Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

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.

Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells.

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

First evidence of Schmallenberg virus infection in domestic ruminants in Kosovo and Albania.

Schmallenberg virus (SBV), a novel Orthobunyavirus, emerged in European domestic ruminants in 2011 causing abortions and malformations in newborns and none or mild clinical symptoms in adult animals. Here, a total of 364 bovine, ovine and caprine serum samples were collected in Kosovo and Albania between May 2014 and August 2016 and analyzed for the presence of anti­SBV antibodies. Sera were tested using an enzyme­linked immunosorbent assay (ELISA), and 48 ELISA­positive samples were subsequently analyzed by serum neutralization test (SNT). The overall percentage of ELISA positive results was 17.9%; 23.1% (53/229) was the prevalence observed in Kosovo (cattle 45.5%, sheep 19.2% and goat 6.8%), while 8.9% (12/135) was that observed in Albania (cattle 11.1%, sheep 0% and goat 20.0%). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested. This is the first study reporting SBV circulation in domestic ruminants in Kosovo and Albania, with indication that this virus has been present in Kosovo and Albania at least since 2014 without being detected.

Orthobunyaviruses in the Caribbean: Melao and Oropouche virus infections in school children in Haiti in 2014.

We report the identification of two orthobunyaviruses, Melao virus (MELV) and Oropouche virus (OROV), in plasma specimens from Haitian children with acute febrile illness who presented during outbreaks caused by alpha- and flaviviruses in 2014. Heretofore not described as a human pathogen, MELV was isolated in cell culture from the plasma of five case patients. OROV RNA was detected in the plasma of an additional child, using an unbiased sequencing approach, with phylogenetic inference suggesting a close relationship with strains from Brazil. Abdominal pain was reported by four case patients with MELV infections, with lymphadenopathy noted in two cases. Our findings document the occurrence of these orthobunyaviruses within the Caribbean region and highlight the critical importance of surveillance with viral genome sequence analyses to identify outbreaks caused by these and other emerging viruses.

Arbovirus serosurvey (Orthobunyavirus, Flavivirus, and Alphavirus) in a draft horse population from Santa Fe, Argentina (2013-2016).

In the present study, we serosurveyed the exposure of 222 draft horses to different arboviruses in the city of Santa Fe, Argentina. Plaque reduction neutralization tests confirmed exposure to Fort Sherman virus (FSV), Saint Louis encephalitis virus (SLEV), West Nile virus (WNV), and Río Negro virus (RNV). Apparently, Western and Eastern equine encephalitis viruses did not circulate in the population tested. The confirmation of five seroconversions for WNV, FSV, and SLEV and the association between prevalence and age are indicative of recent circulation. These results highlight the importance of considering draft horses in arboviral surveillance in urban and rural areas of developing countries.

Vector Competence of Peruvian Mosquitoes for Two Orthobunyaviruses Isolated From Mosquitoes Captured in Peru.

We evaluated the potential for mosquitoes collected in the Amazon Basin, near Iquitos, Peru, to become infected with and transmit Murutucu (MURV) and Itaqui viruses (ITQV) (Order Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus). Viremia levels in Syrian hamsters peaked 2 d after infection with either virus, and both viruses were highly lethal in hamsters with virtually all hamsters dying prior to 3-d postinfection. For almost all of the mosquito species tested some individuals were susceptible to infection and some developed a disseminated infection after oral exposure to either MURV or ITQV. However, only the Culex species (Culex (Culex) coronator Dyar and Knab [Diptera, Culicidae], Culex (Melanoconian) gnomatos Sallum, Huchings, and Ferreira [Diptera, Culicidae], Culex (Mel.) pedroi Sirivanakarn and Belkin [Diptera, Culicidae], and Culex (Mel.) vomerifer Komp [Diptera, Culicidae]) successfully transmitted virus by bite. However, even among these species, only about 37% of the individuals with a disseminated infection successfully transmitted these viruses, indicating a significant salivary gland barrier. Although little is known about the medical or veterinary importance of many members of the genus Orthobunyavirus, we have demonstrated that Culex spp. (Diptera, Culicidae) could be potential vectors.

