Arboviruses in the Astrakhan region of Russia for 2018 season: The development of multiplex PCR assays and analysis of mosquitoes, ticks, and human blood sera.

The Astrakhan region of Russia is endemic for the number of arboviruses. In this paper, we describe the results of the detection of the list of neglected arboviruses in the Astrakhan region for the 2018 season. For the purpose of the study in-house PCR assays for detection of 18 arboviruses have been developed and validated using arboviruses obtained from Russian State Collection of Viruses. Pools of ticks (n = 463) and mosquitoes (n = 312) as well as 420 samples of human patients sera have been collected and analyzed. Using developed multiplex real-time PCR assays we were able to detect RNA of eight arboviruses (Crimean-Congo hemorrhagic fever virus, Dhori (Batken strain) virus, Batai virus, Tahyna virus, Uukuniemi virus, Inkoo virus, Sindbis virus and West Nile fever virus). All discovered viruses are capable of infecting humans causing fever and in some cases severe forms with hemorrhagic or neurologic symptoms. From PCR-positive samples, we were able to recover one isolate each of Dhori (Batken strain) virus and Crimean-Congo hemorrhagic fever virus which were further characterized by next-generation sequencing. The genomic sequences of identified Dhori (Batken strain) virus strain represent the most complete genome of Batken virus strain among previously reported.

Comparative characterization of the reassortant Orthobunyavirus Ngari with putative parental viruses, Bunyamwera and Batai: in vitro characterization and ex vivo stability.

Bunyamwera (BUNV), Batai (BATV) and Ngari (NRIV) are mosquito-borne viruses that are members of the genus Orthobunyavirus in the order Bunyavirales. These three viruses are enveloped with single-stranded, negative-sense RNA genomes consiting of three segments, denoted as Small (S), Medium (M) and Large (L). Ngari is thought to be the natural reassortant progeny of Bunyamwera and Batai viruses. The relationship between these 'parental' viruses and the 'progeny' poses an interesting question, especially given that there is overlap in their respective transmission ecologies, but differences in their infection host ranges and pathogenesis. We compared the in vivo kinetics of these three viruses in a common laboratory system and found no significant difference in growth kinetics. There was, however, a tendency of BATV to have smaller plaques than either BUNV or NRIV. Furthermore, we determined that all three viruses are stable in extracellular conditions and retain infectivity for a week in non-cellular media, which has public health and biosafety implications. The study of this understudied group of viruses addresses a need for basic characterization of viruses that have not yet reached epidemic transmission intensity, but that have the potential due to their infectivity to both human and animal hosts. These results lay the groundwork for future studies of these neglected viruses of potential public and One Health importance.

Identification of Bunyamwera and Possible Other Orthobunyavirus Infections and Disease in Cattle during a Rift Valley Fever Outbreak in Rwanda in 2018.

In 2018, a large outbreak of Rift Valley fever (RVF)-like illness in cattle in Rwanda and surrounding countries was reported. From this outbreak, sera samples from 157 cows and 28 goats suspected to be cases of RVF were tested to confirm or determine the etiology of the disease. Specifically, the hypothesis that orthobunyaviruses-Bunyamwera virus (BUNV), Batai virus (BATV), and Ngari virus (NRIV)-were co-circulating and contributed to RVF-like disease was tested. Using reverse transcriptase-polymerase chain reaction (RT-PCR), RVFV RNA was detected in approximately 30% of acutely ill animals, but in all cases of hemorrhagic disease. Seven cows with experienced abortion had positive amplification and visualization by gel electrophoresis of all three segments of either BUNV or BATV, and three of these were suggested to be coinfected with BUNV and BATV. On sequencing, five of these seven cows were conclusively positive for BUNV. However, in several other animals, sequencing was successful for some but not all segments of targeted viruses BUNV and BATV. In addition, there was evidence of RVFV-orthobunyavirus coinfection, through RT-PCR/gel electrophoresis and subsequent Sanger sequencing. In no cases were we able to definitely identify the specific coinfecting viral species. This is the first time evidence for orthobunyavirus circulation has been molecularly confirmed in Rwanda. Furthermore, RT-PCR results suggest that BUNV and BATV may coinfect cattle and that RVFV-infected animals may be coinfected with other orthobunyaviruses. Finally, we confirm that BUNV and, perhaps, other orthobunyaviruses were co-circulating with RVFV and contributed to the burden of disease attributed to RVFV in Rwanda.

Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of the Batai virus.

Chittoor virus (CHITV), a mosquito-borne bunyavirus (Orthobunyavirus: Bunyaviridae) isolated in India, has been found to be antigenically close to the Batai virus (BATV), which has a wide distribution across Asia, Europe, and Africa. The latter virus causes influenza-like illness in humans and mild illness in sheep and goats. BATV has been involved in genetic reassortment with other bunyaviruses, generating novel genome combinations and causing severe clinical manifestations including hemorrhagic fever. Conversely, CHITV has never been associated with any major outbreaks in India, although neutralizing antibodies have been detected in humans and domestic animals. Repeated isolations and seroprevalence have prompted us to determine the vector competence of three important mosquito species, viz., Culex quinquefasciatus, Culex tritaeniorhynchus, and Aedes aegypti, for CHITV. The three mosquito species replicated CHITV to titers of 6.3, 5.0, and 5.2 log10 TCID50/mL, respectively, and maintained the virus for substantial periods. Both of the Culex species demonstrated vector competence, while A. aegypti did not. Horizontal transmission to infant mice was also demonstrated by both Culex species. Active circulation of the virus and the availability of both susceptible hosts and competent vector mosquitoes pose a serious threat to public health should there be a reassortment.

Cache Valley Virus in Aedes japonicus japonicus Mosquitoes, Appalachian Region, United States.

We detected Cache Valley virus in Aedes japonicus, a widely distributed invasive mosquito species, in an Appalachian forest in the United States. The forest contained abundant white-tailed deer, a major host of the mosquito and virus. Vector competence trials indicated that Ae. j. japonicus mosquitoes can transmit this virus in this region.

Comparison of two zoonotic viruses from the order Bunyavirales.

A comparison of two geographicallly distinct viruses in the order Bunyavirales that are zoonotic and known to cause congenital abnormalities in ruminant livestock was performed. One of these viruses, Cache Valley fever virus, is found in the Americas and is primarily associated with disease in sheep. The other, Rift Valley fever virus, is found in Sub-Saharan Africa and is associated with disease in camels, cattle, goats and sheep. Neither virus has been associated with teratogenicity in humans to date. These two viruses are briefly reviewed and potential for genetic changes especially if introduced into new ecology that could affect pathogenicity are discussed.

Characterization of Batai virus isolated from a domestic Muscovy duck (Cairina moschate).

Batai virus (BATV) belongs to the genus Orthobunyavirus of the family Bunyaviridae. It has been isolated from mosquitos, pigs, cattle, and humans throughout Africa, Asia, and Europe, and causes clinical signs in domestic animals and humans. Here, we report the isolation of BATV from a domestic duck flock. Genome sequence analysis revealed clustering of this isolate in the Africa-Asia lineage. The virus replicated in mosquitos and vertebrate host cells, showing different phenotypic characteristics, and showed the potential to infect mice. This is the first report of BATV in domestic birds and indicates the wide circulation of BATV in China.

Phylogenetic analysis of Bunyamwera and Ngari viruses (family Bunyaviridae, genus Orthobunyavirus) isolated in Kenya.

