Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, Massachusetts, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein, which was demonstrated to have fusion activity, and also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated as VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined.
Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that>95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.
Host-range shifts in influenza virus are a major risk factor for pandemics. A key question in the study of emerging zoonoses is how the evolution of transmission efficiency interacts with heterogeneity in contact patterns in the new host species, as this interplay influences disease dynamics and prospects for control. Here we use a synergistic mixture of models and data to tease apart the evolutionary and demographic processes controlling a host-range shift in equine H3N8-derived canine influenza virus (CIV). CIV has experienced 15 years of continuous transfer among dogs in the United States, but maintains a patchy distribution, characterized by sporadic short-lived outbreaks coupled with endemic hotspots in large animal shelters. We show that CIV has a high reproductive potential in these facilities (mean R(0)?=?3.9) and that these hotspots act as refugia from the sparsely connected majority of the dog population. Intriguingly, CIV has evolved a transmission efficiency that closely matches the minimum required to persist in these refugia, leaving it poised on the extinction/invasion threshold of the host contact network. Corresponding phylogenetic analyses show strong geographic clustering in three US regions, and that the effective reproductive number of the virus (R(e)) in the general dog population is close to 1.0. Our results highlight the critical role of host contact structure in CIV dynamics, and show how host contact networks could shape the evolution of pathogen transmission efficiency. Importantly, efficient control measures could eradicate the virus, in turn minimizing the risk of future sustained transmission among companion dogs that could represent a potential new axis to the human-animal interface for influenza.
European brown hare syndrome virus (EBHSV) is the aetiological agent of European brown hare syndrome (EBHS), a disease affecting Lepus europaeus and Lepus timidus first diagnosed in Sweden in 1980. To characterize EBHSV evolution we studied hare samples collected in Sweden between 1982 and 2008. Our molecular clock dating is compatible with EBHSV emergence in the 1970s. Phylogenetic analysis revealed two lineages: Group A persisted until 1989 when it apparently suffered extinction; Group B emerged in the mid-1980s and contains the most recent strains. Antigenic differences exist between groups, with loss of reactivity of some MAbs over time, which are associated with amino acid substitutions in recognized epitopes. A role for immune selection is also supported by the presence of positively selected codons in exposed regions of the capsid. Hence, EBHSV evolution is characterized by replacement of Group A by Group B viruses, suggesting that the latter possess a selective advantage.
The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution.
The capacity of influenza A viruses to cross species barriers presents a continual threat to human and animal health. Knowledge of the human-swine interface is particularly important for understanding how viruses with pandemic potential evolve in swine hosts. We sequenced the genomes of 141 influenza viruses collected from North American swine during 2002 to 2011 and identified a swine virus that possessed all eight genome segments of human seasonal A/H3N2 virus origin. A molecular clock analysis indicates that this virus--A/sw/Saskatchewan/02903/2009(H3N2)--has likely circulated undetected in swine for at least 7 years. For historical context, we performed a comprehensive phylogenetic analysis of an additional 1,404 whole-genome sequences from swine influenza A viruses collected globally during 1931 to 2013. Human-to-swine transmission occurred frequently over this time period, with 20 discrete introductions of human seasonal influenza A viruses showing sustained onward transmission in swine for at least 1 year since 1965. Notably, human-origin hemagglutinin (H1 and H3) and neuraminidase (particularly N2) segments were detected in swine at a much higher rate than the six internal gene segments, suggesting an association between the acquisition of swine-origin internal genes via reassortment and the adaptation of human influenza viruses to new swine hosts. Further understanding of the fitness constraints on the adaptation of human viruses to swine, and vice versa, at a genomic level is central to understanding the complex multihost ecology of influenza and the disease threats that swine and humans pose to each other.
Bacteria of the order Rickettsiales (Alphaproteobacteria) are obligate intracellular parasites that infect species from virtually every major eukaryotic lineage. Several rickettsial genera harbor species that are significant emerging and re-emerging pathogens of humans. As species of Rickettsiales are associated with an extremely diverse host range, a better understanding of the historical associations between these bacteria and their hosts will provide important information on their evolutionary trajectories and, particularly, their potential emergence as pathogens.
Hemorrhagic fever with renal syndrome (HFRS) was first recognized in far eastern Asia in the 1930s, and has been highly prevalent in this region ever since. To reveal the molecular epidemiology of hantaviruses in this region, a total of 374 small mammals (eight species of rodents and one species of shrew) were captured in the Chinese part of the Bolshoy Ussuriysky Island (Heilongjiang Province). Hantavirus sequences were recovered from three striped field mice (Apodemus agrarius), 11 Maximowicz's voles (Microtus maximowiczii), and one flat-skulled shrew (Sorex roboratus). Genetic and phylogenetic analysis revealed the presence of three viruses: Hantaan virus (HTNV), Khabarovsk virus (KHAV), and Kenkeme virus (KKMV). HTNV sequences recovered from A. agrarius were closely related to those identified in Apodemus mice from the surrounding areas, while a new lineage of KHAV was present in M. maximowiczii. Additionally, while the viral sequences recovered from one flat-skulled shrew were most closely related to KKMV, their divergence to the prototype strain suggests that they represent a new viral subtype. Overall, these results suggest that Bolshoy Ussuriysky Island harbors considerable hantavirus diversity.