Zoo animals as sentinels for Schmallenberg virus monitoring in Spain.

Schmallenberg virus (SBV) is a newly emerged vector-borne pathogen that affects many domestic and wild animal species. A serosurvey was carried out to assess SBV exposure in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals. Between 2002 and 2019, sera from 278 animals belonging to 73 different species were collected from five zoos (A-E). Thirty-one of these animals were longitudinally sampled at three of these zoo parks during the study period. Seropositivity was detected in 28 (10.1 %) of 278 animals analyzed by blocking ELISA. Specific anti-SBV antibodies were confirmed in 20 (7.2 %; 95 %CI: 4.2-10.3) animals of six different species using virus neutralization test (VNT). The multiple logistic regression model showed that "order" (Artiodactyla) and "zoo provenance" (zoo B; southern Spain) were risk factors potentially associated with SBV exposure. Two (8.7 %) of the 31 longitudinally-sampled individuals showed specific antibodies against SBV at all samplings whereas seroconversion was detected in one mouflon (Ovis aries musimon) and one Asian elephant (Elephas maximus) in 2016 and 2019, respectively. To the best of the author's knowledge, this is the first surveillance conducted on SBV in zoos in Spain. The results confirm SBV exposure in zoo animals in this country and indicate circulation of the virus before the first Schmallenberg disease outbreak was reported in Spain. Surveillance in zoological parks could be a complementary approach to monitoring SBV activity. Further studies are warranted to assess the impact of this virus on the health status of susceptible zoo animals.

Ngari virus (Orthobunyavirus, Peribunyaviridae) in ixodid ticks collected from cattle in Guinea.

Ngari virus is a mosquito-borne virus belonging to the genus Orthobunyavirus (Peribunyaviridae family). This virus is pathogenic to humans and causes severe illness. Ngari virus is present in several African countries, including Madagascar. Here, we report the detection of Ngari virus in ixodid ticks collected from cows in Guinea. A tick survey was conducted in March-November of 2018 in six regions of Guinea. The sample comprised 710 pools, with a total of 2067 ticks belonging to five species collected from 197 cows. At the initial stage, we screened a subsample of tick pools of vector-borne viruses with a multiplex genus-specific primer panel. In the second stage of the study, we narrowed the search and screened all the samples by qPCR for the detection of Ngari virus. All positive samples were sequenced with primers flanking Ngari virus-specific fragments on the S and M segments. We found Ngari virus in 12 pools that were formed from engorged ticks collected from livestock in three villages of the Kindia and Kankan regions. Sequencing of the S and M segments confirmed that the detected viruses belong to Ngari virus, and the viruses were most similar to the strain Adrar, which was isolated in Mauritania. We detected viral RNA in ticks of the following species: Amblyomma variegatum, Rhipicephalus geigyi, and Rh. (Boophilus) spp. There is no evidence that ixodid ticks are competent vectors of the Ngari virus. Most likely, the ticks obtained the virus through blood from an infected host. The study of engorged ticks can be recommended as a simpler approach for the wide screening of the Ngari virus and subsequent testing of cattle and mosquitos in those locations where the PCR-positive ticks were collected.

Emerging orthobunyaviruses associated with CNS disease.

