Six novel insect-specific viruses, isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, the United States, Ivory Coast, Israel, and Indonesia, are described. Their genomes consist of single-stranded, positive-sense RNAs with poly(A) tails. By electron microscopy, the virions appear as spherical particles with diameters of ∼45 to 55 nm. Based on their genome organization and phylogenetic relationship, the six viruses, designated Negev, Ngewotan, Piura, Loreto, Dezidougou, and Santana, appear to form a new taxon, tentatively designated Negevirus. Their closest but still distant relatives are citrus leposis virus C (CiLV-C) and viruses in the genus Cilevirus, which are mite-transmitted plant viruses. The negeviruses replicate rapidly and to high titer (up to 1010 PFU/ml) in mosquito cells, producing extensive cytopathic effect and plaques, but they do not appear to replicate in mammalian cells or mice. A discussion follows on their possible biological significance and effect on mosquito vector competence for arboviruses.
Evolutionary insights into the phleboviruses are limited because of an imprecise classification scheme based on partial nucleotide sequences and scattered antigenic relationships. In this report, the serologic and phylogenetic relationships of the Uukuniemi group viruses and their relationships with other recently characterized tick-borne phleboviruses are described using full-length genome sequences. We propose that the viruses currently included in the Uukuniemi virus group be assigned to five different species as follows: Uukuniemi virus, EgAn 1825-61 virus, Fin V707 virus, Chizé virus, and Zaliv Terpenia virus would be classified into the Uukuniemi species; Murre virus, RML-105-105355 virus, and Sunday Canyon virus would be classified into a Murre virus species; and Grand Arbaud virus, Precarious Point virus, and Manawa virus would each be given individual species status. Although limited sequence similarity was detected between current members of the Uukuniemi group and Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus, a clear serological reaction was observed between some of them, indicating that SFTSV and Heartland virus should be considered part of the Uukuniemi virus group. Moreover, based on the genomic diversity of the phleboviruses and given the low correlation observed between complement fixation titers and genetic distance, we propose a system for classification of the Bunyaviridae based on genetic as well as serological data. Finally, the recent descriptions of SFTSV and Heartland virus also indicate that the public health importance of the Uukuniemi group viruses must be reevaluated.
Farmington virus (FARV) is a rhabdovirus that was isolated from a wild bird during an outbreak of epizootic eastern equine encephalitis on a pheasant farm in Connecticut, USA.
Analysis of the nearly complete genome sequence of the prototype CT AN 114 strain indicates that it encodes the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (> 180 nt) in the N and G genes. Phenotypic and genetic characterization of FARV has confirmed that it is a novel rhabdovirus and probably represents a new species within the family Rhabdoviridae.
In sum, our analysis indicates that FARV represents a new species within the family Rhabdoviridae.
Farmington virus (FARV); Family Rhabdoviridae; Next generation sequencing; Phylogeny
Globally, yellow fever virus infects nearly 200,000 people, leading to 30,000 deaths annually. Although the virus is endemic to Latin America, only a single genome from this region has been sequenced. Here, we report 12 Brazilian yellow fever virus complete genomes, their genetic traits, phylogenetic characterization, and phylogeographic dynamics. Variable 3′ noncoding region (3′NCR) patterns and specific mutations throughout the open reading frame altered predicted secondary structures. Our findings suggest that whereas the introduction of yellow fever virus in Brazil led to genotype I-predominant dispersal throughout South and Central Americas, genotype II remained confined to Bolivia, Peru, and the western Brazilian Amazon.
Hantaviruses are important contributors to disease burden in the New World, yet many aspects of their distribution and dynamics remain uncharacterized. To examine the patterns and processes that influence the diversity and geographic distribution of hantaviruses in South America, we performed genetic and phylogeographic analyses of all available South American hantavirus sequences. We sequenced multiple novel and previously described viruses (Anajatuba, Laguna Negra-like, two genotypes of Castelo dos Sonhos, and two genotypes of Rio Mamore) from Brazilian Oligoryzomys rodents and hantavirus pulmonary syndrome cases and identified a previously uncharacterized species of Oligoryzomys associated with a new genotype of Rio Mamore virus. Our analysis indicates that the majority of South American hantaviruses fall into three phylogenetic clades, corresponding to Andes and Andes-like viruses, Laguna Negra and Laguna Negra-like viruses, and Rio Mamore and Rio Mamore-like viruses. In addition, the dynamics and distribution of these viruses appear to be shaped by both the geographic proximity and phylogenetic relatedness of their rodent hosts. The current system of nomenclature used in the hantavirus community is a significant impediment to understanding the ecology and evolutionary history of hantaviruses; here, we suggest strict adherence to a modified taxonomic system, with species and strain designations resembling the numerical system of the enterovirus genus.
