Old World group.

Old World Pathogenic Non-Pathogenic

pN pS pN pS Epitope 1 0.000 ±0.000 0.072 ±0.045 0.005 ±0.005 0.232 ±0.185 Epitope 2 0.000 ±0.000 0.063 ±0.024a 0.000 ±0.000 0.151 ±0.107 Epitope 3 0.005 ±0.004 0.059 ±0.032 0.000 ±0.000 0.125 ±0.071 Epitope 4 0.003 ±0.003 0.062 ±0.030 0.000 ±0.000 0.081 ±0.037a Epitope 5 0.003 ±0.003 0.067 ±0.039 0.010 ±0.007 0.210 ±0.090a Epitope 6 0.000 ±0.000 0.154 ±0.072a 0.000 ±0.000 0.085 ±0.034a Epitope 7 0.002 ±0.002 0.123 ±0.060 0.000 ±0.000 0.170 ±0.070a Epitope 8 0.002 ±0.002 0.094 ±0.053 0.000 ±0.000 0.123 ±0.082 Epitope 9 0.005 ±0.003 0.100 ±0.058 0.000 ±0.000 0.395 ±0.219 Epitope 10 0.000 ±0.000 0.087 ±0.044 0.000 ±0.000 0.291 ±0.157 Epitope 11 0.010 ±0.007 0.051 ±0.028 0.026 ±0.026 0.173 ±0.077 Epitope 12 0.009 ±0.009 0.063 ±0.046 0.050 ±0.050 0.087 ±0.065 Epitope 13 0.004 ±0.004 0.084 ±0.051 0.000 ±0.000 0.076 ±0.051 Epitope 14 0.002 ±0.002 0.067 ±0.021b 0.006 ±0.006 0.216 ±0.097a Epitope 15 0.010 ±0.006 0.071 ±0.042 0.024 ±0.024 0.247 ±0.159 Epitope 16 0.000 ±0.000 0.066 ±0.051 0.000 ±0.000 0.180 ±0.110 Remainder 0.003 ±0.001 0.090 ±0.032b 0.002 ±0.001 0.113 ±0.040a a b p<0.05, p<0.01 test of hypothesis pS = pN

Figure 1. Phylogenetic tree of sister pairs of complete nucleocapsid protein sequences. Boot strap values shown were taken from the NJ Poisson phylogeny. Old World and New World groups are indicated, as well as hantavirus species pathogenic to humans (+).

Figure 2. Results from the Chi Square test of independence designed to test whether the distribution of the number of incidences where pS was greater than pN among each of the groups (ie, New World pathogenic, New World non-pathogenic, Old World pathogenic, and Old World non-pathogenic) differed.