Time-scales of viral evolution and emergence have been studied widely, but are often poorly understood. Molecular analyses of viral evolutionary time-scales generally rely on estimates of rates of nucleotide substitution, which vary by several orders of magnitude depending on the timeframe of measurement. We analysed data from all major groups of viruses and found a strong negative relationship between estimates of nucleotide substitution rate and evolutionary timescale. Strikingly, this relationship was upheld both within and among diverse groups of viruses. A detailed case study of primate lentiviruses revealed that the combined effects of sequence saturation and purifying selection can explain this time-dependent pattern of rate variation. Therefore, our analyses show that studies of evolutionary time-scales in viruses require a reconsideration of substitution rates as a dynamic, rather than as a static, feature of molecular evolution. Improved modelling of viral evolutionary rates has the potential to change our understanding of virus origins.
Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus--Jingmen tick virus (JMTV)--that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.
Virions vary in size by at least 4 orders of magnitude, yet the evolutionary forces responsible for this enormous diversity are unknown. We document a significant allometric relationship, with an exponent of approximately 1.5, between the genome length and virion volume of viruses and find that this relationship is not due to geometric constraints. Notably, this allometric relationship holds regardless of genomic nucleic acid, genome structure, or type of virion architecture and therefore represents a powerful scaling law. In contrast, no such relationship is observed at the scale of individual genes. Similarly, after adjusting for genome length, no association is observed between virion volume and the number of proteins, ruling out protein number as the explanation for the relationship between genome and virion sizes. Such a fundamental allometric relationship not only sheds light on the constraints to virus evolution, in that increases in virion size but not necessarily structure are associated with concomitant increases in genome size, but also implies that virion sizes in nature can be broadly predicted from genome sequence data alone.
Early methods for estimating divergence times from gene sequence data relied on the assumption of a molecular clock. More sophisticated methods were created to model rate variation and used auto-correlation of rates, local clocks, or the so called "uncorrelated relaxed clock" where substitution rates are assumed to be drawn from a parametric distribution. In the case of Bayesian inference methods the impact of the prior on branching times is not clearly understood, and if the amount of data is limited the posterior could be strongly influenced by the prior.
The recent discovery of numerous hantaviruses in insectivores has provided a new view of hantavirus biodiversity and evolution. To determine the presence and genetic diversity of Imjin virus (MJNV) and Thottapalayam virus (TPMV) in insectivores in Zhejiang Province, China, we captured and performed virus screening of 32 Ussuri white-toothed shrews (Crocidura lasiura) and 105 Asian house shrews (Suncus murinus) in different coastal regions. Hantavirus genome (S, M, and L segments) sequences were successfully recovered from one Ussuri white-toothed shrew and seven Asian house shrews. Phylogenetic analysis revealed that the virus carried by the Ussuri white-toothed shrew was most closely related to MJNV, but with >15% nucleotide sequence difference, suggesting that it represents a new subtype. The hantaviruses carried by Asian house shrews were closely related to the TPMV variants found in the same geographic area, but more distantly related to those sampled in India and Nepal. Additionally, the TPMV sequences obtained in this study, as well as those found previously in this area, could be divided into three lineages reflecting their geographic origins, indicative of largely allopatric evolution. Overall, our data highlights the high genetic diversity of insectivore-borne hantaviruses in China, suggesting that more may be discovered in the future.
Norovirus is the leading cause of acute gastroenteritis with most infections caused by GII.4 variants. To understand the evolutionary processes that contribute to the emergence of GII.4 variants, we examined the molecular epidemiology of norovirus-associated acute gastroenteritis in Australia and New Zealand from 893 outbreaks between 2009 and 2012. Throughout the study GII.4 New Orleans 2009 was predominant; however, during 2012 it was replaced by an emergent GII.4 variant, Sydney 2012. An evolutionary analysis of capsid gene sequences was performed to determine the origins and selective pressures driving the emergence of these recently circulating GII.4 variants. This revealed that both New Orleans 2009 and Sydney 2012 share a common ancestor with GII.4 Apeldoorn 2007. Furthermore, pre-epidemic ancestral variants of each virus were identified up to two years before their pandemic emergence. Adaptive changes at known blockade epitopes in the viral capsid were also identified that likely contributed to their emergence.
Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown.
Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low-pathogenic (LPAI) progenitor viruses currently is poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultradeep-sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high- and low-pathogenicity viruses undertaken to date and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties.
Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic.
Our understanding of the global ecology of influenza viruses is impeded by historically low levels of viral surveillance in Africa. Increased genetic sequencing of African A/H1N1 pandemic influenza viruses during 2009-2013 revealed multiyear persistence of 2 viral lineages within West Africa, raising questions about the roles of reduced air traffic and the asynchrony of seasonal influenza epidemics among West African countries in the evolution of independent lineages. The potential for novel influenza virus lineages to evolve within Africa warrants intensified influenza surveillance in Africa and other understudied areas.
Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11?482 nt (ABTV) and 11?876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.
In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.