Orthobunyaviruses, tri-segmented, negative-sense RNA viruses, have long been associated with mild to severe human disease in Africa, but not haemorrhagic fever. However, during a Rift Valley fever outbreak in East Africa in 1997-1998, Ngari virus was isolated from two patients and antibody detected in several others with haemorrhagic fever. The isolates were used to identify Ngari virus as a natural Orthobunyavirus reassortant. Despite their potential to reassort and cause severe human disease, characterization of orthobunyaviruses is hampered by paucity of genetic sequences. Our objective was to obtain complete gene sequences of two Bunyamwera virus and three Ngari virus isolates from recent surveys in Kenya and to determine their phylogenetic positioning within the Bunyamwera serogroup. Newly sequenced Kenyan Bunyamwera virus isolates clustered closest to a Bunyamwera virus isolate from the same locality and a Central African Republic isolate indicating that similar strains may be circulating regionally. Recent Kenyan Ngari isolates were closest to the Ngari isolates associated with the 1997-1998 haemorrhagic fever outbreak. We observed a temporal/geographical relationship among Ngari isolates in all three gene segments suggesting a geographical/temporal association with genetic diversity. These sequences in addition to earlier sequences can be used for future analyses of this neglected but potentially deadly group of viruses.

Spatial-temporal analysis of Cache Valley virus (Bunyaviridae: Orthobunyavirus) infection in anopheline and culicine mosquitoes (Diptera: Culicidae) in the northeastern United States, 1997-2012.

Cache Valley virus (CVV) is a mosquito-borne bunyavirus (family Bunyaviridae, genus Orthobunyavirus) that is enzootic throughout much of North and Central America. White-tailed deer (Odocoileus virginianus) have been incriminated as important reservoir and amplification hosts. CVV has been found in a diverse array of mosquito species, but the principal vectors are unknown. A 16-year study was undertaken to identify the primary mosquito vectors in Connecticut, quantify seasonal prevalence rates of infection, and define the spatial geographic distribution of CVV in the state as a function of land use and white-tailed deer populations, which have increased substantially over this period. CVV was isolated from 16 mosquito species in seven genera, almost all of which were multivoltine and mammalophilic. Anopheles (An.) punctipennis was incriminated as the most consistent and likely vector in this region on the basis of yearly isolation frequencies and the spatial geographic distribution of infected mosquitoes. Other species exhibiting frequent temporal and moderate spatial geographic patterns of virus isolation within the state included Ochlerotatus (Oc.) trivittatus, Oc. canadensis, Aedes (Ae.) vexans, and Ae. cinereus. New isolation records for CVV were established for An. walkeri, Culiseta melanura, and Oc. cantator. Other species from which CVV was isolated included An. quadrimaculatus, Coquillettidia perturbans, Culex salinarius, Oc. japonicus, Oc. sollicitans, Oc. taeniorhynchus, Oc. triseriatus, and Psorophora ferox. Mosquitoes infected with CVV were equally distributed throughout urban, suburban, and rural locales, and infection rates were not directly associated with the localized abundance of white-tailed deer, possibly due to their saturation throughout the region. Virus activity in mosquitoes was episodic with no consistent pattern from year-to-year, and fluctuations in yearly seasonal infection rates did not appear to be directly impacted by overall mosquito abundance. Virus infection in mosquitoes occurred late in the season that mostly extended from mid-August through September, when adult mosquito populations were visibly declining and were comparatively low. Findings argue for a limited role for vertical transmission for the perpetuation of CVV as occurs with other related bunyaviruses.

Isolation and complete nucleotide sequence of a Batai virus strain in Inner Mongolia, China.