We report the characterization of three novel flaviviruses isolated in Spain. Marisma Mosquito virus, a novel mosquito borne virus, was isolated from Ochlerotatus caspius mosquitoes; Spanish Ochlerotatus flavivirus and Spanish Culex flavivirus, two novel insect flaviviruses, were isolated from Oc. caspius and Culex pipiens, respectively. During this investigation, we designed a sensitive RT-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus NS5 gene and could be directly used in clinical or environmental samples for flavivirus characterization and surveillance. Analysis of the sequence generated from that amplicon contains enough phylogenetic information for proper taxonomic studies. Moreover, the use of this tool allowed the detection of additional flavivirus DNA forms in Culex, Culiseta, and Ochlerotatus mosquitoes.
Flavivirus; Mosquitoes; Mosquito-only flavivirus
Moraxella macacae is a recently described bacterial species that has been associated with at least two outbreaks of epistaxis in macaques. Here we present the first genome sequence of this novel species, isolated from a symptomatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases.
Novel Weissella sp. bacteria have recently been reported to be associated with disease outbreaks in cultured rainbow trout (Oncorhynchus mykiss) at commercial farms in China, Brazil, and the United States. Here we present the first genome sequence of this novel Weissella species, isolated from the southeastern United States.
Jos virus (JOSV), originally isolated in Jos, Nigeria in 1967, has remained unclassified despite cultivation in tissue culture, development of animal models of infection and implementation of seroprevalence surveys for infection. Here, we report genetic, ultrastructural and serological evidence that JOSV is an orthomyxovirus distinct from but phylogenetically related to viruses of the genus Thogotovirus.
To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.
An aborted mid-gestational male Steller sea lion fetus with an attached placenta was recovered on the floor of an open floating capture trap located off Norris Rock near Denman Island, British Columbia. Viral culture of the placenta demonstrated cytopathic effect. Although no specific signal was obtained in microarray experiments using RNA obtained from viral culture, elution and sequence analysis revealed the presence of a reovirus. Complete genome pyrosequencing led to the identification of an orthoreovirus that we have tentatively named Steller sea lion reovirus (SSRV). Phylogenetic analysis revealed similarities between SSRV and orthoreoviruses of birds, bats and other mammals that suggests potential for interspecies transmission.
Leanyer virus (LEAV), currently classified as a member of the genus Orthobunyavirus, in the family Bunyaviridae, was originally isolated from a pool of Anopheles meraukensis mosquitoes, collected at Leanyer, Northern Territory, Australia in 1974. When it failed to react in serological tests with antisera from other known viruses, full-length genomic sequencing was pursued to determine the relationship of LEAV to other orthobunyavirus species. Genetic and serological characterization confirmed its antigenic distance from other orthobunyaviruses, including to its closest genetic neighbours, the Simbu group viruses, suggesting that it may represent a new antigenic complex.
Genomic and antigenic characterization of Aguacate virus, a tentative species of the genus Phlebovirus, and three other unclassified viruses, Armero virus, Durania virus and Ixcanal virus, demonstrate a close relationship to one another. They are distinct from the other nine recognized species within the genus Phlebovirus. We propose to designate them as a new (tenth) serogroup or species (Aguacate virus) within the genus. The four viruses were all isolated from phlebotomine sandflies (Lutzomyia sp.) collected in Central and South America. Aguacate virus appears to be a natural reassortant and serves as one more example of the high frequency of reassortment in this genus.
Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory.
We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008.
Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples.
This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.
In Argentina, successful vaccination and control of terrestrial rabies in the 1980s revealed the importance of the aerial route in RABV transmission. Current distribution of cases shows a predominance of rabies by hematophagous bats in the Northern regions where rabies is a major public health concern; in contrast, in Central and Southern regions where rabies is not a major public health concern, little surveillance is performed. Based on the analysis of insectivorous bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats in these regions ranged from 3.1 to 5.4%. This rate is comparable to other nations such as the United States (9–10%) where insectivorous bats are an important cause of concern for RABV surveillance systems. Antigenic and genetic analysis of a wide collection of rabies strains shows the presence of multiple endemic cycles associated with six bat insectivorous species distributed among an extensive area of the Argentinean territory and several countries of the Americas. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. Increased public education about the relationship between insectivorous bats and rabies are essential to avoid human cases and potential spread to terrestrial mammals.