Hemorrhagic fevers (HF) caused by viruses and bacteria are a major public health problem in China and characterized by variable clinical manifestations, such that it is often difficult to achieve accurate diagnosis and treatment. The causes of HF in 85 patients admitted to Dandong hospital, China, between 2011-2012 were determined by serological and PCR tests. Of these, 34 patients were diagnosed with Huaiyangshan hemorrhagic fever (HYSHF), 34 with Hemorrhagic Fever with Renal Syndrome (HFRS), one with murine typhus, and one with scrub typhus. Etiologic agents could not be determined in the 15 remaining patients. Phylogenetic analyses of recovered bacterial and viral sequences revealed that the causative infectious agents were closely related to those described in other geographical regions. As these diseases have no distinctive clinical features in their early stage, only 13 patients were initially accurately diagnosed. The distinctive clinical features of HFRS and HYSHF developed during disease progression. Enlarged lymph nodes, cough, sputum, and diarrhea were more common in HYSHF patients, while more HFRS cases presented with headache, sore throat, oliguria, percussion pain kidney area, and petechiae. Additionally, HYSHF patients displayed significantly lower levels of white blood cells (WBC), higher levels of creations kinase (CK) and alanine aminotransferase (ALT), while HFRS patients presented with an elevation of blood urea nitrogen (BUN) and creatinine (CREA). These clinical features will assist in the accurate diagnosis of both HYSHF and HFRS. Overall, our data reveal the complexity of pathogens causing HFs in a single Chinese hospital, and highlight the need for accurate early diagnosis and a better understanding of their distinctive clinical features.
Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.
Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes a debilitating disease of cattle in Africa, Asia and Australia. However, its global geodynamics are poorly understood. An evolutionary analysis of G gene (envelope glycoprotein) ectodomain sequences of 97 BEFV isolates collected from Australia during 1956-2012 revealed that all have a single common ancestor and are phylogenetically distinct from BEFV sampled in other geographical regions. The age of the Australian clade is estimated as between 56-65 years, suggesting that BEFV has entered the continent on few occasions since it was first reported in 1936, and that the 1955/56 epizootic was the source of all currently circulating viruses. Notably, the Australian clade has evolved as a single genetic lineage across the continent, and at a high evolutionary rate of ?10(-3) nucleotide substitutions/site/year. Screening of 66 isolates using monoclonal antibodies indicated that neutralizing antigenic sites G1, G2 and G4 have been relatively stable, although variations in site G3a/b defined four antigenic sub-types. A shift in an epitope at site G3a, which occurred in the mid-1970s, was strongly associated with a K218R substitution. Similarly, a shift at site G3b was associated primarily with substitutions at residues 215, 220 and 223 which map to the tip of the spike on the pre-fusion form of the G protein. Finally, we propose that positive selection on residue 215 was likely due to cross-reacting neutralising antibody to Kimberley virus which has a similar geographic distribution and ecology to BEFV in Australia but has not been associated with disease.
Longquan City, Zhejiang province, China, has been seriously affected by hemorrhagic fever with renal syndrome (HFRS) since the first cases were registered in 1974. To understand the epidemiology and emergence of HFRS in Longquan, which may be indicative of large parts of rural China, we studied long-term incidence patterns and performed a molecular epidemiological investigation of the causative hantaviruses in human and rodent populations.
Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.
The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.
Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae.
The epidemiological and evolutionary dynamics of the two cocirculating lineages of influenza B virus, Victoria and Yamagata, are poorly understood, especially in tropical or subtropical areas of Southeast Asia. We performed a phylogenetic analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of influenza B viruses isolated in Guangzhou, a southern Chinese city, during 2009 to 2010 and compared the demographic and clinical features of infected patients. We identified multiple viral introductions of Victoria strains from both Chinese and international sources, which formed two phylogenetically and antigenically distinct clades (Victoria 1 and 2), some of which persisted between seasons. We identified one dominant Yamagata introduction from outside China during 2009. Our phylogenetic analysis reveals the occurrence of reassortment events among the Victoria and Yamagata lineages and also within the Victoria lineage. We found no significant difference in clinical severity by influenza B lineage, with the exceptions that (i) the Yamagata lineage infected older people than either Victoria lineage and (ii) fewer upper respiratory tract infections were caused by the Victoria 2 than the Victoria 1 clade. Overall, our study reveals the complex epidemiological dynamics of different influenza B lineages within a single geographic locality and has implications for vaccination policy in southern China.
The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) that first appeared in Saudi Arabia during the summer of 2012 has to date (20th September 2013) caused 58 human deaths. MERS-CoV utilizes the dipeptidyl peptidase 4 (DPP4) host cell receptor, and analysis of the long-term interaction between virus and receptor provides key information on the evolutionary events that lead to the viral emergence.
Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.
Rabbit Haemorrhagic Disease Virus (RHDV) was introduced into Australia in 1995 as a biological control agent against the wild European rabbit (Oryctolagus cuniculus). We evaluated its evolution over a 16-year period (1995-2011) by examining 50 isolates collected throughout Australia, as well as the original inoculum strains. Phylogenetic analysis of capsid protein VP60 sequences of the Australian isolates, compared with those sampled globally, revealed that they form a monophyletic group with the inoculum strains (CAPM V-351 and RHDV351INOC). Strikingly, despite more than 3000 rereleases of RHDV351INOC since 1995, only a single viral lineage has sustained its transmission in the long-term, indicative of a major competitive advantage. In addition, we find evidence for widespread viral gene flow, in which multiple lineages entered individual geographic locations, resulting in a marked turnover of viral lineages with time, as well as a continual increase in viral genetic diversity. The rate of RHDV evolution recorded in Australia -4.0 (3.3-4.7) × 10(-3) nucleotide substitutions per site per year - was higher than previously observed in RHDV, and evidence for adaptive evolution was obtained at two VP60 residues. Finally, more intensive study of a single rabbit population (Turretfield) in South Australia provided no evidence for viral persistence between outbreaks, with genetic diversity instead generated by continual strain importation.