BACKGROUND: Batai virus (BATV) is a member of the Orthobunyavirus genus of the family Bunyaviridae, and a vector-borne pathogen. Genomic variations of BATV exist in different regions of the world, due to genetic reassortment. Whole-genome sequencing of any isolate is necessary for a phylogenetic analysis. In 1998, a BATV strain was isolated from an Anopheles philippines mosquito in Yunnan Province, China. This strain has not been found to infect any other host. We investigated BATV infection in cattle in Inner Mongolia, China and performed deep sequencing of the genome of the BATV isolate. FINDINGS: Ninety-five blood samples were collected from cattle in Inner Mongolia, China in 2012. The BATV infection rate was 2.1%. Previously, BATV strain NM/12 was isolated from two cattle in Inner Mongolia, China, and the whole genomic sequence of the strain has been available. We determined the complete genomic nucleotide sequences of the small (S), medium (M), and large (L) genome segments using bovine blood obtained in 2012, and the nucleotide homologies of these segments with those from GenBank were 88.7%-97%, 84%-95.4%, and 72.6%-95.8%, respectively. The deduced amino acid identities were 87.2-99.7%, 64.2-96.8%, and 81.1-98.6%. Phylogenetic analyses based on full-length genomic sequences indicated that the M and L segments, and a portion of the S segment, of NM/12 are most closely related to the BATV strains isolated in Asia. The S and M segments of NM/12 were independent of phylogenetic lineages. The L segment was the most closely related to Chittoor/IG-20217 (isolated in India), and distantly related to isolated strains in Italy. Nucleotide substitution rates in the nucleotide sequences that code for the nucleocapsid, envelope glycoprotein, and polymerase protein of NM/12 strain were 2.56%, 4.69%, and 4.21%, respectively, relative to the original strain of MM2222. CONCLUSION: A novel BATV NM/12 strain from bovine serum collected in Inner Mongolia was isolated from cattle in China for the first time. Our findings elucidate the evolutionary status of the BATV NM/12 strain among different orthobunyavirus strains and may provide some clues to prevent the emergence of BATV infection in cattle and humans.

Genome sequence analysis of in vitro and in vivo phenotypes of Bunyamwera and Ngari virus isolates from northern Kenya.

Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP) and large plaque (LP) and determined in vitro growth properties and in vivo pathogenesis in suckling mice. We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I) that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies.

[Complete genome analysis of the Batai virus (BATV) and the new Anadyr virus (ANADV) of the Bunyamwera group (Bunyaviridae, Orthobunyavirus) isolated in Russia].

Almost complete nucleotide sequences for the S, M, and L segments were obtained for three strains of the Batai virus (Bunyamwera serogroup, genus Orthobunyavirus, Bunyaviridae family). Based on the results of the phylogenetic analysis conducted forthe three genomic segments LEIV Ast507 and LEIV-Ast528 strains were grouped with other European BATV isolates and were found to be almost identical to the strain 42 isolated from Volgograd Region, Russia, 2003. Surprisingly, LEIV-13395 strain isolated from the Aedes sp. mosquitos in Magadan Oblast, 1987, turned out to be a novel genotype inside Bunyamwera serogroup. The highest nucleotide identity levels of LEIV-13395 genomicsegments (86.9%, 80.8%, 79.7% for S, M and L segments respectively) were observed with corresponding segments of the Batai virus.

Clarifying Bunyamwera virus riddles of the past.

Pyrosequencing data and phylogenetic analysis for the full genome of Ilesha virus, Ngari virus and Calovo virus are described clarifying their much discussed relationship within the species Bunyamwera virus of the genus Orthobunyavirus of the Bunyaviridae.

Complete genome sequencing of four geographically diverse strains of Batai virus.

Batai virus (BATV) is a widely distributed but poorly studied member of the Orthobunyavirus genus in the family Bunyaviridae and is of particular interest as a known participant in natural reassortment events. Both research and surveillance efforts on this and other related viruses have been hampered by the lack of available full-length sequence data covering all three genomic segments. Here, we report the complete genome sequence of four BATV strains (MM2222, Chittoor/IG-20217, UgMP-6830, and MS50) isolated from various geographical locations. Based on these data, we have determined that strain MS50 is in fact unrelated to BATV and likely represents as a novel genotype in the genus Orthobunyavirus.

Nucleotide sequencing and serologic analysis of Cache Valley virus isolates from the Yucatan Peninsula of Mexico.

Nucleotide sequencing was performed on part of the medium and large genome segments of 17 Cache Valley virus (CVV) isolates from the Yucatan Peninsula of Mexico. Alignment of these sequences to all other sequences in the Genbank database revealed that they have greatest nucleotide identity (97-98 %) with the equivalent regions of Tlacotalpan virus (TLAV), which is considered to be a variety of CVV. Next, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice that had been inoculated with a representative isolate from the Yucatan Peninsula (CVV-478) or the prototype TLAV isolate (61-D-240). The PRNT titers exhibited a twofold difference in one direction and no difference in the other direction suggesting that CVV-478 and 61-D-240 belong to the same CVV subtype. In conclusion, we demonstrate that the CVV isolates from the Yucatan Peninsula of Mexico are genetically and antigenically similar to the prototype TLAV isolate.

