Characterization of five unclassified orthobunyaviruses (Bunyaviridae) from Africa and the Americas.

The Bunyaviridae family is made up of a diverse range of viruses, some of which cause disease and are a cause for concern in human and veterinary health. Here, we report the genomic and antigenic characterization of five previously uncharacterized bunyaviruses. Based on their ultrastructure, antigenic relationships and phylogenomic relationships, the five viruses are classified as members of the Orthobunyavirus genus. Three are viruses in the California encephalitis virus serogroup and are related to Trivittatus virus; the two others are most similar to the Mermet virus in the Simbu serogroup, and to the Tataguine virus, which is not currently assigned to a serogroup. Each of these five viruses was pathogenic to newborn mice, indicating their potential to cause illness in humans and other animals.

Eastern equine encephalitis in Latin America.

BACKGROUND: The eastern equine encephalitis (EEE) and Venezuelan equine encephalitis (VEE) viruses are pathogens that infect humans and horses in the Americas. Outbreaks of neurologic disease in humans and horses were reported in Panama from May through early August 2010. METHODS: We performed antibody assays and tests to detect viral RNA and isolate the viruses in serum samples from hospitalized patients. Additional cases were identified with enhanced surveillance. RESULTS: A total of 19 patients were hospitalized for encephalitis. Among them, 7 had confirmed EEE, 3 had VEE, and 1 was infected with both viruses; 3 patients died, 1 of whom had confirmed VEE. The clinical findings for patients with EEE included brain lesions, seizures that evolved to status epilepticus, and neurologic sequelae. An additional 99 suspected or probable cases of alphavirus infection were detected during active surveillance. In total, 13 cases were confirmed as EEE, along with 11 cases of VEE and 1 case of dual infection. A total of 50 cases in horses were confirmed as EEE and 8 as VEE; mixed etiologic factors were associated with 11 cases in horses. Phylogenetic analyses of isolates from 2 cases of equine infection with the EEE virus and 1 case of human infection with the VEE virus indicated that the viruses were of enzootic lineages previously identified in Panama rather than new introductions. CONCLUSIONS: Cases of EEE in humans in Latin America may be the result of ecologic changes that increased human contact with enzootic transmission cycles, genetic changes in EEE viral strains that resulted in increased human virulence, or an altered host range. (Funded by the National Institutes of Health and the Secretaría Nacional de Ciencia, Tecnología e Innovación, Panama.).

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus.

UNLABELLED: 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, MA, 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 that was demonstrated to have fusion activity; this 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 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. IMPORTANCE: The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

Characterization of the Punta Toro species complex (genus Phlebovirus, family Bunyaviridae).

Punta Toro virus (PTV), a member of the PTV complex, is a relatively common causative agent of febrile illness in Panama that is often misdiagnosed as 'dengue' or 'influenza'. Currently, only two named members make up this species complex, PTV and Buenaventura virus (BUEV). Genomic and antigenic characterization of 17 members of the PTV complex, nine of which were isolated from human acute febrile illness cases, reveals that this species complex is composed of six distant viruses. We propose to add four additional new viruses, designated Leticia virus, Cocle virus, Campana virus and Capira virus.

Characterization of the Sandfly fever Naples species complex and description of a new Karimabad species complex (genus Phlebovirus, family Bunyaviridae).

Genomic and antigenic characterization of members of the Sandfly fever Naples virus (SFNV) complex reveals the presence of five clades that differ in their geographical distribution. Saint Floris and Gordil viruses, both found in Africa, form one clade; Punique, Granada and Massilia viruses, all isolated in the western Mediterranean, constitute a second; Toscana virus, a third; SFNV isolates from Italy, Cyprus, Egypt and India form a fourth; while Tehran virus and a Serbian isolate Yu 8/76, represent a fifth. Interestingly, this last clade appears not to express the second non-structural protein ORF. Karimabad virus, previously classified as a member of the SFNV complex, and Gabek Forest virus are distinct and form a new species complex (named Karimabad) in the Phlebovirus genus. In contrast with the high reassortment frequency observed in some South American phleboviruses, the only virus of the SFNV complex with evidence of reassortment was Granada virus.

Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil.

The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analysis by electron microscopy and gel electrophoresis, respectively, revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.

Characterization of the Bhanja serogroup viruses (Bunyaviridae): a novel species of the genus Phlebovirus and its relationship with other emerging tick-borne phleboviruses.