Mycobacterium tuberculosis (M.tb), the cause of tuberculosis (TB), is estimated to infect a new host every second. While analyses of genetic data from natural populations of M.tb have emphasized the role of genetic drift in shaping patterns of diversity, the influence of natural selection on this successful pathogen is less well understood. We investigated the effects of natural selection on patterns of diversity in 63 globally extant genomes of M.tb and related pathogenic mycobacteria. We found evidence of strong purifying selection, with an estimated genome-wide selection coefficient equal to -9.5 × 10(-4) (95% CI -1.1 × 10(-3) to -6.8 × 10(-4)); this is several orders of magnitude higher than recent estimates for eukaryotic and prokaryotic organisms. We also identified different patterns of variation across categories of gene function. Genes involved in transport and metabolism of inorganic ions exhibited very low levels of non-synonymous polymorphism, equivalent to categories under strong purifying selection (essential and translation-associated genes). The highest levels of non-synonymous variation were seen in a group of transporter genes, likely due to either diversifying selection or local selective sweeps. In addition to selection, we identified other important influences on M.tb genetic diversity, such as a 25-fold expansion of global M.tb populations coincident with explosive growth in human populations (estimated timing 1684 C.E., 95% CI 1620-1713 C.E.). These results emphasize the parallel demographic histories of this obligate pathogen and its human host, and suggest that the dominant effect of selection on M.tb is removal of novel variants, with exceptions in an interesting group of genes involved in transportation and defense. We speculate that the hostile environment within a host imposes strict demands on M.tb physiology, and thus a substantial fitness cost for most new mutations. In this respect, obligate bacterial pathogens may differ from other host-associated microbes such as symbionts.
In 2009 to 2010, there was a marked increase in the number of infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in China. Through phylogenetic analysis, we show that viruses from this outbreak originated from a single recombination event, illustrating the potential importance of this process for disease emergence.
The emergence and spread of Type 2 Porcine Reproductive and Respiratory Syndrome virus (Type 2 PRRSV) in North America is heavily influenced by the multiple site production system used in the hog industry. However, it is unclear how anthropogenic factors such has this have shaped the current spatial distribution of PRRSV genotypes. We employed Bayesian phylogeographic analyses of 7040 ORF5 sequences to reveal the recent geographical spread of Type 2 PRRSV in North America. The directions and intensities in our inferred virus traffic network closely mirror the hog transportation. Most notably, we reveal multiple viral introductions from Canada into the United States causing a major shift in virus genetic composition in the Midwest USA that went unnoticed by the regular surveillance and field epidemiological studies. Overall, these findings provide important insights into the dynamics of Type 2 PRRSV evolution and spread that will facilitate programs for control and prevention.
Flanders virus (FLAV) and Hart Park virus (HPV) are rhabdoviruses that circulate in mosquito-bird cycles in the eastern and western United States, respectively, and constitute the only two North American representatives of the Hart Park serogroup. Previously, it was suggested that FLAV is unique among the rhabdoviruses in that it contains two pseudogenes located between the P and M genes, while the cognate sequence for HPV has been lacking. Herein, we demonstrate that FLAV and HPV do not contain pseudogenes in this region, but encode three small functional proteins designated as U1-U3 that apparently arose by gene duplication. To further investigate the U1-U3 region, we conducted the first large-scale evolutionary analysis of a member of the Hart Park serogroup by analyzing over 100 spatially and temporally distinct FLAV isolates. Our phylogeographic analysis demonstrates that although FLAV appears to be slowly evolving, phylogenetically divergent lineages co-circulate sympatrically.
Gene overlapping is widely employed by RNA viruses to generate genetic novelty while retaining a small genome size. However, gene overlapping also increases the deleterious effect of mutations as they affect more than one gene, thereby reducing the evolutionary rate of RNA viruses and hence their adaptive capacity. Although there is general agreement on the benefits of gene overlapping as a mechanism of genomic compression for rapidly evolving organisms, its effect on the pace of RNA virus evolution remains a source of debate. To address this issue, we collected sequence data from 117 instances of gene overlapping across 19 families, 30 genera, and 55 species of RNA viruses. On these data, we analyzed how genetic distances, selective pressures, and the distribution of RNA secondary structures and conserved protein functional domains vary between overlapping (OV) and nonoverlapping (NOV) regions. We show that gene overlapping generally results in a decrease in the rate of RNA virus evolution through a reduction in the frequency of synonymous mutations. However, this effect is less pronounced in genes with a terminal rather than an internal gene overlap, which might result from a greater proportion of protein functional conserved domains in NOV than in OV regions, in turn reducing the number of nonsynonymous mutations in the former. Overall, our analyses clarify the role of gene overlapping as a modulator of the evolutionary rates exhibited by RNA viruses and shed light on the factors that shape the genetic diversity of this important group of pathogens.
Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.
Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.
Although there are over 1,150 bat species worldwide, the diversity of viruses harbored by bats has only recently come into focus as a result of expanded wildlife surveillance. Such surveys are of importance in determining the potential for novel viruses to emerge in humans, and for optimal management of bats and their habitats. To enhance our knowledge of the viral diversity present in bats, we initially surveyed 415 sera from African and Central American bats. Unbiased high-throughput sequencing revealed the presence of a highly diverse group of bat-derived viruses related to hepaciviruses and pegiviruses within the family Flaviridae. Subsequent PCR screening of 1,258 bat specimens collected worldwide indicated the presence of these viruses also in North America and Asia. A total of 83 bat-derived viruses were identified, representing an infection rate of nearly 5%. Evolutionary analyses revealed that all known hepaciviruses and pegiviruses, including those previously documented in humans and other primates, fall within the phylogenetic diversity of the bat-derived viruses described here. The prevalence, unprecedented viral biodiversity, phylogenetic divergence, and worldwide distribution of the bat-derived viruses suggest that bats are a major and ancient natural reservoir for both hepaciviruses and pegiviruses and provide insights into the evolutionary history of hepatitis C virus and the human GB viruses.
Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease.
Gene duplication generates genetic novelty and redundancy and is a major mechanism of evolutionary change in bacteria and eukaryotes. To date, however, gene duplication has been reported only rarely in RNA viruses. Using a conservative BLAST approach we systematically screened for the presence of duplicated (i.e., paralogous) proteins in all RNA viruses for which full genome sequences are publicly available. Strikingly, we found only nine significantly supported cases of gene duplication, two of which are newly described here--in the 25 and 26 kDa proteins of Beet necrotic yellow vein virus (genus Benyvirus) and in the U1 and U2 proteins of Wongabel virus (family Rhabdoviridae). Hence, gene duplication has occurred at a far lower frequency in the recent evolutionary history of RNA viruses than in other organisms. Although the rapidity of RNA virus evolution means that older gene duplication events will be difficult to detect through sequence-based analyses alone, it is likely that specific features of RNA virus biology, and particularly intrinsic constraints on genome size, reduce the likelihood of the fixation and maintenance of duplicated genes.
Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (?ERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized ?ERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses.
Intra-host sequence data from RNA viruses have revealed the ubiquity of defective viruses in natural viral populations, sometimes at surprisingly high frequency. Although defective viruses have long been known to laboratory virologists, their relevance in clinical and epidemiological settings has not been established. The discovery of long-term transmission of a defective lineage of dengue virus type 1 (DENV-1) in Myanmar, first seen in 2001, raised important questions about the emergence of transmissible defective viruses and their role in viral epidemiology. By combining phylogenetic analyses and dynamical modeling, we investigate how evolutionary and ecological processes at the intra-host and inter-host scales shaped the emergence and spread of the defective DENV-1 lineage. We show that this lineage of defective viruses emerged between June 1998 and February 2001, and that the defective virus was transmitted primarily through co-transmission with the functional virus to uninfected individuals. We provide evidence that, surprisingly, this co-transmission route has a higher transmission potential than transmission of functional dengue viruses alone. Consequently, we predict that the defective lineage should increase overall incidence of dengue infection, which could account for the historically high dengue incidence reported in Myanmar in 2001-2002. Our results show the unappreciated potential for defective viruses to impact the epidemiology of human pathogens, possibly by modifying the virulence-transmissibility trade-off, or to emerge as circulating infections in their own right. They also demonstrate that interactions between viral variants, such as complementation, can open new pathways to viral emergence.
Fox rabies re-emerged in north-eastern Italy at the end of 2008 and circulated until early 2011. As with previous rabies epidemics, the Italian cases were linked to the epidemiological situation in adjacent regions. To obtain a comprehensive picture of the dynamics of the recent Italian epidemic, we performed a detailed evolutionary analysis of RABVs circulating in north-eastern Italy. Sequences were obtained for the hyper-variable region of the nucleoprotein gene, the complete glycoprotein gene, and the intergenic region G-L from 113 selected fox rabies cases. We identified two viral genetic groups, here referred to as Italy-1 and Italy-2. Phylogenetic and phylogeographic analyses revealed that both groups had been circulating in the Western Balkans and Slovenia in previous years and were only later introduced into Italy (into the Friuli Venezia Giulia region-FVG), occupying different areas of the Italian territories. Notably, viruses belonging to the Italy-1 group remained confined to the region of introduction and their spread was minimised by the implementation of oral fox vaccination campaigns. In contrast, Italy-2 viruses spread westward over a territory of 100 km from their first identification in FVG, likely crossing the northern territories where surveillance was inadequate. A genetic sub-group (Italy-2A), characterised by a unique amino acid mutation (D106A) in the N gene, was also observed to occupy a distinct geographic cluster. This molecular epidemiological analysis of the 2008-2011 fox rabies epidemic will contribute to future control programmes both at national and regional levels. In particular, our findings highlight the weaknesses of the national surveillance strategy in the period preceding rabies re-emergence, and of control plans implemented immediately after rabies notification, and underline the need of a coordinated approach at the regional level for both the surveillance and control of wildlife rabies.
Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.
There are a number of RNA virus pathogens that represent a serious threat to the health of managed honey bees (Apis mellifera). That some of these viruses are also found in the broader pollinator community suggests the wider environmental spread of these viruses, with the potential for a broader impact on ecosystems. Studies on the ecology and evolution of these viruses in the arthropod community as a whole may therefore provide important insights into these potential impacts. We examined managed A. mellifera colonies, nearby non-Apis hymenopteran pollinators, and other associated arthropods for the presence of five commonly occurring picorna-like RNA viruses of honey bees - black queen cell virus, deformed wing virus, Israeli acute paralysis virus, Kashmir bee virus and sacbrood virus. Notably, we observed their presence in several arthropod species. Additionally, detection of negative-strand RNA using strand-specific RT-PCR assays for deformed wing virus and Israeli acute paralysis virus suggests active replication of deformed wing virus in at least six non-Apis species and active replication of Israeli acute paralysis virus in one non-Apis species. Phylogenetic analysis of deformed wing virus also revealed that this virus is freely disseminating across the species sampled in this study. In sum, our study indicates that these viruses are not specific to the pollinator community and that other arthropod species have the potential to be involved in disease transmission in pollinator populations.
