Nucelotide Substitution Rates in Hantavirus CD8+ T-Cell Epitopes
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Winthrop University
Digital Commons @ Winthrop
University
Graduate Theses The Graduate School
12-2016
Nucelotide Substitution Rates in Hantavirus CD8+
T-Cell Epitopes
Brian Gerald Sikes
Winthrop University
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Recommended Citation
Sikes, Brian Gerald, "Nucelotide Substitution Rates in Hantavirus CD8+ T-Cell Epitopes" (2016). Graduate Theses. 41.
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bramed@winthrop.edu.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 SikesAbstract
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 non-
synonymous 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.
iiAcknowledgements
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.
iiiTABLE 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
ivLIST 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
vLIST 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
viINTRODUCTION
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 &
1Sironen 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
2non-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
3vesicle 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
4kidneys 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.
5Relatively 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
6very 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:
7 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
8(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
9synonymous 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.
10RESULTS 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
11group 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
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
13with 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
14Puumala 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
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
16the 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
17considerable 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.
18REFERENCES
Ali, H. et al. 2015. Complete genome of a Puumala virus strain from Central Europe.
Virus Genes 50 (2), p292-298.
Allen, T. et al. 2000. Tat-specific cytotoxic T lymphocytes select for SIV escape variants
during resolution of primary viraemia. Nature 407, p386-390.
Arai, S. et al. 2008. Molecular phylogeny of a newfound hantavirus in the Japanese shrew
mole (Urotrichus talpoides). Proceedings of the National Academy of Science U.S.A.
105 (42), p16296-16301.
Arikawa, J. et al. 1990. Coding properties of the S and the M genome segments of
Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal
syndrome. Virology 176 (1), p114-125.
Asikainen, K. rt al. 2000. Molecular evolution of Puumala hantavirus in Fennoscandia:
phylogenetic analysis of strains from two recolonization routes, Karelia and Denmark.
Journal of General Virology 81 (PT 12), p2833-2841.
Avsic-Zupanc, T. et al. Genetic and antigenic properties of Dobrava virus: a unique
member of the Hantavirus genus, family Bunyaviridae. Journal of General Virology 76
(Pt 11), p2801-2808.
Baek, L. et al. 2006. Soochong virus: an antigenically and genetically distinct hantavirus
isolated from Apodemus peninsulae in Korea. Journal of Medical Virology 78 (2), p290-
297.
Bagamian, K. et al. 2012. Transmission ecology of Sin Nombre hantavirus in naturally
infected North American deermouse populations in outdoor enclosures. PLoS ONE 7
(10), E47731.
Bennett, S. et al. 2014. Reconstructing the evolutionary origins and phylogeography of
hantaviruses. Trends in Microbiology 22 (8), p473-482.
Benson, D. et al. 2013. GenBank. Nucleic Acids Research 41, p36-42.
Bharadwaj, M. 1997. Rio Mamore virus: genetic characterization of a newly recognized
hantavirus of the pygmy rice rat, Oligoryzomys microtis, from Bolivia. American Journal
of Tropical Medicine and Hygiene 57 (3), p368-374.
Bi, Z. et al. 2008. Hantavirus infection: a review and global update. Journal of Infection
in Developing Countries 2 (1), p3-23.
Bohlman, M. et al. 2002. Analysis of hantavirus genetic diversity in Argentina: S
segment-derived phylogeny. Journal of Virology 76 (8), p3765-3773.
Bowen, M. et al. 1995. Genetic characterization of a human isolate of Puumala
hantavirus from France. Virus Research 38 (2-3), p279-289.
19Cao, Z. et al. 2010. Genetic analysis of a hantavirus strain carried by Niviventer
confucianus in Yunnan province, China. Virus Research 153 (1), p157-160.
Capron, A., Dessaint, J. 1989. Molecular basis of host-parasite relationships: towards the
definition of protective antigens. Immunological Reviews 112, p27-48.
Carvalho de Oliveira, R. et al. 2014. Rio Mamore virus and hantavirus pulmonary
syndrome, Brazil. Emerging Infectious Diseases 20 (9), p1568-1570.
Castel, G. et al. 2015. Complete genome and phylogeny of Puumala Hantavirus isolates
circulating in France. Viruses 7 (10), p5476-5488.
Chu,Y. et al. 2006. Phylogenetic and geographical relationships of hantavirus strains in
eastern and western Paraguay. American Journal of Tropical Medicine and Hygiene 75
(6), p1127-1134.
