Orbiviruses in biting midges and mosquitoes from the Zambezi region, Namibia.

The genus Orbivirus includes a variety of pathogenic viruses that are transmitted by biting midges, mosquitoes and ticks. Some of the economically most relevant orbiviruses are endemic to Namibia, like the livestock-pathogenic Bluetongue and African horse sickness viruses. Here, we assessed the diversity of orbiviruses circulating in the Zambezi region of north-eastern Namibia. A total of 10 250 biting midges and 10 206 mosquitoes were collected and screened for orbivirus infections. We identified Palyam virus (PALV) in a pool of biting midges (Culicoides sp.) sampled in the Wuparo Conservancy and three strains of Corriparta virus (CORV) in Culex sp. mosquitoes sampled in Mudumu National Park and the Mashi Conservancy. This is, to our knowledge, the first detection of PALV and CORV in Namibia. Both viruses infect vertebrates but only PALV has been reported to cause disease. PALV can cause foetal malformations and abortions in ruminants. Furthermore, a novel orbivirus, related to Kammavanpettai virus from India and Umatilla virus from North America, was discovered in biting midges (Culicoides sp.) originating from Mudumu National Park and tentatively named Mudumu virus (MUMUV). Complete genomes of PALV, CORV and MUMUV were sequenced and genetically characterized. The Namibian CORV strain showed 24.3 % nucleotide divergence in its subcore shell gene to CORV strains from Australia, indicating that African CORV variants vary widely from their Australian relatives. CORV was isolated in cell culture and replicated to high titres in mosquito and duck cells. No growth was found in rodent and primate cells. The data presented here show that diverse orbiviruses are endemic to the Zambezi region. Further studies are needed to assess their effects on wildlife and livestock.

Genome-scale molecular and phylogenetic characterization of Middle Point orbiviruses from Australia.

Middle Point orbivirus (MPOV) is an Australian arbovirus, belongs to the Yunnan orbivirus species found in China. First detected and reported from Beatrice Hill, Northern Territory (NT), MPOV has to date, only been exclusively reported from the NT, Australia. Whilst genetic characterization of MPOV has been previously described, only restricted to sequence information for segments 2 and 3 coding core protein VP2 and outer capsid protein VP3, respectively. This study presents for the first time nearly full-length genome sequences of MPOV, which represent 24 isolates collected over a span of more than 20 years from 1997 to 2018. Whilst the majority of isolates were sampled at Beatrice Hill, NT where MPOV is most frequently isolated, this report also describes the first two isolations of MPOV from Queensland (QLD), Australia. One of which is the first non-bovine isolate obtained from the mosquito vector Aedes vittiger. We further compared these MPOV sequences with known sequences of the Yunnan orbivirus and other known orbivirus sequences of mosquito origin found in Australia. The phylogenetic analyses indicate the Australian MPOV sequences are more closely related to each other than other known sequences of Yunnan orbivirus. Furthermore, MPOV sequences are closely related to sequences from the Indonesian isolate JKT-8650. The clustering of Australian sequences in the phylogenetic tree suggests the monophyletic lineage of MPOV circulating in Australia. Further, ongoing surveillance is required to assess the existence and prevalence of this or other yet undetected lineages of MPOV and other orbiviruses in Australia.

Characterization of a novel reassortment Tibet orbivirus isolated from Culicoides spp. in Yunnan, PR China.

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.

Genomic analysis of putative novel serotypes of Tibet orbivirus isolated in Japan.

Tibet orbivirus (TIBOV) was initially isolated in Tibet in 2009 and subsequently in Guangdong, Hunan, and Yunnan, China. We document the first isolation of TIBOV outside of China: two TIBOV isolates from Culicoides collected in 2009 and 2010 in Kagoshima, Japan. Their complete genome sequences were also determined. Our results suggest that the two virus isolates are of novel serotypes, evident by variability within genome segment 2 encoding VP2. These new putative TIBOV serotypes will help with future virus surveillance and with the evaluation of its potential to cause disease in domestic ruminants.

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

Identification and characterization of a novel orbivirus, Yonaguni orbivirus, isolated from cattle on the westernmost island of Japan.