The Orthobunyavirus genus comprises a wide range of arthropod-borne viruses which are prevalent worldwide and commonly associated with central nervous system (CNS) disease in humans and other vertebrates. Several orthobunyaviruses have recently emerged and increasingly more will likely do so in the future. Despite this large number, an overview of these viruses is currently lacking, making it challenging to determine importance from a One Health perspective. Causality is a key feature of determining importance, yet classical tools are unfit to evaluate the causality of orthobunyaviral CNS disease. Therefore, we aimed to provide an overview of orthobunyaviral CNS disease in vertebrates and objectify the causality strength of each virus. In total, we identified 27 orthobunyaviruses described in literature to be associated with CNS disease. Ten were associated with disease in multiple host species of which seven included humans. Seven viruses were associated with both congenital and postnatal CNS disease. CNS disease-associated orthobunyaviruses were spread across all known Orthobunyavirus serogroups by phylogenetic analyses. Taken together, these results indicate that orthobunyaviruses may have a common tendency to infect the CNS of vertebrates. Next, we developed six tailor-made causality indicators and evaluated the causality strength of each of the identified orthobunyaviruses. Nine viruses had a 'strong' causality score and were deemed causal. Eight had a 'moderate' and ten a 'weak' causality score. Notably, there was a lack of case-control studies, which was only available for one virus. We, therefore, stress the importance of proper case-control studies as a fundamental aspect of proving causality. This comprehensible overview can be used to identify orthobunyaviruses which may be considered causal, reveal research gaps for viruses with moderate to low causality scores, and provide a framework to evaluate the causality of orthobunyaviruses that may newly emerge in the future.

Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus.

Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.

Cross-sectional study of British wild deer for evidence of Schmallenberg virus infection.

BACKGROUND: Schmallenberg virus (SBV) is an orthobunyavirus, carried by Culicoides biting midges, that causes reproductive problems in adult ruminants when infected during their gestation period. SBV was first detected in ruminants in the UK in 2011/2012 and then again in 2016. The reason behind the 2016 re-emergence of SBV is unknown, but one possibility is that it can be maintained in wildlife, such as deer. SBV has been detected at high seroprevalence in deer in a number of European countries, but only once in the UK in a single region. METHODS: The purpose of this study was to survey wild deer across Great Britain for recent evidence of SBV. Deer hunters were recruited for the purpose of providing postmortem blood samples to be tested for SBV antibodies. RESULTS: The seroprevalence of SBV in the British wild deer population was 13.8 per cent; found in red, roe, muntjac and fallow deer species, with more in deer further south. CONCLUSION: These results support the growing concern that SBV is now endemic in Great Britain and highlight the need to know the role of wildlife in SBV transmission.

Re-circulation of Schmallenberg virus, Germany, 2019.

Schmallenberg virus (SBV), an insect-transmitted orthobunyavirus that induces severe foetal malformation in calves and lambs, was detected for the first time in late summer 2011 in Central Europe. Thereafter, the virus spread rapidly across the continent causing a large epidemic in the ruminant population. In 2019, detection of virus was again reported more frequently in Germany. From March to November, infections of viremic adult animals were noticed. In September, SBV genome was also detected in newborn lambs. Altogether, affected species included cattle, sheep, a goat and a fallow deer. M-segment sequences were generated from viruses detected in viremic cattle and compared to viral sequences from previous years. The genome of viruses detected in the blood of acutely infected adult cattle and sheep, which represent the circulating SBV strains, seems very stable over the course of nine years and in various European countries. The nucleotide similarities of these viruses are as high as 99.4%-100%. The renewed SBV circulation in 2019 in the country, in which the virus was first detected in 2011 and where it circulated again in 2014 and 2016, suggests the establishment of an enzootic status in Central Europe with regular larger waves in a cycle of around 3 years. Therefore, it has to be anticipated that SBV will re-emerge at similar intervals in future, and hence, it represents a constant threat for the continent's ruminant population.

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.

Oropouche virus detection in saliva and urine.

Oropouche virus (OROV) is an arthropod-borne virus of the Peribunyaviridae family, transmitted to humans primarily by Culicoides paraensis. It is one of the main arboviruses infecting humans in Brazil, primarily in the Amazon Region. Here, we report the detection of OROV in the saliva and urine of a patient whose samples were collected five days after the onset of symptoms. Nucleotide sequencing and phylogenetic analysis further confirmed the results. To our knowledge, this is the first study reporting the detection of OROV in the saliva and urine of an infected patient. In addition, the results of our study expand the current knowledge pertaining to the natural history of Oropouche fever.

Identification of animal hosts of Fort Sherman virus, a New World zoonotic orthobunyavirus.