Chu, Y. et al. 2008. Genetic characterization and phylogeny of a hantavirus from Western
Mexico. Virus Research 131 (2), p180-188.
Cruz, C. et al. 2012. Novel strain of Andes virus associated with fatal human infection,
central Bolivia. Emerging Infectious Disease 18 (5), p750-757.
Dekonenko, A. et al. 2003. Genetic similarity of Puumala viruses found in Finland and
western Siberia and of the mitochondrial DNA of their rodent hosts suggests a common
evolutionary origin. Infection, Genetics, & Evolution 3 (4), p245-257.
Dzagurova, T. et al. 2012. Isolation of Sochi virus from a fatal case of hantavirus disease
with fulminant clinical course. Clinical Infectious Disease 54 (1), pE1-E4.
Elliott, R., Schmaljohn, R. 2013. Bunyaviridae. In Fields Virology 6th Ed. Lippincott
Williams & Wilkins. Philadelphia, p1244.
Ennis, F. et al. 1997. Hantavirus pulmonary syndrome: CD8+ and CD+4 cytotoxic T
lymphocytes to epitopes on Sin Nombre virus nucleocapsid protein isolated during acute
illness. Virology 238, p380-390.
Escutenaire, S. et al. Genetic characterization of Puumala hantavirus strains from
Belgium: evidence for a distinct phylogenetic lineage. Virus Research 74 (1-2), p1-15.
Essbauer, S. et al. 2006. A new Puumala hantavirus subtype in rodents associated with an
outbreak of Nephropathia epidemica in South-East Germany in 2004. Epidemiology &
Infection 134 (6), p1333-1344.
Ettinger, J. et al. 2012. Multiple synchronous outbreaks of Puumala virus, Germany,
2010. Emerging Infectious Disease 18 (9), p1461-1464.
Fang, L. et al. 2015. Reservoir host expansion of hantavirus, China. Emerging Infectious
Diseases 21 (1), p170-171.
20Felsenstein, J. 1985. Phylogenies and the comparative method. American Naturalist 125
(1), p1-15.
Figueiredo, L. et al. 2014. Hantaviruses and cardiopulmonary syndrome in South
America. Virus Research 187, p43-54.
Firth, C. 2012. Diversity and distribution of hantaviruses in South America. Journal of
Virology 86 (24), p13756-13766.
Fulhorst, C. et al. 2004. Maporal virus, a hantavirus associated with the fulvous pygmy
rice rat (Oligoryzomys fulvescens) in western Venezuela. Virus Research 104 (2), p139-
144.
Gonzalez Della Valle, M. 2002. Andes virus associated with hantavirus pulmonary
syndrome in northern Argentina and determination of the precise site of infection.
American Journal of Tropical Medicine and Hygiene 66 (6), p713-720.
Gu, S. et al. 2013. Complete genome sequence and molecular phylogeny of a newfound
hantavirus harbored by the Doucet's musk shrew (Crocidura douceti) in Guinea.
Infection, Genetics, & Evolution 20C, p118-123.
Gu, S. et al. 2014. Co-circulation of soricid- and talpid-borne hantaviruses in Poland.
Infection, Genetics & Evolution 28, p296-303.
Guterres, A. et al. 2013. Phylogenetic analysis of the S segment from Juquitiba
hantavirus: identification of two distinct lineages in Oligoryzomys nigripes. Infection,
Genetics & Evolution 18, p262-268.
Guterres, A. 2015. Detection of different South American hantaviruses. Virus Research
210, p106-113.
Hall, B. 2013. Building phylogenetic trees from molecular data with MEGA. Molecular
Biology and Evolution. Downloaded from http://mbe.oxfordjournals.org on March 13,
2013.
Heiske, A., et al. 1999. A new Clethrionomys-derived hantavirus from Germany:
evidence for distinct genetic sublineages of Puumala viruses in Western Europe. Virus
Research 61 (2), p101-112.
Hepojoki, J. et al. 2012. Hantavirus structure – molecular interactions behind the scene.
Journal of General Viriology 93, p1631-1644.
Hepojoki, J. et al. 2014. The fundamental role of endothelial cells in hantavirus
pathogenesis. Frontier Microbiology 5 Article 727.
Hjelle, B. et al. 1994. Genetic identification of a novel hantavirus of the harvest mouse
Reithrodontomys megalotis. Journal Virolology 68 (10), p6751-6754.
