Monoclonal antibodies to Cache Valley virus for serological diagnosis.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.

Serological and Molecular Investigation of Batai Virus Infections in Ruminants from the State of Saxony-Anhalt, Germany, 2018.

Arthropod-borne Batai virus (BATV) is an Orthobunyavirus widely distributed throughout European livestock and has, in the past, been linked to febrile diseases in humans. In Germany, BATV was found in mosquitoes and in one captive harbor seal, and antibodies were recently detected in various ruminant species. We have, therefore, conducted a follow-up study in ruminants from Saxony-Anhalt, the most affected region in Eastern Germany. A total of 325 blood samples from apparently healthy sheep, goats, and cattle were tested using a BATV-specific qRT-PCR and SNT. Even though viral RNA was not detected, the presence of antibodies was confirmed in the sera of all three species: sheep (16.5%), goats (18.3%), and cattle (41.4%). Sera were further analyzed by a glycoprotein Gc-based indirect ELISA to evaluate Gc-derived antibodies as a basis for a new serological test for BATV infections. Interestingly, the presence of neutralizing antibodies was not directly linked to the presence of BATV Gc antibodies. Overall, our results illustrate the high frequency of BATV infections in ruminants in Eastern Germany.

Seroprevalance of Batai virus in ruminants from East Germany.

Batai virus (BATV), a mosquito-transmitted Orthobunyavirus, was first detected in Southwest Germany in anopheline and culicine mosquitoes in 2009. However, little is known about the exposure to BATV infections for farm animals and humans in Germany as almost no systematic surveillance or infection studies have been carried out to date. This may explain why clinical symptoms in animals or humans have not been reported so far. Therefore and since BATV has meanwhile been detected repeatedly in different mosquito species in several regions of Germany, we performed a surveillance study by assaying more than 1300 blood samples from ruminants (goats, bovines, sheep) from six different federal states covering the years 2013 to 2016. Samples were investigated by BATV-specific real-time polymerase chain reaction as well as by virus neutralisation test. BATV-specific RNA was not detected, whereas BATV-specific antibodies were found in livestock from various geographic regions. We have determined the seroprevalence of 38.8% for goats, 44.7% for sheep and 36.4% for bovines in Saxony-Anhalt. The seroprevalence of goats from Brandenburg was 38.6% and of goats from Saxony 28.4%. These results confirm the levels of seroprevalence to BATV, suggesting endemic circulation, in different regions and indicate that ruminants are potential hosts of BATV in East Germany. Furthermore, the role of BATV as segment donor in disease emergence events should not be overlooked.

Seroprevalence of Cache Valley virus and related viruses in sheep and other livestock from Saskatchewan, Canada.

Cache Valley virus, an orthobunyavirus, is an important cause of ovine neonatal malformations. Information on the seroprevalence of this virus in Saskatchewan livestock populations is lacking. The objectives of this study were to determine the seroprevalence of Cache Valley virus and closely related viruses in sheep, cattle, goats, horses, and mule deer in Saskatchewan by performing a plaque-reduction neutralization test using Cache Valley virus. In total, sera from 130 sheep from 50 flocks were tested. Seroprevalence in sheep was 64.6% (84/130) and 94.0% (47/50) of flocks had 1 or more seropositive sheep. Antibodies to Cache Valley virus or closely related viruses were also detected in serum samples collected from cattle, goats, horses, and mule deer with seroprevalences of 20.0% (5/25), 33.3% (8/24), 69.0% (40/58), and 50.8% (33/65), respectively. These results suggest widespread exposure to Cache Valley virus or closely related viruses in domestic animals and mule deer in Saskatchewan.

Séroprevalence du virus de la Vallée Cache ou de virus connexes chez les moutons et d'autres animaux de cheptel en Saskatchewan, Canada. Le virus de la Vallée Cache, un orthobunyavirus, est une cause importante de malformations néonatales ovines. Il manque des renseignements sur la séroprévalence de ce virus dans les populations des cheptels de la Saskatchewan. Les objectifs de cette étude consistaient à déterminer la séroprévalence du virus de la Vallée Cache et des virus étroitement apparentés chez les moutons, les bovins, les chèvres, les chevaux et les cerfs mulets en Saskatchewan en réalisant un test de séro-neutralisation par réduction des plages en utilisant le virus de la Vallée Cache. Au total, le sérum provenant de 130 moutons dans 50 troupeaux a été testé. Chez les moutons, la séroprévalence était de 64,6 % (84/130) et 94,0 % (47/50) des troupeaux avaient un mouton ou plusieurs moutons séropositifs. Les anticorps pour le virus de la Vallée Cache ou les virus étroitement apparentés ont aussi été détectés dans les échantillons de sérum prélevés auprès des bovins, des chèvres, des chevaux et des cerfs mulets avec une séroprévalence de 20,0 % (5/25), de 33,3 % (8/24), de 69,0 % (40/58) et de 50,8 % (33/65), respectivement. Ces résultats suggèrent une vaste exposition au virus de la Vallée Cache ou à des virus étroitement apparentés chez les animaux domestiques et les cerfs mulets en Saskatchewan.(Traduit par Isabelle Vallières).

Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

Management Factors Associated with Operation-Level Prevalence of Antibodies to Cache Valley Virus and Other Bunyamwera Serogroup Viruses in Sheep in the United States.

A cross-sectional study was performed to identify operation-level risk factors associated with prevalence of antibody to Bunyamwera (BUN) serogroup viruses in sheep in the United States. Sera were obtained from 5150 sheep in 270 operations located in 22 states (three in the west, nine central states, and 10 in the east) and tested at a dilution of 1:20 by a plaque reduction neutralization test (PRNT) using Cache Valley virus (CVV). Antibodies that neutralized CVV were identified in 1455 (28%) sheep. Animal-level seroprevalence was higher in the east (49%) than the central (17%) and western (10%) states. A convenient subset (n = 509) of sera with antibodies that neutralized CVV was titrated and further analyzed by PRNT using all six BUN serogroup viruses that occur in the United States: CVV, Lokern virus (LOKV), Main Drain virus (MDV), Northway virus (NORV), Potosi virus (POTV), and Tensaw virus (TENV). Antibodies to CVV and LOKV were identified in sheep in all three geographic regions; MDV and POTV activity was detected in the central and eastern states, NORV activity was restricted to the west, and antibodies to TENV were not detected in any sheep. Several management factors were significantly associated with the presence of antibodies to BUN serogroup viruses. For instance, sheep housed during the lambing season inside structures that contained four walls and a roof and a door closed most of the time were more likely to be seropositive than other sheep. In contrast, herded/open-range sheep were less likely to be seropositive than their counterparts. These data can be used by producers to implement strategies to reduce the likelihood of BUN serogroup virus infection and improve the health and management practices of sheep.

Orthobunyavirus antibodies among humans in selected parts of the Rift Valley and northeastern Kenya.

Ngari, Bunyamwera, Ilesha, and Germiston viruses are among the mosquito-borne human pathogens in the Orthobunyavirus genus, family Bunyaviridae, associated with febrile illness. Although the four orthobunyaviruses have been isolated from mosquito and/or tick vectors sampled from different geographic regions in Kenya, little is known of human exposure in such areas. We conducted a serologic investigation to determine whether orthobunyaviruses commonly infect humans in Kenya. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization tests in 89 (25.8%) of 345 persons tested. Multivariable analysis revealed age and residence in northeastern Kenya as risk factors. Implementation of acute febrile illness surveillance in northeastern Kenya will help to detect such infections.

Surveillance of Batai virus in bovines from Germany.

To estimate the veterinary importance of Batai virus (BATV), we investigated the presence of BATV-specific antibodies and BATV RNA in 548 bovines from southwest Germany, and we demonstrated that 3 cattle serum samples contained BATV-neutralizing antibodies, resulting in a seroprevalence of 0.55%. Thus, our results confirm local transmission and indicate cattle as potential hosts of BATV in southwest Germany.

Serological evidence of Batai virus infections, bovines, northern Italy, 2011.

Batai virus (BATV) was identified in mosquitoes in the Caltignaga region of Novarra, northern Italy in 2009. Here, we report the identification of antibodies to BATV in serum samples that were taken from healthy bovines in that region in 2011. BATV has been associated with a mild febrile human illness and identified as the likely parental segment donor in a reassortment event that resulted in the generation of the virulent progeny, Ngari virus. The possible veterinary disease associations of BATV are unknown. The presence of antibodies to BATV in bovine populations confirms local transmission in northern Italy. Given its likely role as a segment donor, an understanding of the geographic and host distributions of BATV is of veterinary and human public health interest.

Ovine fetal immune response to Cache Valley virus infection.