A novel orbivirus (genus Orbivirus, family Reoviridae), designated Yonaguni orbivirus (YONOV), was isolated from bovine blood collected on a subtropical island of Japan in 2015. The YONOV genome (20,054 nucleotides in total) has a coding arrangement similar to those of mosquito-borne orbiviruses. YONOV has a close genetic relationship to mosquito-borne orbiviruses, especially to Mobuck virus (MBV), which was isolated in North America. However, YONOV and MBV share less than 74% nucleotide sequence identity in the major subcore protein (T2) coding sequence, which satisfies the criterion for species demarcation. It is still uncertain whether YONOV should be assigned to a novel species in the genus Orbivirus.

Genomic characterization of Changuinola viruses from Panama: evidence for multiple genome segment reassortment.

The complete coding sequences of five divergent strains of Changuinola virus (CGLV), collected over a 16-year period in Panama, were determined, using viral metagenomics. Each strain had 10 RNA segments that encoded structural and non-structural proteins with amino acid identities ranging from 33 to 99% with sequences of other 15 members of the Changuinola virus (Reoviridae: Orbivirus) species group. Genetic analyses of the five Panamanian virus strains revealed probable reassortment among multiple segments of the viruses.

Skunk River virus, a novel orbivirus isolated from Aedes trivittatus in the United States.

The genomic organization and in vitro host range of a novel mosquito-associated orbivirus, designated Skunk River virus, is described. The virus was isolated from Aedes trivittatus collected in Iowa in the United States. Three recognized viruses were also recovered: Culex flavivirus (family Flaviviridae), Houston virus (family Mesoniviridae) and Umatilla virus (family Reoviridae). The genome of Skunk River virus contains 10 segments and its organization is characteristic of viruses in the genus Orbivirus (family Reoviridae). The coding region of each segment was fully sequenced, revealing that the greatest nucleotide identity was to the corresponding regions of Big Cypress orbivirus and Sathuvachari virus, two recently described mosquito-associated orbiviruses. The phylogenetic inference is in agreement with these findings. In vitro host range experiments revealed that Aedes, Anopheles and Culex cell lines, and select lepidopteran and rodent cell lines, are permissive to Skunk River virus replication. In conclusion, we provide evidence of a novel mosquito-associated orbivirus in Iowa.

Equine Encephalosis Virus in India, 2008.

A virus isolated from a sick horse from India in 2008 was confirmed by next-generation sequencing analysis to be equine encephalosis virus (EEV). EEV in India is concerning because several species of Culicoides midge, which play a major role in EEV natural maintenance and transmission, are present in this country.

Full genome sequence of a Sathuvachari virus strain isolated in the southwestern-most archipelago of Japan.

Two virus strains, tentatively designated as ON-6/P/05 and ON-7/E/05, were isolated from blood samples of healthy cattle in the Yaeyama Islands, located in the southwestern-most region of Japan, in 2005. Ultrastructural observations of infected baby hamster (BHK-21) cells revealed that the viruses had features consistent with those of orbivirus. As with other orbiviruses, the viral genome consists of 10 double-stranded RNA segments. The full genome sequence of ON-6/P/05 was determined and shared high nucleotide and amino acid identities (90.07-98.22% nucleotide identity; 96.16-99.72% amino acid identity) with that of Sathuvachari virus (SVIV), a member of the species Sathuvachari virus of the genus Orbivirus, originally isolated from starlings collected in southern India in 1963. The sequence of segment two of ON-7/E/05 was identical to that of ON-6/P/05. The isolation of SVIV from cattle also indicated that the virus has a wider host range than previously thought. The potential pathogenicity of SVIV in domestic animals should be considered in future disease surveillance within its distribution range.

Isolation of Tibet orbivirus from Culicoides and associated infections in livestock in Yunnan, China.