21Hjelle, B., 1995. Prevalence and geographic genetic variation of hantaviruses of New
World harvest mice (Reithrodontomys): identification of a divergent genotype from a
Costa Rican Reithrodontomys mexicanus. Virology 207 (2), p452-459.
Honeycutt, R., Faulde, M. Genetic evidence for Tula virus in Microtus arvalis and
Microtus agrestis populations in Croatia. Vector Borne Zoonotic Diseases 2 (1), p19-27.
Horling, J. et al. 1996. Khabarovsk virus: a phylogenetically and serologically distinct
hantavirus isolated from Microtus fortis trapped in far-east Russia. Journal of General
Virology 77 (Pt 4), p687-694.
Hugot, J. et al. 2006. Genetic analysis of Thailand hantavirus in Bandicota indica
trapped in Thailand. Virology Journal 3, p72.athogenesis. Frontiers in Microbiology 5,
Article 727.
Hughes, A. 1999. Adaptive Evolution of Genes and Genomes, Oxford University Press,
Oxford, UK.
Hughes, A. Friedman, R. 2000. Evolutionary diversification of protein-coding genes of
hantaviruses. Molecular Biology and Evolution 17 (10), p1558-1568.
International Committee on Taxonomy of Viruses. 2015. Virus Taxonomy: 2015 Release.
Downloaded from http://www.ictvonline.org/virustaxonomy.asp on November 2, 2016.
Isegawa, Y. et al. 1994. Association of serine in position 1124 of Hantaan virus
glycoprotein with virulence in mice. Journal of General Virology 75 (PT 11), p3273-
3278.
Jameson, L. et al 2013. Pet rats as a source of hantavirus in England and Wales, 2013.
Euro Surveillance 18 (9).
Jiang, J. et al. 2007. A new Hantaan-like virus in rodents (Apodemus peninsulae) from
Northeastern China. Virus Research 130 (1-2), p292-295.
Jin, M. et al. 2002. Hantaan virus enters cells by clathrin-dependent receptor-mediated
endocytosis. Virology 294, p60-69.
Johansson, P. et al. 2004. Complete gene sequence of a human Puumala hantavirus
isolate, Puumala Umea/hu: sequence comparison and characterization of encoded gene
products. Virus Research 105 (2), p147-155.
Johansson, P. et al. 2010. Molecular characterization of two hantavirus strains from
different Rattus species in Singapore. Virology Journal 7, p15.
Johnson, A. et al. 1997. Laguna Negra virus associated with HPS in western Paraguay
and Bolivia. Virology 238 (1), p115-127.
22Jonsson, C. et al. 2008. Treatment of hantavirus pulmonary syndrome. Antiviral Research
78 (1), p162.
Jonsson, C. et al. 2010. A global perspective on hantavirus ecology, epidemiology, and
disease. Clinical Microbiology Reviews 23 (2), p412-441.
Kallio, E. et al. 2006. Prolonged survival of Puumala hantavirus outside the host:
evidence for indirect transmission via the environment. Journal of General Virology 87,
p2127-2134.
Kang, H. et al. 2010. Novel Hantavirus in the Flat-Skulled Shrew (Sorex roboratus)
Vector Borne Zoonotic Diseases 10 (6), p593-597.
Kang, H. et al. 2011. Shared ancestry between a Newfound mole-borne hantavirus and
hantaviruses harbored by Cricetid rodents. Journal of Virology 85 (15), p7496-7503.
Kang, H. et al. 2014. Expanded host diversity and geographic distribution of hantaviruses
in sub-Saharan Africa. Journal of Virology 88 (13), p7663-7667.
Kang, Y. et al. 2012. Dynamics of hantavirus infections in humans and animals in Wuhan
city, Hubei, China. Infections, Genetics & Evolution 12 (8), p1614-1621.
Kariwa, H. et al. 1999. Genetic diversities of hantaviruses among rodents in Hokkaido,
Japan and Far East Russia. Virus Research 59 (2), p219-228.
Kariwa, H. et al. 2012. Isolation and characterization of hantaviruses in far East Russia
and etiology of hemorrhagic Fever with renal syndrome in the region. American Journal
of Tropical Medicine & Hygiene 86 (3), p545-553.
Kaukinen, P. et al. 2005. Hantavirus nucleocapsid protein: a multifunctional molecule
with both housekeeping and ambassadorial duties. Archives of Virology 150 (9), p1693-
1713.