An orthobunyavirus termed Fort Sherman virus (FSV) was isolated in 1985 from a febrile US soldier in Panama, yet potential animal reservoirs remained unknown. We investigated sera from 192 clinically healthy peri-domestic animals sampled in northeastern Brazil during 2014-2018 by broadly reactive RT-PCR for orthobunyavirus RNA, including 50 cattle, 57 sheep, 35 goats and 50 horses. One horse sampled in 2018 was positive (0.5%; 95% CI, 0.01-3.2) at 6.2 × 103 viral RNA copies/mL. Genomic comparisons following virus isolation in Vero cells and deep sequencing revealed high identity of translated amino acid sequences between the new orthobunyavirus and the Panamanian FSV prototype (genes: L, 98.8%; M, 83.5%; S, 100%), suggesting these viruses are conspecific. Database comparisons revealed even higher genomic identity between the Brazilian FSV and taxonomically unassigned Argentinian mosquito- and horse-derived viruses sampled in 1965, 1982 and 2013 with only 1.1% maximum translated amino acid distances across viral genes, suggesting the Argentinian viruses were also distinct FSV strains. The Panamanian FSV strain was an M gene reassortant relative to all Southern American FSV strains, clustering phylogenetically with Cache Valley virus (CVV). Mean dN/dS ratios among FSV genes ranged from 0.03 to 0.07, compatible with strong purifying selection. FSV-specific neutralizing antibodies occurred at relatively high end-point titres in the range of 1:300 in 22.0% of horses (11 out of 50 animals), 8.0% of cattle (4/50 animals), 7.0% of sheep (4/57 animals) and 2.9% of goats (1/35 animals). High specificity of serologic testing was suggested by significantly higher overall FSV-specific compared to CVV- and Bunyamwera virus-specific end-point titres (p = .009), corroborating a broad vertebrate host range within peri-domestic animals. Growth kinetics using mosquito-, midge- and sandfly-derived cell lines suggested Aedes mosquitos as potential vectors. Our findings highlight the occurrence of FSV across a geographic range exceeding 7,000 km, surprising genomic conservation across a time span exceeding 50 years, M gene-based reassortment events, and the existence of multiple animal hosts of FSV.

Post-epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia.

Schmallenberg virus (SBV) is a vector-borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA-positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re-emergence of SBV in Europe is predicted to occur when population-level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV-positive wild ruminants suggest that these species might be possible sources for the re-emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017-2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real-time RT-PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.

Oropouche virus cases identified in Ecuador using an optimised qRT-PCR informed by metagenomic sequencing.

Oropouche virus (OROV) is responsible for outbreaks of Oropouche fever in parts of South America. We recently identified and isolated OROV from a febrile Ecuadorian patient, however, a previously published qRT-PCR assay did not detect OROV in the patient sample. A primer mismatch to the Ecuadorian OROV lineage was identified from metagenomic sequencing data. We report the optimisation of an qRT-PCR assay for the Ecuadorian OROV lineage, which subsequently identified a further five cases in a cohort of 196 febrile patients. We isolated OROV via cell culture and developed an algorithmically-designed primer set for whole-genome amplification of the virus. Metagenomic sequencing of the patient samples provided OROV genome coverage ranging from 68-99%. The additional cases formed a single phylogenetic cluster together with the initial case. OROV should be considered as a differential diagnosis for Ecuadorian patients with febrile illness to avoid mis-diagnosis with other circulating pathogens.

Real-time RT-PCR for the detection and quantitation of Oropouche virus.

Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/µL. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/µL were detected in both the OROV rRT-PCR and a comparator molecular assay, but the OROV rRT-PCR detected more samples with ≤10 copies/µL (8/14 vs 0/13, respectively, P = 0.002). In a set of 100 acute-phase clinical samples from Paraguay patients with a suspected arboviral illness, no patients tested positive for OROV RNA using either assay. The OROV rRT-PCR provides a sensitive molecular assay for the study of this important yet neglected tropical arboviral infection.