BACKGROUND: Culicoides-borne orbiviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), are important pathogens that cause animal epidemic diseases leading to significant loss of domestic animals. This study was conducted to identify Culicoides-borne arboviruses and to investigate the associated infections in local livestock in Yunnan, China. METHODS: Culicoides were collected overnight in Mangshi City using light traps during August 2013. A virus was isolated from the collected Culicoides and grown using baby hamster kidney (BHK-21), Vero, Madin-Darby bovine kidney (MDBK) and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by polyacrylamide gel (PAGE) analysis. A full-length cDNA copy of the genome was amplified and sequenced. Serological investigations were conducted in local cattle, buffalo and goat using plaque-reduction neutralization tests. RESULTS: We isolated a viral strain (DH13C120) that caused cytopathogenic effects in BHK-21, Vero, MDBK and C6/36 cells. Suckling mice inoculated intracerebrally with DH13C120 showed signs of fatal neurovirulence. PAGE analysis indicated a genome consisting of 10 segments of double-stranded RNA that demonstrated a 3-3-3-1 pattern, similar to the migrating bands of Tibet orbivirus (TIBOV). Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), sub-core-shell (T2, and outer core (T13) proteins revealed that DH13C120 clustered with TIBOV, and the amino acid sequences of DH13C120 virus shared more than 98% identity with TIBOV XZ0906. However, outer capsid protein VP2 and outer capsid protein VP5 shared only 43.1 and 79.3% identity, respectively, indicating that the DH13C120 virus belongs to TIBOV, and it may represent different serotypes with XZ0906. A serosurvey revealed the presence of neutralizing antibodies with 90% plaque-reduction neutralization against TIBOV DH13C120 in local cattle (44%), buffalo (20%), and goat (4%). Four-fold or higher levels of TIBOV-2-neutralizing antibody titers were detected between the convalescent and acute phases of infection in local livestock. CONCLUSIONS: A new strain of TIBOV was isolated from Culicoides. This study provides the first evidence of TIBOV infection in livestock in Yunnan, China, and suggests that TIBOV could be a potential pathogen in livestock.

Discovery of new orbiviruses and totivirus from Anopheles mosquitoes in Eastern Australia.

Three new viruses classifiable within the Totivirus and Orbivirus genera were detected from Anopheles mosquito species collected in Eastern Australia. The viruses could not be isolated in C6/36 mosquito cell cultures but were shown to replicate in their mosquito hosts by small RNA analysis. The viruses grouped phylogenetically with other viruses recently detected in insects. These discoveries contribute to a better understanding of commensal viruses in Australian mosquitoes and the evolution of these viruses.

Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan.

Among the tick-borne orbiviruses (genus Orbivirus, family Reoviridae), 36 serotypes are currently classified within a single virus species, Great Island virus. In this study, we report the first characterization of a tick-borne orbivirus isolated from the tick Ixodes turdus in Japan, which we identified as a new member of the species Great Island virus. The virus isolate, designated Muko virus (MUV), replicated and induced cytopathic effects in BHK-21, Vero E6, and CCL-141 cells and caused high mortality in suckling mice after intracerebral inoculation. Full genome sequence analysis showed that MUV shared the greatest phylogenetic similarity with Tribec virus in terms of the amino acid sequences of all viral proteins except for outer capsid protein 1 (OC1; VP4 of MUV). Analysis of genome segment 9 in MUV detected an uninterrupted open reading frame that overlaps with VP6 (Hel), which putatively encodes a molecular and functional equivalent of NS4 from Great Island virus. Our study provides new insights into the geographic distribution, genetic diversity, and evolutionary history of the members of the species Great Island virus.

Mobuck virus genome sequence and phylogenetic analysis: identification of a novel Orbivirus isolated from a white-tailed deer in Missouri, USA.

The genus Orbivirus includes a diverse group of segmented dsRNA viruses that are transmitted via arthropods, have a global distribution and affect a wide range of hosts. A novel orbivirus was co-isolated with epizootic haemorrhagic disease virus (EHDV) from a white-tailed deer (Odocoileus virginianus) exhibiting clinical signs characteristic of EHDV. Using antiserum generated against EHDV, a pure isolate of the novel non-cytopathic orbivirus was obtained in Aedes albopictus cell culture. Genomic sequencing and phylogenetic analysis of predicted ORFs showed that eight of the ten ORFs were most homologous to Peruvian horse sickness virus (PHSV), with amino acid identities of 44.3-73.7 %. The remaining two ORFs, VP3 and VP5, were most similar to Middle Point orbivirus (35.9 %) and Yunnan orbivirus (59.8 %), respectively. Taxonomic classification of orbiviruses is largely based on homology of the major subcore structural protein VP2(T2), encoded by segment 2 for mobuck virus. With only 69.1 % amino acid identity to PHSV, we propose mobuck virus as the prototype of a new species of Orbivirus.