Bhanja virus (BHAV) and its antigenically close relatives Forecariah virus (FORV), Kismayo virus (KISV), and Palma virus (PALV) are thought to be members of the family Bunyaviridae, but they have not been assigned to a genus or species. Despite their broad geographical distribution and reports that BHAV causes sporadic cases of febrile illness and encephalitis in humans, the public health importance of the Bhanja serogroup viruses remains unclear, due in part to the lack of sequence and biochemical information for the virus proteins. In order to better define the molecular characteristics of this group, we determined the full-length sequences of the L, M, and S genome segments of multiple isolates of BHAV as well as FORV and PALV. The genome structures of these Bhanja viruses are similar to those of viruses belonging to the genus Phlebovirus. Functional domains and amino acid motifs in the viral proteins that are conserved among other known phleboviruses were also identified in proteins of the BHAV group. Phylogenetic and serological analyses revealed that the BHAVs are most closely related to the novel emerging tick-borne phleboviruses severe fever with thrombocytopenia syndrome virus and Heartland virus, which have recently been implicated as causing severe acute febrile illnesses associated with thrombocytopenia in humans in China and the United States. Our results indicate that the Bhanja serogroup viruses constitute a single novel species in the genus Phlebovirus. The results of this study should facilitate epidemiological surveillance for other, similar tick-borne phleboviruses that may represent unrecognized causes of febrile illness in humans.

A novel mosquito-borne Orbivirus species found in South-east Asia.

The genus Orbivirus of the family Reoviridae includes a genetically diverse group of dsRNA arthropod-borne viruses that infect a wide variety of animal species. Here, we report the complete genome and phylogenetic analysis of a novel orbivirus (IAn-66411 or Sathuvachari virus, SVIV) isolated in 1963 from starlings (Brahminy myna) collected in Vellore, Tamil Nadu, India. Comparative genetic analysis of the SVIV polymerase (VP1 protein), core protein (VP3) and outer core protein (VP7) confirmed that SVIV is most closely related to the mosquito-borne orbiviruses, but that it is equally divergent from all known species. Therefore, SVIV should be tentatively considered as the prototype of a novel mosquito-associated Orbivirus species. These findings will aid in the development of molecular reagents that can identify genetically similar orbiviruses and help elucidate their geographical distribution, epidemiology, species tropism and possible disease association.

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea.

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.

Characterization of Farmington virus, a novel virus from birds that is distantly related to members of the family Rhabdoviridae.

BACKGROUND: 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. FINDINGS: 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. CONCLUSIONS: In sum, our analysis indicates that FARV represents a new species within the family Rhabdoviridae.

Identification of a new Newcastle disease virus isolate from Indonesia represents an ancestral lineage of class II genotype XIII.

An unknown virus was isolated from a mosquito pool collected in Jakarta during routine surveillance in 1979. Analysis of the sample using the Illumina platform resulted in the identification of a Newcastle disease virus (NDV) isolate. The sequence of the isolate indicated that it is an ancestral lineage of class II, genotype XIII. The source of the isolate is unusual, as newcastle disease virus is not believed to be vector-borne, although this mosquito pool was processed in a laboratory also handling samples for avian influenza surveillance and it is possible that this resulted in cross-contamination. This NDV isolate is still ancestral to most extant genotype XIII strains and provides a useful insight into historic NDV evolution.

Emergence potential of mosquito-borne arboviruses from the Florida Everglades.

The Greater Everglades Region of South Florida is one of the largest natural wetlands and the only subtropical ecosystem found in the continental United States. Mosquitoes are seasonally abundant in the Everglades where several potentially pathogenic mosquito-borne arboviruses are maintained in natural transmission cycles involving vector-competent mosquitoes and reservoir-competent vertebrate hosts. The fragile nature of this ecosystem is vulnerable to many sources of environmental change, including a wetlands restoration project, climate change, invasive species and residential development. In this study, we obtained baseline data on the distribution and abundance of both mosquitos and arboviruses occurring in the southern Everglades region during the summer months of 2013, when water levels were high, and in 2014, when water levels were low. A total of 367,060 mosquitoes were collected with CO2-baited CDC light traps at 105 collection sites stratified among the major landscape features found in Everglades National Park, Big Cypress National Preserve, Fakahatchee State Park Preserve and Picayune State Forest, an area already undergoing restoration. A total of 2,010 pools of taxonomically identified mosquitoes were cultured for arbovirus isolation and identification. Seven vertebrate arboviruses were isolated: Everglades virus, Tensaw virus, Shark River virus, Gumbo Limbo virus, Mahogany Hammock virus, Keystone virus, and St. Louis encephalitis virus. Except for Tensaw virus, which was absent in 2013, the remaining viruses were found to be most prevalent in hardwood hammocks and in Fakahatchee, less prevalent in mangroves and pinelands, and absent in cypress and sawgrass. In contrast, in the summer of 2014 when water levels were lower, these arboviruses were far less prevalent and only found in hardwood hammocks, but Tensaw virus was present in cypress, sawgrass, pinelands, and a recently burned site. Major environmental changes are anticipated in the Everglades, many of which will result in increased water levels. How these might lead to the emergence of arboviruses potentially pathogenic to both humans and wildlife is discussed.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Experimental infection of potential reservoir hosts with Venezuelan equine encephalitis virus, Mexico.