Members of the family Rhabdoviridae have been assigned to eight genera but many remain unassigned. Rhabdoviruses have a remarkably diverse host range that includes terrestrial and marine animals, invertebrates and plants. Transmission of some rhabdoviruses often requires an arthropod vector, such as mosquitoes, midges, sandflies, ticks, aphids and leafhoppers, in which they replicate. Herein we characterize Niakha virus (NIAV), a previously uncharacterized rhabdovirus isolated from phebotomine sandflies in Senegal. Analysis of the 11,124 nt genome sequence indicates that it encodes the five common rhabdovirus proteins with alternative ORFs in the M, G and L genes. Phylogenetic analysis of the L protein indicate that NIAVs closest relative is Oak Vale rhabdovirus, although in this analysis NIAV is still so phylogenetically distinct that it might be classified as distinct from the eight currently recognized Rhabdoviridae genera. This observation highlights the vast, and yet not fully recognized diversity, of this family.
Hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. Despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. Documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) sampled from bats and shrews in China, and which are distinct from other known hantaviruses. Huangpi virus was found in Pipistrellus abramus, Lianghe virus in Anourosorex squamipes, Longquan virus in Rhinolophus affinis, Rhinolophus sinicus, and Rhinolophus monoceros, and Yakeshi virus in Sorex isodon, respectively. A phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. Notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. Our phylogenetic analysis also suggests that hantaviruses might have first appeared in Chiroptera (bats) or Soricomorpha (moles and shrews), before emerging in rodent species. Overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses.
Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo, we studied the intra- and interhost evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity were similar to those in naïve horses, intrahost bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.
Revealing the patterns and determinants of the spread of dengue virus (DENV) at local scales is central to understanding the epidemiology and evolution of this major human pathogen. We performed a phylogenetic analysis of the envelope (E) genes of DENV-1, -2, -3, and -4 isolates (involving 97, 23, 5, and 74 newly collected sequences, respectively) sampled from school-based cohort and village-based cluster studies in Kamphaeng Phet, Thailand, between 2004 and 2007. With these data, we sought to describe the spatial and temporal patterns of DENV spread within a rural population where a future vaccine efficacy trial is planned. Our analysis revealed considerable genetic diversity within the study population, with multiple lineages within each serotype circulating for various lengths of time during the study period. These results suggest that DENV is frequently introduced into both semi-urban and rural areas in Kamphaeng Phet from other populations. In contrast, the persistence of viral lineages across sampling years was observed less frequently. Analysis of phylogenetic clustering indicated that DENV transmission was highly spatially and temporally focal, and that it occurred in homes rather than at school. Overall, the strength of temporal clustering suggests that seasonal bottlenecks in local DENV populations facilitate the invasion and establishment of viruses from outside of the study area, in turn reducing the extent of lineage persistence.
Endogenous gammaretroviruses (EGVs) have been widely studied in terrestrial mammals but seldom so in marine species. A genomic mining of the bottlenose dolphin (Tursiops truncatus) genome revealed a new EGV, termed Tursiops truncatus endogenous retrovirus (TTEV), which is divergent from extant mammalian EGVs. Molecular clock dating estimated the invasion time of TTEV into the host genome to be approximately 10-19 million years ago (MYA), while a previously identified killer whale endogenous gammaretrovirus (KWERV) was estimated to have invaded the host genome approximately 3-5 MYA. Using a PCR-based technique, we then verified that similar endogenous viruses exist in nine cetacean genomes. Phylogenetic analysis revealed that these cetacean EGVs are highly divergent from their counterparts in other mammals, including KWERV from the killer whale. In sum, we conclude that there have been at least two invasion episodes of EGVs into cetaceans during their evolutionary history.
Malpais Spring virus (MSPV) is a mosquito-borne rhabdovirus that infects a variety of wild and feral ungulates in New Mexico, including horses and deer. Although, initial serologic tests and electron microscopy at the time of isolation nearly 25 years ago provided evidence that MSPV is a novel virus, possibly related to vesiculoviruses, the virus still has not been approved as a new species.
Recombination plays an important role in shaping the genetic diversity of a number of DNA and RNA viruses. Although some recent studies have reported bioinformatic evidence of mosaic sequences in a variety of influenza A viruses, it remains controversial as to whether these represent bona fide natural recombination events or laboratory artifacts. Importantly, mosaic genome structures can create significant topological incongruence during phylogenetic analyses, which can mislead additional phylogeny-based molecular evolutionary analyses such as molecular clock dating, the detection of selection pressures and phylogeographic inference. As a result, there is a strong need for systematic screenings for mosaic structures within the influenza virus genome database. We used a combination of sequence-based and phylogeny-based methods to identify 388 mosaic influenza genomic segments, of which 332 are previously unreported and are significantly supported by phylogenetic methods. It is impossible, however, to ascertain whether these represent natural recombinants. To facilitate the future identification of recombinants, reference sets of non-recombinant sequences were selected for use in an automatic screening protocol for detecting mosaic sequences. Tests using real and simulated mosaic sequences indicate that our screening protocol is both sensitive (average >90%) and accurate (average >77%) enough to identify a range of different mosaic patterns. The relatively high prevalence of mosaic influenza virus sequences implies that efficient systematic screens, such as that proposed here, should be performed routinely to detect natural recombinant strains, potential laboratory artifacts, and sequencing contaminants either prior to sequences being deposited in GenBank or before they are used for phylogenetic analyses.