Lymphocytic choriomeningitis virus (LCMV) was detected in 2 patients with acute meningitis in southern Spain within a 3-year period. Although the prevalence of LCMV infection was low (2 [1.3%] of 159 meningitis patients), it represents 2.9% of all pathogens detected. LCMV is a noteworthy agent of neurologic illness in immunocompetent persons.
Lymphocytic choriomeningitis virus; meningitis; Spain; encephalitis; viruses
Identifying emerging viral pathogens and characterizing their transmission is essential to developing effective public health measures in response to an epidemic. Phylogenetics, though currently the most popular tool used to characterize the likely host of a virus, can be ambiguous when studying species very distant to known species and when there is very little reliable sequence information available in the early stages of the outbreak of disease. Motivated by an existing framework for representing biological sequence information, we learn sparse, tree-structured models, built from decision rules based on subsequences, to predict viral hosts from protein sequence data using popular discriminative machine learning tools. Furthermore, the predictive motifs robustly selected by the learning algorithm are found to show strong host-specificity and occur in highly conserved regions of the viral proteome.
The taxonomy of the family Filoviridae (marburgviruses and ebolaviruses) has changed several times since the discovery of its members, resulting in a plethora of species and virus names and abbreviations. The current taxonomy has only been partially accepted by most laboratory virologists. Confusion likely arose for several reasons: species names that consist of several words or which (should) contain diacritical marks, the current orthographic identity of species and virus names, and the similar pronunciation of several virus abbreviations in the absence of guidance for the correct use of vernacular names. To rectify this problem, we suggest (1) to retain the current species names Reston ebolavirus, Sudan ebolavirus, and Zaire ebolavirus, but to replace the name Cote d'Ivoire ebolavirus [sic] with Taï Forest ebolavirus and Lake Victoria marburgvirus with Marburg marburgvirus; (2) to revert the virus names of the type marburgviruses and ebolaviruses to those used for decades in the field (Marburg virus instead of Lake Victoria marburgvirus and Ebola virus instead of Zaire ebolavirus); (3) to introduce names for the remaining viruses reminiscent of jargon used by laboratory virologists but nevertheless different from species names (Reston virus, Sudan virus, Taï Forest virus), and (4) to introduce distinct abbreviations for the individual viruses (RESTV for Reston virus, SUDV for Sudan virus, and TAFV for Taï Forest virus), while retaining that for Marburg virus (MARV) and reintroducing that used over decades for Ebola virus (EBOV). Paying tribute to developments in the field, we propose (a) to create a new ebolavirus species (Bundibugyo ebolavirus) for one member virus (Bundibugyo virus, BDBV); (b) to assign a second virus to the species Marburg marburgvirus (Ravn virus, RAVV) for better reflection of now available high-resolution phylogeny; and (c) to create a new tentative genus (Cuevavirus) with one tentative species (Lloviu cuevavirus) for the recently discovered Lloviu virus (LLOV). Furthermore, we explain the etymological derivation of individual names, their pronunciation, and their correct use, and we elaborate on demarcation criteria for each taxon and virus.
Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.
Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS.
Initially in 2007, and again between 2008 and 2010, cases of a life-threatening disease with sudden fever, thrombocytopenia, and leukopenia were reported in Henan Province, China. Patient reports of tick bites suggested that this disease could be infectious or tick-transmitted. Many patients were provisionally diagnosed with human granulocytic anaplasmosis (HGA). However, only 24 of 285 (8%) had objective evidence of HGA, suggesting that other pathogens likely contributed to fever, thrombocytopenia and leukopenia syndrome (FTLS). Illumina sequencing was used for direct detection in clinical samples of pathogens possibly associated with FTLS. A novel bunyavirus was found only in samples from FTLS patients. Further epidemiologic and laboratory investigation confirmed that the novel bunyavirus was associated with FTLS. The results illustrate that metagenomic analysis is a powerful method for the discovery of novel pathogenic agents. Combined with epidemiologic investigation, it could assist in rapid diagnosis of unknown diseases and distinguish them from other diseases with similar symptoms caused by known pathogens.
Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.
A novel filovirus, provisionally named Lloviu virus (LLOV), was detected during the investigation of Miniopterus schreibersii die-offs in Cueva del Lloviu in southern Europe. LLOV is genetically distinct from other marburgviruses and ebolaviruses and is the first filovirus detected in Europe that was not imported from an endemic area in Africa. Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification of genetically distinct filovirus in dead insectivorous bats in caves in Spain.
A new member of the phlebovirus genus, tentatively named Granada virus, was detected in sandflies collected in Spain. By showing the presence of specific neutralizing antibodies in human serum collected in Granada, we show that Granada virus infects humans. The analysis of the complete genome of Granada virus revealed that this agent is likely to be a natural reassortant of the recently described Massilia virus (donor of the long and short segments) with a yet unidentified phlebovirus (donor of the medium segment).
The genus Phlebovirus of the family Bunyaviridae consists of approximately 70 named viruses, currently assigned to nine serocomplexes (species) based on antigenic similarities. Sixteen other named viruses that show little serologic relationship to the nine recognized groups are also classified as tentative species in the genus. In an effort to develop a more precise classification system for phleboviruses, we are attempting to sequence most of the named viruses in the genus with the goal of clarifying their phylogenetic relationships. In this report, we describe the serologic and phylogenetic relationships of 13 viruses that were found to be members of the Candiru serocomplex; 6 of them cause disease in humans. Analysis of full genome sequences revealed branching inconsistencies that suggest five reassortment events, all involving the M segment, and thus appear to be natural reassortants. This high rate of reassortment illustrates the inaccuracy of a classification system based solely on antigenic relationships.
Dengue virus (DENV) circulates in human and sylvatic cycles. Sylvatic strains are both ecologically and evolutionarily distinct from endemic viruses. Although sylvatic dengue cycles occur in West African countries and Malaysia, only a few cases of mild human disease caused by sylvatic strains and one single case of dengue hemorrhagic fever in Malaysia have been reported. Here we report a case of dengue hemorrhagic fever (DHF) with thrombocytopenia (13000/µl), a raised hematocrit (32% above baseline) and mucosal bleeding in a 27-year-old male returning to Spain in November 2009 after visiting his home country Guinea Bissau. Sylvatic DENV-2 West African lineage was isolated from blood and sera. This is the first case of DHF associated with sylvatic DENV-2 in Africa and the second case worldwide of DHF caused by a sylvatic strain.
Dengue viruses are mosquito borne pathogens that cause up to 100 million infections annually. Dengue viruses circulate in either humans or non-human primates in two separate cycles: human and sylvatic. All four different serotypes infect humans causing frequent epidemics and severe disease, including hemorrhagic fever. Until a few years ago, viruses circulating in the sylvatic cycle had been associated with asymptomatic to mild disease only in humans. For example, retrospective analysis of human serum samples collected in Nigeria from the 1960s proved that sylvatic dengue was able to cause an outbreak of dengue fever in human population. Recently a case of DHF in Asia was linked to sylvatic DENV-2 (strain DKD811) infection. Here, we report the first case of DHF associated with sylvatic DENV-2 infection in Africa caused by a complete different lineage of the virus. All these events underscore the need to be vigilant about the re-emergence of DHF from sylvatic cycles.
Merino Walk virus (MWV), a proposed novel tentative species of the family Arenaviridae, was isolated from a rodent, Myotomys unisulcatus, collected at Merino Walk, Eastern Cape, South Africa, in 1985. Full-length genomic sequence confirmed MWV as an arenavirus related distantly to Mobala, Mopeia and Ippy viruses, all members of the Old World arenavirus complex. We propose MWV as a tentative novel species in the Lassa–lymphocytic choriomeningitis virus complex, based on its isolation from a novel rodent species and its genetic and serological characteristics.
The genetic relatedness of mountain gorillas and humans has led to concerns about interspecies transmission of infectious agents. Human-to-gorilla transmission may explain human metapneumovirus in 2 wild mountain gorillas that died during a respiratory disease outbreak in Rwanda in 2009. Surveillance is needed to ensure survival of these critically endangered animals.
Viral disease; respiratory tract diseases; pneumonia; human metapneumovirus; gorilla; Gorilla beringei beringei; viruses; Rwanda; dispatch