Kilpatrick, E. et al. 2004. Role of specific CD8+ T cells in the severity of a fulminant
zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome. Journal of
Immunology 172, p3297.
Kirsanovs, S. et al. 2010. Genetic reassortment between high-virulent and low-virulent
Dobrava-Belgrade virus strains. Virus Genes 41 (3), p319-328.
Klempa, B. et al. 2003. Occurrence of renal and pulmonary syndrome in a region of
northeast Germany where Tula hantavirus circulates. Journal of Clinical Microbiology 41
(10), p4894-4897.
Klempa, B. et al. 2004. First molecular identification of human Dobrava virus infection
in central Europe. Journal of Clinical Microbiology 42 (3), p1322-1325.
23Klempa, B. et al. 2005. Central European Dobrava Hantavirus isolate from a striped field
mouse (Apodemus agrarius). Journal of Clinical Microbiolology. 43 (6), p2756-2763.
Klempa, B. et al. 2008. Hemorrhagic fever with renal syndrome caused by 2 lineages of
Dobrava hantavirus, Russia. Emerging Infectious Diseases 14 (4), p617-625.
Klempa, B. et al. 2012. Sangassou virus, the first hantavirus isolate from Africa, displays
genetic and functional properties distinct from those of other murinae-associated
hantaviruses. Journal Virology 86 (7), p3819-3827.
Koma, T. et al. 2012. Development of a serotyping enzyme-linked immunosorbent assay
system based on recombinant truncated hantavirus nucleocapsid proteins for New World
hantavirus infection. J. Virol. Methods 185 (1), 74-81 (2012)
Kruger, D. 2015. Life-Threatening Sochi Virus Infections, Russia. Emerging Infectious
Diseases 21 (12), p2204-2208.
Kruger, D. et al. 2015. Hantaviruses – globally emerging pathogens. Journal of Clinical
Virology 64, p128-136.
Laenen, L. et al. 2015. Complete Genome Sequence of Nova Virus, a Hantavirus
Circulating in the European Mole in Belgium. Genome Announcement 3 (4), e00770-15.
Lee, H. et al. 1978. Isolation of the etiologic agent of Korean hemorrhagic fever. Journal
of Infectious Dieases 137, p298-308.
Lee, H. et al. 2014. Discovery of hantaviruses and of the hantavirus genus: personal and
historical perspectives of the presidents of the International Society of Hantaviruses.
Virus Research 187, p2-5.
Lee, J. et al. 2014. Whole-genome sequence of Muju virus, an arvicolid rodent-borne
Hantavirus. Genome Announcement 2 (1), e00099-14.
Lee, K. 2002. Characterization of HLA-A2.1 restricted epitopes, conserved in both
Hantaan and Sin Nombre viruses, in Hantaan virus-infected patients. Journal of General
Virology 83, p1131-1136.
Lee, S. et al.1997. Nucleotide sequence of nucleocapsid protein (N) of Hantaan virus
isolated from a Korean hemorrhagic fever patient. DNA Sequence 7 (6), p349-352.
Levine, J. et al. 2010. Antagonism of Type I interferon responses by new world
hantaviruses. Journal of Virology 84 (22), p11790-11801.
Lin, X. et al. 2012. Cross-species transmission in the speciation of the currently known
murinae-associated hantavirus. Journal of Virology 86 (20), p11171.
Lin, X. et al. 2014. Biodiversity and evolution of Imjin virus and Thottapalayam virus in
Crocidurinae shrews in Zhejiang Province, China. Virus Research 189C, p114-120.
24Lokugamage, K. et al. 2004. Genetic and antigenic characterization of the Amur virus
associated with hemorrhagic fever with renal syndrome. Virus Research 101 (2), p127-
134.
Lopez, N. et al. 1997. Genetic characterization and phylogeny of Andes virus and
variants from Argentina and Chile. Virus Research 50 (1), p77-84.
Lundkvist, A. et al. 1997. Cell culture adaptation of Puumala hantavirus changes the
infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of
mutants with altered genomic RNA S segment. Journal Virology 71 (12), p9515-9523.
Ma, C. et al. 2012. Hantaviruses in rodents and humans, Xi'an, PR China. Journal of
General Virology 93 (PT 10), p2227-2236.
Ma, Y. et al. 2013. HLA-A2 and B35 restricted Hantaan virus nucleoprotein CD+8 T-
cell epitope specific immune response correlates with milder disease in hemorrhagic
fever with renal syndrome. PLOS Neglected Tropical Diseases 7 (2), Article e20176.
