Bluetongue Virus Serotype 3 and Schmallenberg Virus in Culicoides Biting Midges, Western Germany, 2023.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

Hefer valley virus: a novel ephemerovirus detected in the blood of a cow with severe clinical signs in Israel in 2022.

A novel ephemerovirus was identified in a Holstein-Friesian cow in the Hefer Valley, Israel, that showed severe and fatal clinical signs resembling an arboviral infection. A sample taken during the acute phase tested negative for important endemic arboviral infectious cattle diseases. However, sequencing from blood revealed the full genome sequence of Hefer Valley virus, which is likely to represent a new species within the genus Ephemerovirus, family Rhabdoviridae. Archived samples from cattle with comparable clinical signs collected in Israel in 2021 and 2022 tested negative for the novel virus, and therefore, the actual distribution of the virus is unknown. As this is a recently identified new viral infection, the viral vector and the prevalence of the virus in the cattle population are still unknown but will be the subject of future investigations.

First evidence of schmallenberg virus infection in southern Italy.

BACKGROUND: Schmallenberg virus (SBV) is a vector-borne pathogen that mainly affects ruminants. Schmallenberg disease has never been described in southern Italy, although this geographic area displays climatic features suitable for Culicoides biting midges, which transmit the pathogen. An observational study was carried out in the Campania region in 2020 to evaluate the seroprevalence in cattle and water buffalo as well as to identify the risk factors involved in the distribution of SBV. RESULTS: Relatively high seroprevalences of 38.2% (cattle) and 43% (water buffalo) were found by using a commercial SBV ELISA, which is comparable to the prevalence obtained in other countries under post-epidemic conditions. A virus neutralization assay performed on positive samples showed high titers in a large percentage of animals which is assumed to indicate recent exposure. Bivariate analysis of several variables revealed some environmental factors associated with higher seroprevalence, such as mean annual temperature, distance from the coast, and altitude. Multivariate logistic regression confirmed the statistical association only for mean annual temperature, that was found to be the main factor responsible for the distribution of the virus in southern Italy. In addition, molecular diagnosis attempts were performed on serum samples and resulted in the detection of SBV RNA in two herds and six animals. CONCLUSIONS: In this work we have demonstrated the circulation of SBV in southern Italy using both molecular and serological assays. This study emphasized the essential role of monitoring in preventing the re-emergence of vector-borne diseases in ruminants.

Characterization of experimental Shuni virus infection in the mouse.

Shuni virus (SHUV), an orthobunyavirus of the Simbu serogroup, was initially isolated in Nigeria in the 1960s, further detected in other African countries and in the Middle East, and is now endemic in Israel. Transmitted by blood-sucking insects, SHUV infection is associated with neurological disease in cattle and horses, and with abortion, stillbirth, or the birth of malformed offspring in ruminants. Surveillance studies also indicated a zoonotic potential. This study aimed to test the susceptibility of the well-characterized interferon (IFN)-α/ß receptor knock-out mouse model (Ifnar-/-), to identify target cells, and to describe the neuropathological features. Ifnar-/-mice were subcutaneously infected with two different SHUV strains, including a strain isolated from the brain of a heifer showing neurological signs. The second strain represented a natural deletion mutant exhibiting a loss of function of the S-segment-encoded nonstructural protein NSs, which counteracts the host's IFN response. Here it is shown that Ifnar-/-mice are susceptible to both SHUV strains and can develop fatal disease. Histological examination confirmed meningoencephalomyelitis in mice as described in cattle with natural and experimental infections. RNA in situ hybridization was applied using RNA Scope™ for SHUV detection. Target cells identified included neurons and astrocytes, as well as macrophages in the spleen and gut-associated lymphoid tissue. Thus, this mouse model is particularly beneficial for the evaluation of virulence determinants in the pathogenesis of SHUV infection in animals.

Antibodies against SARS-CoV-2 Suggestive of Single Events of Spillover to Cattle, Germany.

Human infection with SARS-CoV-2 poses a risk for transmission to animals. To characterize the risk for cattle, we serologically investigated 1,000 samples collected from cattle in Germany in late 2021. Eleven antibody-positive samples indicated that cattle may be occasionally infected by contact with SARS-CoV-2-positive keepers, but we found no indication of further spread.

International proficiency trial for bovine viral diarrhea virus (BVDV) antibody detection: limitations of milk serology.