In 1993, an outbreak of encephalitis among 125 affected equids in coastal Chiapas, Mexico, resulted in a 50% case-fatality rate. The outbreak was attributed to Venezuelan equine encephalitis virus (VEEV) subtype IE, not previously associated with equine disease and death. To better understand the ecology of this VEEV strain in Chiapas, we experimentally infected 5 species of wild rodents and evaluated their competence as reservoir and amplifying hosts. Rodents from 1 species (Baiomys musculus) showed signs of disease and died by day 8 postinoculation. Rodents from the 4 other species (Liomys salvini, Oligoryzomys fulvescens, Oryzomys couesi, and Sigmodon hispidus) became viremic but survived and developed neutralizing antibodies, indicating that multiple species may contribute to VEEV maintenance. By infecting numerous rodent species and producing adequate viremia, VEEV may increase its chances of long-term persistence in nature and could increase risk for establishment in disease-endemic areas and amplification outside the disease-endemic range.

Characterization of Three Novel Viruses from the Families Nyamiviridae, Orthomyxoviridae, and Peribunyaviridae, Isolated from Dead Birds Collected during West Nile Virus Surveillance in Harris County, Texas.

This report describes and characterizes three novel RNA viruses isolated from dead birds collected during West Nile virus surveillance in Harris County, TX, USA (the Houston metropolitan area). The novel viruses are identified as members of the families Nyamaviridae, Orthomyxoviridae, and Peribunyaviridae and have been designated as San Jacinto virus, Mason Creek virus, and Buffalo Bayou virus, respectively. Their potential public health and/or veterinary importance are still unknown.

Biological and phylogenetic characterization of pigeon paramyxovirus serotype 1 circulating in wild North American pigeons and doves.

As part of West Nile virus surveillance programs in Rhode Island and eastern Texas between 2000 and 2007, brain tissue was collected from 5,608 dead birds representing 21 avian orders found in public places or reported by homeowners. Fifteen Newcastle disease virus isolates were recovered only from birds of the order Columbiformes and were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene and more specifically as pigeon paramyxovirus serotype 1 (PPMV-1) by hemagglutinin inhibition with monoclonal antibodies. Based upon partial genomic sequencing and phylogenetic analysis, the newly isolated viruses represent a distinct sublineage within class II genotype VIb. All of the viruses (15/15) were classified as virulent based upon their fusion cleavage site motif ((112)RRKKRF(117)) and intracerebral pathogenicity indices of >0.7 (ranging from 0.98 to 1.35); however, these viruses escaped detection by the fusion gene-based real-time PCR test for virulence. Modifications introduced to the probe site of the fusion gene-based assay allowed rapid virulence detection within this distinct sublineage.

Human and Equine Infection with Alphaviruses and Flaviviruses in Panamá during 2010: A Cross-Sectional Study of Household Contacts during an Encephalitis Outbreak.

Members of the genera Alphavirus (family Togaviridae) and Flavivirus (family Flaviridae) are important zoonotic human and equine etiologic agents of neurologic diseases in the New World. In 2010, an outbreak of Madariaga virus (MADV; formerly eastern equine encephalitis virus) and Venezuelan equine encephalitis virus (VEEV) infections was reported in eastern Panamá. We further characterized the epidemiology of the outbreak by studying household contacts of confirmed human cases and of equine cases with neurological disease signs. Serum samples were screened using a hemagglutination inhibition test, and human results were confirmed using plaque reduction neutralization tests. A generalized linear model was used to evaluate the human MADV and VEEV seroprevalence ratios by age (in tercile) and gender. Overall, antibody prevalence for human MADV infection was 19.4%, VEEV 33.3%, and Mayaro virus 1.4%. In comparison with individuals aged 2-20 years, people from older age groups (21-41 and > 41 years) were five times more likely to have antibodies against VEEV, whereas the MADV prevalence ratio was independent of age. The overall seroprevalence of MADV in equids was 26.3%, VEEV 29.4%, West Nile virus (WNV) 2.6%, and St. Louis encephalitis virus (SLEV) was 63.0%. Taken together, our results suggest that multiple arboviruses are circulating in human and equine populations in Panamá. Our findings of a lack of increase in the seroprevalence ratio with age support the hypothesis of recent MADV exposure to people living in the affected region.

Characterization of Three New Insect-Specific Flaviviruses: Their Relationship to the Mosquito-Borne Flavivirus Pathogens.

Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.