Cache Valley virus (CVV)-induced malformations have been previously reproduced in ovine fetuses. To evaluate the development of the antiviral response by the early, infected fetus, before the development of immunocompetency, ovine fetuses at 35 days of gestation were inoculated in utero with CVV and euthanized at 7, 10, 14, 21, and 28 days postinfection. The antiviral immune response in immature fetuses infected with CVV was evaluated. Gene expression associated with an innate, immune response was quantified by real-time quantitative PCR. The upregulated genes in infected fetuses included ISG15, Mx1, Mx2, IL-1, IL-6, TNF-α, TLR-7, and TLR-8. The amount of Mx1 protein, an interferon-stimulated GTPase capable of restricting growth of bunyaviruses, was elevated in the allantoic and amniotic fluid in infected fetuses. ISG15 protein expression was significantly increased in target tissues of infected animals. B lymphocytes and immunoglobulin-positive cells were detected in lymphoid tissues and in the meninges of infected animals. These results demonstrated that the infected ovine fetus is able to initiate an innate and adaptive immune response much earlier than previously known, which presumably contributes to viral clearance in infected animals.

Viperin, MTAP44, and protein kinase R contribute to the interferon-induced inhibition of Bunyamwera Orthobunyavirus replication.

The first line of defense against viral infection is the interferon (IFN) response, which culminates in the expression of hundreds of proteins with presumed antiviral activity, and must be overcome by a virus for successful replication. The nonstructural NSs protein is the primary IFN antagonist encoded by Bunyamwera virus (BUNV), the prototype of the Orthobunyavirus genus and the family Bunyaviridae. The NSs protein interferes with RNA polymerase II-mediated transcription, thereby inhibiting cellular mRNA production, including IFN mRNAs. A recombinant virus, rBUNdelNSs, that is unable to express the NSs protein does not inhibit cellular transcription and is a strong IFN inducer. We report here that cells stimulated into the antiviral state by IFN-ß treatment were protected against wild-type BUNV and rBUNdelNSs infection but addition of IFN-ß after infection had little effect on the replication cycle of either virus. By screening a panel of cell lines that overexpressed individual IFN-stimulated genes, we found that protein kinase R (PKR), MTAP44, and particularly viperin appreciably restricted BUNV replication. The enzymatic activities of PKR and viperin were required for their inhibitory activities. Taken together, our data show that the restriction of BUNV replication mediated by IFN is an accumulated effect of at least three IFN-stimulated genes that probably act on different stages of the viral replication cycle.

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.

Identification of the target cells and sequence of infection during experimental infection of ovine fetuses with Cache Valley virus.

Cache Valley virus-induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the course of infection of cells and tissues with Cache Valley virus. To address these questions, ovine fetuses at 35 days of gestation were inoculated in utero with Cache Valley virus and euthanized at 7, 10, 14, 21, and 28 days postinfection. On postmortem examination, arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphological studies showed necrosis in the central nervous system and skeletal muscle of infected fetuses evaluated after 7 to 14 days postinfection, and hydrocephalus, micromyelia, and muscular loss were observed in infected fetuses after 21 to 28 days postinfection. Using immunohistochemistry and in situ hybridization, intense Cache Valley virus antigen and RNA staining was detected in the brain, spinal cord, skeletal muscle, and, to a lesser degree, in fetal membranes and other tissues of infected fetuses. Viral antigen and RNA staining decreased in targeted and infected tissues with the progression of the infection.

Self-protection and survival of arbovirus-infected mosquito cells.

Arboviruses are serious pathogens for men but cause little damage to their arthropod vectors. We have studied how a mosquito cell line derived from one of the relevant vectors for arboviruses responds to Bunyamwera virus, a well-characterized arbovirus. Confocal, live cell microscopy and electron microscopy showed that Bunyamwera virus induces deep changes in mosquito cells. Early in infection these cells develop long projections and create new intercellular connections where cell organelles and viral proteins are detected. Live cell microscopy shows that these connections are developed before viral protein can be detected by immunofluorescence. Interestingly, their proliferation is accompanied by a progressive trapping of the nucleocapsid and RNA polymerase viral proteins into large cytoplasmic aggregates. A significant drop in the release of infectious virions then follows. Before that, numerous viruses assemble in peripheral Golgi stacks and they apparently exit the cells immediately since they do not accumulate intracellularly. This mechanism of assembly seems to cause little damage to the integrity of cell endomembranes. The characterization of the antiviral mechanisms operating in mosquito cells can be of great help in the fight against pathogenic arboviruses.

Heterocellular induction of interferon by negative-sense RNA viruses.

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism.

Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

Evaluation of the eastern cottontail Sylvilagus floridanus as an amplifying vertebrate host for Cache Valley virus (Bunyaviridae) in Indiana.