Sequence and phylogenetic data indicate that an orthobunyavirus recently detected in the Yucatan Peninsula of Mexico is a novel reassortant of Potosi and Cache Valley viruses.

We determined the complete nucleotide sequences of the small (S) and medium (M) RNA segments of an orthobunyavirus isolated from mosquitoes in the Yucatan Peninsula of Mexico. A 528-nt region of the large (L) RNA segment was also sequenced. The S RNA segment has greatest nucleotide identity to the homologous region of Cache Valley virus (CVV; 98%) followed by Potosi virus (POTV; 89%) and Northway virus (86%). The M RNA segment has 96% nucleotide identity to the homologous region of POTV, and less than 74% nucleotide identity to the homologous regions of all other orthobunyaviruses for which M segment sequence data are available. The L RNA segment has greatest nucleotide identity to the homologous region of POTV (98%) followed by CVV (82%) and Tensaw virus (77%). These data indicate that the virus, tentatively named Cholul virus (CHLV), is a novel reassortant that acquired its S RNA segment from CVV and its M and L RNA segments from POTV. Phylogenetic data support this conclusion.

Molecular characterization of Chittoor (Batai) virus isolates from India.

BACKGROUND & OBJECTIVES: Chittoor virus (CHITV) belongs to genus Orthobunyavirus, family Bunyaviridae. It has been isolated from various species of mosquitoes and pig from different parts of India. Five isolates of CHITV were characterized at the molecular level and compared with other Batai viruses (BATV) to find out any kind of reassortment in their genome. METHODS: Complete nucelocapsid (S), glycoprotein (M) and partial RNA polymerase (L) segments of CHITV were amplified and sequenced. These sequences were compared with those of Batai viruses, isolated from different geographical locations in Asia, Africa and Europe. RESULTS: Phylogenetic analysis revealed CHITV as a variant of BATV. High level of conservation was seen among the CHITV isolates studied. The CHITV sequences showed clustering in one lineage with the sequences from Japan and Malaysia, however, BATV sequences from Europe and Africa formed a separate phylogenetic lineage. INTERPRETATION & CONCLUSIONS: The study indicates the presence of a single genotype of CHITV circulating in India, despite the involvement of different hosts in the natural cycle by this virus. Analysis of the sequences of the S, M and L segments of genome indicated that the virus has not undergone any reassortment. This virus has not caused any epidemic involving humans, however, replication of the virus in different mosquito and vertebrate hosts species suggests that it is a cause of concern.

[Molecular-genetic analysis of the Batai virus strains isolated from mosquitoes in Volgograd Region of the Russian Federation, West Ukraine, and Czech Republic].

Partial nucleotide sequence of the M-segment from five Batai virus strains was determined. These strains were isolated in Volgograd Region, West Ukraine, and Czech Republic. Our data based on the partial sequence of the M-segment of Batai virus strains demonstrated that the strains isolated in Russian Federation, Ukraine, and Czech Republic grouped together into an European genetic group that was distinct from Asian and African strains of Batai virus.

Molecular characterization of medically important viruses of the genus Orthobunyavirus.

We have characterized the full-length S segment RNA sequences of five human pathogens of the virus family Bunyaviridae, genus Orthobunyavirus. S segment sequences of Fort Sherman, Shokwe and Xingu viruses of the Bunyamwera serogroup, as well as those of Bwamba and Pongola viruses of the Bwamba serogroup, are described. S segment sequences of Bwamba and Pongola viruses represent the first nucleotide sequences characterized for viruses of the Bwamba serogroup. The described molecular and phylogenetic analyses of these and other selected viruses of the genus Orthobunyavirus reveal that a close sequence similarity is shared between the African Bwamba and the predominantly North American and European California serogroups of the genus Orthobunyavirus.