Maeda, K. et al. 2004. Identification and analysis for cross-reactivity among hantaviruses
of H-2b-restricted CTL epitopes in Sin Nombre virus nucleocapsid protein. Journal of
General Virology 85, p1909-1919.
Mackow, E., Gavrilovskaya, I. 2009. Hantavirus regulation of endothelial cell functions.
Thrombosis & Haemostasis 102, p1030.
Matheus, S. et al. 2012. Complete genome sequence of a novel hantavirus variant of Rio
Mamore virus, Maripa virus, from French Guiana. Journal of Virology 86 (9), p5399.
Melo-Silva, C. et al. 2009. Characterization of hantaviruses circulating in Central Brazil
Infection, Genetics & Evolution 9 (2), p241-247.
Milazzo, M. et al. 2006. Catacamas virus, a hantaviral species naturally associated with
Oryzomys couesi (Coues' oryzomys) in Honduras. American Journal of Tropical
Medicine & Hygiene 75 (5), p1003-1010.
Miles, R. et al. 2016. Complete genome sequence of Seoul virus strain Tchoupitoulas
Genome Announcement 4 (3).
Montoya-Ruiz,C. et al. 2015. Phylogenetic relationship of Necocli Virus to other South
American hantaviruses (Bunyaviridae: Hantavirus). Vector Borne Zoonotic Disease 15
(7), p438-445.
Morzunov, S. et al. 1995. A newly recognized virus associated with a fatal case of
hantavirus pulmonary syndrome in Louisiana. Journal of Virology 69 (3), p1980-1983.
Muyangwa, M. et al. 2015. Hantaviral proteins: structure, functions, and role in
hantavirus infection. Frontiers in Microbiology 6, Article 1326.
25Nei, M., Gojobori, T. 1986. Simple methods for estimating the numbers of synonymous
and nonsynonymous nucleotide substitutions. Molecular Biology and Evolution 3 (5),
p418-426.
Nemirov, K. et al. 1999. Isolation and characterization of Dobrava hantavirus carried by
the striped field mouse (Apodemus agrarius) in Estonia. Journal of General Virology 80
(PT 2), p371-379.
Nemirov, K. et al. 2003. Genetic characterization of new Dobrava hantavirus isolate
from Greece. Journal of Medical Virology 69 (3), p408-416.
Nemirov, K. et al. 2004. Saaremaa hantavirus in Denmark. Journal of Clinical Virology
30 (3), p254-257.
Nemirov, K. et al. 2010. Puumala hantavirus and Myodes glareolus in northern Europe:
no evidence of co-divergence between genetic lineages of virus and host. Journal of
General Virology 91 (PT 5), p1262-1274.
Nichol, S. et al. 1993. Genetic identification of a hantavirus associated with an outbreak
of acute respiratory illness. Science 262, p914-917.
O’Connor, D. et al. 2004. A dominant role for CD8+ T lymphocyte selection in simian
immunodeficiency virus sequence variation. Journal of Virology 78 (24), p14012-14022.
Padula, P. et al. 1998.Hantavirus pulmonary syndrome outbreak in Argentina: molecular
evidence for person-to-person transmission of Andes virus. Virology 241, p323-330.
Parrington, M., Kang, C. 1990. Nucleotide sequence analysis of the S genomic segment
of Prospect Hill virus: comparison with the prototype Hantavirus. Virology 175 (1),
p167-175.
Pattamadilok, S. et al. 2006. Geographical distribution of hantaviruses in Thailand and
potential human health significance of Thailand virus. American Journal of Tropical
Medicine & Hygiene 75 (5), p994-1002.
Piotkivska, H., Hughes, A. 2004. Between-host evolution of cytotoxic T-lymphocyte
epitopes in human immunodeficiency virus type 1: an approach based on
phylogenetically independent comparisons. Journal of Virology 78 (24), p11758-11765.
Piotkivska, H., Hughes, A. 2006. Patterns of sequence evolution at epitopes for host
antibodies and cytotoxic T-lymphocytes in human immunodeficiency virus type 1. Virus
Research 116, p98-105.
Plyusnin, A. et al. 1994. Sequences of wild Puumala virus genes show a correlation of
genetic variation with geographic origin of the strains. Journal of General Virology 75
(PT 2), p405-409.
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