In the past decade, rapid increases in the availability of high-resolution molecular and epidemiological data, combined with developments in statistical and computational methods to simulate and infer migration patterns, have provided key insights into the spatial dynamics of influenza A viruses in humans. In this review, we contrast findings from epidemiological and molecular studies of influenza virus transmission at different spatial scales. We show that findings are broadly consistent in large-scale studies of inter-regional or inter-hemispheric spread in temperate regions, revealing intense epidemics associated with multiple viral introductions, followed by deep troughs driven by seasonal bottlenecks. However, aspects of the global transmission dynamics of influenza viruses are still debated, especially with respect to the existence of tropical source populations experiencing high levels of genetic diversity and the extent of prolonged viral persistence between epidemics. At the scale of a country or community, epidemiological studies have revealed spatially structured diffusion patterns in seasonal and pandemic outbreaks, which were not identified in molecular studies. We discuss the role of sampling issues in generating these conflicting results, and suggest strategies for future research that may help to fully integrate the epidemiological and evolutionary dynamics of influenza virus over space and time.
The adaptation of viruses to new hosts is a poorly understood process likely involving a variety of viral structures and functions that allow efficient replication and spread. Canine parvovirus (CPV) emerged in the late 1970s as a host-range variant of a virus related to feline panleukopenia virus (FPV). Within a few years of its emergence in dogs, there was a worldwide replacement of the initial virus strain (CPV type 2) by a variant (CPV type 2a) characterized by four amino acid differences in the capsid protein. However, the evolutionary processes that underlie the acquisition of these four mutations, as well as their effects on viral fitness, both singly and in combination, are still uncertain. Using a comprehensive experimental analysis of multiple intermediate mutational combinations, we show that these four capsid mutations act in concert to alter antigenicity, cell receptor binding, and relative in vitro growth in feline cells. Hence, host adaptation involved complex interactions among both surface-exposed and buried capsid mutations that together altered cell infection and immune escape properties of the viruses. Notably, most intermediate viral genotypes containing different combinations of the four key amino acids possessed markedly lower fitness than the wild-type viruses.
Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia.
Populations of seasonal influenza virus experience strong annual bottlenecks that pose a considerable extinction risk. It has been suggested that an influenza source population located in tropical Southeast or East Asia seeds annual temperate epidemics. Here we investigate the seasonal dynamics and migration patterns of influenza A H3N2 virus by analysis of virus samples obtained from 2003 to 2006 from Australia, Europe, Japan, New York, New Zealand, Southeast Asia, and newly sequenced viruses from Hong Kong. In contrast to annual temperate epidemics, relatively low levels of relative genetic diversity and no seasonal fluctuations characterized virus populations in tropical Southeast Asia and Hong Kong. Bayesian phylogeographic analysis using discrete temporal and spatial characters reveal high rates of viral migration between urban centers tested. Although the virus population that migrated between Southeast Asia and Hong Kong persisted through time, this was dependent on virus input from temperate regions and these tropical regions did not maintain a source for annual H3N2 influenza epidemics. We further show that multiple lineages may seed annual influenza epidemics, and that each region may function as a potential source population. We therefore propose that the global persistence of H3N2 influenza A virus is the result of a migrating metapopulation in which multiple different localities may seed seasonal epidemics in temperate regions in a given year. Such complex global migration dynamics may confound control efforts and contribute to the emergence and spread of antigenic variants and drug-resistant viruses.
Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.
Endogenous retroviruses are a common component of the eukaryotic genome, and their evolution and potential function have attracted considerable interest. More surprising was the recent discovery that eukaryotic genomes contain sequences from RNA viruses that have no DNA stage in their life cycle. Similarly, several single-stranded DNA viruses have left integrated copies in their host genomes. This review explores some major evolutionary aspects arising from the discovery of these endogenous viral elements (EVEs). In particular, the reasons for the bias toward EVEs derived from negative-sense RNA viruses are considered, as well as what they tell us about the long-term "arms races" between hosts and viruses, characterized by episodes of selection and counter-selection. Most dramatically, the presence of orthologous EVEs in divergent hosts demonstrates that some viral families have ancestries dating back almost 100 million years, and hence are far older than expected from the phylogenetic analysis of their exogenous relatives.
There has been an explosion in the discovery of insect-specific flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all insect-specific flavivirus sequences currently available and conduct phylogenetic analyses of both the insect-specific flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus-mosquito co-divergence, suggesting that the insect-specific flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on insect-specific flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its effect on the emergence and maintenance of the flaviviruses.
Despite the importance of migratory birds in the ecology and evolution of avian influenza virus (AIV), there is a lack of information on the patterns of AIV spread at the intra-continental scale. We applied a variety of statistical phylogeographic techniques to a plethora of viral genome sequence data to determine the strength, pattern and determinants of gene flow in AIV sampled from wild birds in North America. These analyses revealed a clear isolation-by-distance of AIV among sampling localities. In addition, we show that phylogeographic models incorporating information on the avian flyway of sampling proved a better fit to the observed sequence data than those specifying homogeneous or random rates of gene flow among localities. In sum, these data strongly suggest that the intra-continental spread of AIV by migratory birds is subject to major ecological barriers, including spatial distance and avian flyway.