First isolation and characterization of a mosquito-borne orbivirus belonging to the species Umatilla virus in East Asia.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

First Isolation and Molecular Characterization of Umatilla Virus (Sedoreoviridae, Orbivirus) in Brazil.

In this study, we provide a genomic description of the first isolation of the Umattila virus (UMAV) in Brazil. The virus was obtained from the blood of a bird (Turdus fumigatus) and isolated in a C6/36 cell culture. The viral genome contains ten segments, and its organization is characteristic of viruses of the genus Orbivirus (family Sedoreoviridae). The coding region of each segment was sequenced, demonstrating the nucleotide identity with UMAV. The phylogenetic inference results were in line with these findings and demonstrated the formation of two distinct monophyletic clades containing strains isolated around the world, where our isolate, belonging to the same clade as the prototype strain, was allocated to a different subclade, highlighting the genetic divergence between them. This work reports the first isolation of UMAV in Brazil, and due to the scarcity of information on this viral agent in the scientific literature, it is essential to carry out further studies to better understand its epidemiology, dispersion, and, in particular, its interactions with vertebrate hosts, vectors, and the environment.

Purification of Epizootic Hemorrhagic Disease Virus (and Other Orbiviruses) Particles from Infected Mammalian or Insect Cells.

Epizootic hemorrhagic disease virus (EHDV), like other orbiviruses, infects and replicates in mammalian and insect vector cells. Within its ruminant hosts EHDV, like bluetongue virus (BTV), it has mainly been associated with infection of endothelial cells of capillaries as well as leukocyte subsets. Furthermore, EHDV infects and replicates within its biological vector, Culicoides biting midges and Culicoides-derived cells. A wide range of common laboratory cell lines such as BHK, BSR, and Vero cells are susceptible to infection with certain EHDV strains. Cell culture supernatants of infected cells are commonly used for both in vivo and in vitro infection studies. For specific virological or immunological studies, using highly purified virus particles, however, might be beneficial or even required. Here we describe a purification method for EHDV particles, which had been originally developed for certain strains of BTV.

Evidence of Active Orbivirus Transmission in 2016 in Kansas and Nebraska.

Retrospective serological and case diagnostic data of endemic bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) provide evidence of viral transmission among livestock and wildlife from 2016 in Kansas and Nebraska. Serological testing of mature cattle in nine distinct regional zones of Kansas revealed 76% to 100% had detectable antibodies to BTV and/or EHDV. Specimens tested in the Kansas Veterinary Diagnostic Laboratory (55 submissions) were 51% test positive for antibodies to BTV and/or EHDV. Specimens tested in the Nebraska Veterinary Diagnostic Center (283 submissions) were 25% test positive for antibodies to BTV and/or EHDV. Low disease incidence in white-tailed deer and other susceptible wild ungulates was observed during 2016. However, there were no confirmed reports of disease in livestock in either state. The reasons for emergence of significant clinical disease in livestock and wildlife populations remain undefined.

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.

[The taxonomy of the Baku virus (BAKV; Reoviridae, Orbivirus) isolated from the birds obligate parasites Argasidae ticks in Azerbaijan, Turkmenistan, and Uzbekistan].

The Baku virus (BAKV) was originally isolated from the ticks Ornithodoros capensis Neumann, 1901 (Acari: Argasidae) collected from the seagull (Larus argentatus) seating nests on the islands of the Baku archipelago, the Caspian sea. BAKV was assigned to Kemerovo group (KEMV) (Orbivirus, Reoviridae). The BAKV was frequently isolated from the ticks O. coniceps Canestrini, 1980, collected from L. argentatus and tern (Sterna hirundo) nests in Turkmenia and pigeon (Columba livia neglecta) nests in Uzbekistan. In this work, the genome of the BAKV was sequenced using the next-generation sequencing technology. The BAKV Pol protein has 48.6% identity level with the viruses of the Great Island Virus group and at average 41% with non-tick orbiviruses. The BAKV T2 protein level identity with the orbiviruses ranges from 23.7% to 64.8%. The maximum identity level of the T2 protein (64.8%) is observed for the tick-borne viruses of the GIV (KEMV) group. According to the conducted molecular-genetic and phylogenetic analysis, the BAKV is a novel species of the genus Orbivirus. It forms a phylogenetic group distinctly related to the GIV group.