To evaluate the importance of eastern cottontails (Sylvilagus floridanus) as amplifying hosts for Cache Valley virus (CVV), we tested hunter-provided blood samples from northern Indiana for specific neutralizing (N) antibodies against this mosquito-borne bunya-virus. Samples were collected during the winter of 1994-95. Two seronegative eastern cottontails, captured in July 1995, were also infected with CVV by subcutaneous inoculation, and two others were infected by allowing CVV-infected mosquitoes to feed on them. The results indicate that eastern cottontails probably are not important amplifying hosts for CVV. The prevalence of N antibodies against CVV was low (6.0%, n=82) among the hunter-killed animals. Low viremia (<1.8 log10 plaque-forming units/ml) of short duration (1-3 days) were seen in three of four experimentally infected eastern cottontails. The viremias were insufficient for infecting Coquillettidia perturbans, a mosquito species commonly found naturally infected with CVV.

[Complex environmental and virological monitoring in the Primorye Territory in 2003-2006].

The paper presents the results of monitoring of viruses of Western Nile (WN), Japanese encephalitis (JE), tick-borne encephalitis (TBE), Geta, Influenza A, as well as avian paramicroviruses type I (virus of Newcastle disease (ND)) and type 6 (APMV-6) in the Primorye Territory in 2003-2006. Totally throughout the period, specific antibodies to the viruses were detected by neutralization test in wild birds (7.3%, WN; 8.0%, Geta; 0.7% Batai; 2.8%, Alpine hare (Lepus timidus); by hemagglutination-inhibition test in cattle (11.4% WN; 5.9%, JE; j 3.0%, TBE; 11.6%, Geta), horses (6.1, 6.8, 0, and 25.3%, respectively), and pigs (5.4, 1.5, 0, and 5.9%, respectively) by enzyme immunoassay (IgG) in human beings (0.8, 0.5, 6.8, and 3.2%, respectively. Reverse-transcription polymerase chain reaction (RT-PCR) was used to reveal RNA of the NP segment of influenza A virus in 57.9 and 65% of the cloacal swabs from wild and domestic birds, respectively; and the HA-segment of subtype HH was not detected in 2005. HA/H5 RNA was recorded in 5.5 and 6.7% of the swabs from wild and domestic birds, respectively; 6% of the specimens from domestic birds were M-segment positive in 2006. RNA of influenza A virus NA/H7 and RNA was not detected throughout the years. In 2004, the cloacal swabs 8 isolated influenza A strains: two H3N8 and two H4N8 strains from European teals (Anas crecca), two (H3N8 and H6N2) strains from Baikal teals (A. formosa), one (H10N4) strain from shovelers (A. clypeata), and one (H4N8) from garganeys (A. querquedula). In 2004, one ND virus strain was isolated from the cloacal swabs from European teals (A. crecca). RT-PCR revealed RNA of this virus in some 8 more cloacal swabs from black ducks (A. poecilorhyncha) (3 positive specimens), pheasants (Phasianus colchicus) (n = 2), garganeys (A. querquedula) (n = 1), gadwalls (A. strepera) (n = 1), and geese (Anser anser domesticus) (n = 1). Sequencing of the 374-member fragment of the ND virus F gene, which included a proteolytic cleavage site, could assign two samples to the weakly pathogenetic variants of genotype 1, one sample to highly pathogenic variants of genotype 3a, five to highly pathogenic ones of genotype 5b. Isolation of APMV-6 (2003) from common egrets (Egretta alba) and geese (Ans. anser domesticus) is first described.

[The ecology and prevalence of arboviruses on the territory of the Saratov Region].

The territorial spread of Tahyna, Batai, Sindbis, West Nile fever and Crimean-Congo hemorrhagic fever viruses throughout the Saratov region in 1998 - 2000 was analyzed. The characteristics of the epizootic activity of the natural foci of these arboviruses in different landscape zones (temperate forest-steppes, steppes and semi-deserts) were calculated. The species composition of small mammals, the natural reservoirs of the causative agents of arbovirus infections, was determined.

Mosquitoborne viruses, Czech Republic, 2002.

Specimens from residents (N = 497) of an area affected by the 2002 flood were examined serologically for mosquitoborne viruses. Antibodies were detected against Tahyna (16%), Sindbis (1%), and Batai (0.2%) viruses, but not West Nile virus. An examination of paired serum samples showed 1 Tahyna bunyavirus (California group) infection.