Diarrhea is a major cause of childhood morbidity and mortality in developing countries, and the majority of infections are of viral etiology. We aimed to compare the etiological prevalence of the major enteric viruses in an urban and a rural setting in southern Vietnam. We simultaneously screened fecal specimens from 362 children in Ho Chi Minh City and Dong Thap province that were hospitalized with acute diarrhea over a 1-month-long period for four viral gastrointestinal pathogens. Rotavirus was the most common pathogen identified, but there was a differential prevalence of rotavirus and norovirus between the urban and rural locations. Furthermore, rotavirus genotyping and phylogenetic analysis again differentiated the genotypes by the sampling location. Our data show a disproportional distribution of enteric viral pathogens in urban and rural locations, and we provide evidence of continual importation of new rotavirus strains into southern Vietnam and report the emergence of rotavirus genotype G12.
Co-divergence between host and parasites suggests that evolutionary processes act across similar spatial and temporal scales. Although there has been considerable work on the extent and correlates of co-divergence of RNA viruses and their mammalian hosts, relatively little is known about the extent to which virus evolution is determined by the phylogeographic history of host species. To test hypotheses related to co-divergence across a variety of spatial and temporal scales, we explored phylogenetic signatures in Andes virus (ANDV) sampled from Chile and its host rodent, Oligoryzomys longicaudatus. ANDV showed strong spatial subdivision, a phylogeographic pattern also recovered in the host using both spatial and genealogical approaches, and despite incomplete lineage sorting. Lineage structure in the virus seemed to be a response to current population dynamics in the host at the spatial scale of ecoregions. However, finer scale analyses revealed contrasting patterns of genetic structure across a latitudinal gradient. As predicted by their higher substitution rates, ANDV showed greater genealogical resolution than the rodent, with topological congruence influenced by the degree of lineage sorting within the host. However, despite these major differences in evolutionary dynamics, the geographic structure of host and virus converged across large spatial scales.
That pigs may play a pivotal role in the emergence of pandemic influenza was indicated by the recent H1N1/2009 human pandemic, likely caused by a reassortant between viruses of the American triple-reassortant (TR) and Eurasian avian-like (EA) swine influenza lineages. As China has the largest human and pig populations in the world and is the only place where both TR and EA viruses have been reported to cocirculate, it is potentially the source of the H1N1/2009 pandemic virus. To examine this, the genome sequences of 405 swine influenza viruses from China were analyzed. Thirty-six TR and EA reassortant viruses were identified before and after the occurrence of the pandemic. Several of these TR-EA reassortant viruses had genotypes with most segments having the same lineage origin as the segments of the H1N1/2009 pandemic virus. However, these viruses were generated from independent reassortment events throughout our survey period and were not associated with the current pandemic. One TR-EA reassortant, which is least similar to the pandemic virus, has persisted since 2007, while all the other variants appear to be transient. Despite frequent reassortment events between TR and EA lineage viruses in China, evidence for the genesis of the 2009 pandemic virus in pigs in this region is still absent.
Viral encephalopathy and retinopathy (VER) is one of the most devastating diseases for marine aquaculture, and similarly represents a threat to wild fish populations because of its high infectivity and broad host range. Betanodavirus, the causative agent of VER, is a small non-enveloped virus with a bipartite RNA genome comprising the RNA1 and RNA2 segments. We partially sequenced both RNA1 and RNA2 from 120 viral strains isolated from 2000 to 2009 in six different countries in Southern Europe. Phylogenetic analysis revealed the presence of the red-spotted grouper nervous necrosis virus (RGNNV) (n=96) and striped jack nervous necrosis virus (SJNNV) (n=1) genotypes in Southern Europe, with 23/120 samples classified as RGNNV/SJNNV reassortants. Viruses sampled from individual countries tended to cluster together suggesting a major geographic subdivision among betanodaviruses, although some phylogenetic evidence for viral gene flow was also obtained. Rates of nucleotide substitution were similar to those observed in a broad array of RNA viruses, and revealed a significantly higher evolutionary rate in the polymerase compared to the coat protein gene. This may reflect temperature adaptation of betanodaviruses, although a site-specific analysis of selection pressures identified relatively few selected sites in either gene. Overall, our analyses yielded novel data on the evolutionary dynamics and phylogeography of betanodaviruses and therein provides a more complete understanding of the distribution and evolution of different genotypes in Southern Europe.
Hao (2011) reported that the PB2 genes of three swine influenza A viruses were likely generated through homologous recombination between two closely related parental strains. However, we show that Haos observation is an artifact of incorrect taxon sampling arising through the lack of an appropriate evolutionary context. Through rigorous phylogenetic analyses we explain the evolutionary origins of these stains and confirm the lack of any statistical support for intra-segmental recombination.
Recombination occurs in many RNA viruses and can be of major evolutionary significance. However, rates of recombination vary dramatically among RNA viruses, which can range from clonal to highly recombinogenic. Here, we review the factors that might explain this variation in recombination frequency and show that there is little evidence that recombination is favoured by natural selection to create advantageous genotypes or purge deleterious mutations, as predicted if recombination functions as a form of sexual reproduction. Rather, recombination rates seemingly reflect larger-scale patterns of viral genome organization, such that recombination may be a mechanistic by-product of the evolutionary pressures acting on other aspects of virus biology.
Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.
The four dengue virus (DENV) serotypes that circulate among humans emerged independently from ancestral sylvatic progenitors that were present in non-human primates, following the establishment of human populations that were large and dense enough to support continuous inter-human transmission by mosquitoes. This ancestral sylvatic-DENV transmission cycle still exists and is maintained in non-human primates and Aedes mosquitoes in the forests of Southeast Asia and West Africa. Here, we provide an overview of the ecology and molecular evolution of sylvatic DENV and its potential for adaptation to human transmission. We also emphasize how the study of sylvatic DENV will improve our ability to understand, predict and, ideally, avert further DENV emergence.
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