BACKGROUND: Control programs were implemented in several countries against bovine viral diarrhea (BVD), one of the most significant cattle diseases worldwide. Most of the programs rely on serological diagnostics in any phase of the program. For the detection of antibodies against BVD virus (BVDV), neutralization tests as well as a variety of (commercially available) ELISAs are used. Here, test systems applied in various laboratories were evaluated in the context of an international interlaboratory proficiency trial. A panel of standardized samples comprising five sera and five milk samples was sent to veterinary diagnostic laboratories (n=51) and test kit manufacturers (n=3). RESULTS: The ring trial sample panel was investigated by nine commercially available antibody ELISAs as well as by neutralization tests against diverse BVDV-1, BVDV-2 and/or border disease virus (BDV) strains. The negative serum and milk sample as well as a serum collected after BVDV-2 infection were mostly correctly tested regardless of the applied test system. A serum sample obtained from an animal immunized with an inactivated BVDV-1 vaccine tested positive by neutralization tests or by total antibody or Erns-based ELISAs, while all applied NS3-based ELISAs gave negative results. A further serum, containing antibodies against the ovine BDV, reacted positive in all applied BVDV ELISAs, a differentiation between anti-BDV and anti-BVDV antibodies was only enabled by parallel application of neutralization tests against BVDV and BDV isolates. For the BVDV antibody-positive milk samples (n=4), which mimicked prevalences of 20% (n=2) or 50% (n=2), considerable differences in the number of positive results were observed, which mainly depended on the ELISA kit and the sample incubation protocols used. These 4 milk samples tested negative in 43.6%, 50.9%, 3.6% and 56.4%, respectively, of all investigations. Overall, negative results occurred more often, when a short sample incubation protocol instead of an over-night protocol was applied. CONCLUSIONS: While the seronegative samples were correctly evaluated in most cases, there were considerable differences in the number of correct evaluations for the seropositive samples, most notably when pooled milk samples were tested. Hence, thorough validation and careful selection of ELISA tests are necessary, especially when applied during surveillance programs in BVD-free regions.

Experimental SARS-CoV-2 Infection of Bank Voles.

After experimental inoculation, severe acute respiratory syndrome coronavirus 2 infection was confirmed in bank voles by seroconversion within 8 days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Thus, bank voles are unlikely to establish effective transmission cycles in nature.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

Autonomously Replicating RNAs of Bungowannah Pestivirus: ERNS Is Not Essential for the Generation of Infectious Particles.

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.

Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples.

BACKGROUND: As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5' untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. RESULTS: To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19-32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. CONCLUSIONS: Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

Experimental Infection of Cattle with SARS-CoV-2.

We inoculated 6 cattle with severe acute respiratory syndrome coronavirus 2 and kept them together with 3 uninoculated cattle. We observed viral replication and specific seroreactivity in 2 inoculated animals, despite high levels of preexisting antibody titers against a bovine betacoronavirus. The in-contact animals did not become infected.

Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection.

Raccoon dogs might have been intermediate hosts for severe acute respiratory syndrome-associated coronavirus in 2002-2004. We demonstrated susceptibility of raccoon dogs to severe acute respiratory syndrome coronavirus 2 infection and transmission to in-contact animals. Infected animals had no signs of illness. Virus replication and tissue lesions occurred in the nasal conchae.

Novel Picornavirus in Lambs with Severe Encephalomyelitis.

Using metagenomic analysis, we identified a novel picornavirus in young preweaned lambs with neurologic signs associated with severe nonsuppurative encephalitis and sensory ganglionitis in 2016 and 2017 in the United Kingdom. In situ hybridization demonstrated intralesional neuronotropism of this virus, which was also detected in archived samples of similarly affected lambs (1998-2014).

Misinterpretation of Schmallenberg virus sequence variations: the sample material makes the difference.

In recent reports about the molecular epidemiology of Schmallenberg virus (SBV), an orthobunyavirus affecting ruminants, it was proposed that the observed sequence variability within the viral M-segment might be higher in sheep than in cattle. However, these analyses are highly biased by the sample material from which the publicly available sequences were generated. While from cattle predominantly blood samples from acutely infected animals were studied, the vast majority of ovine samples originate from malformed fetuses or newborn lambs. Therefore, the observed sequence variability is misinterpreted since the samples from malformed fetuses and lambs do not reflect circulating SBV.

Bungowannah virus in the affected pig population: a retrospective genetic analysis.

Bungowannah virus, which belongs to the genus Pestivirus within the family Flaviviridae, has been associated with myocarditis and a high incidence of stillbirths in pigs. In 2003, the virus was initially detected in a large pig farming complex on two separate sites in New South Wales, Australia. Until now, it has not been detected at other locations. Despite a program of depopulation and disinfection, the virus could be only eradicated from one of the affected farm complexes, the Bungowannah unit, but became endemic on the second complex, the Corowa unit. In the present study, the genetic variability of virus isolates collected between 2003 and 2014 in the endemically infected population has been retrospectively investigated. Phylogenetic analysis carried out based on sequences of the E2 and NS5B coding regions and the full-length open-reading frame revealed that the isolates from the different farm sites are closely related, but that samples collected between 2010 and 2014 at the Corowa farm site clustered in a different branch of the phylogenetic tree. Since 2010, a high-genetic stability of this RNA virus within the Corowa farm complex, probably due to an effective adaptation of the virus to the affected pig population, could be observed.

Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE: Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce highly attenuated, safer vaccines was evident after the development of the BTV reverse-genetics system that allows the introduction of targeted mutations in the virus genome. We targeted an essential gene to develop disabled virus strains as vaccine candidates. The results presented in this report further substantiate our previous evidence and support the suitability of these virus strains as the next-generation BTV vaccines.

Neutralizing antibodies against Simbu serogroup viruses in cattle and sheep, Nigeria, 2012-2014.

BACKGROUND: Simbu serogroup viruses of the Orthobunyavirus genus (Family Peribunyaviridae) include teratogenic pathogens that cause severe economic losses, abortions, stillbirths and congenital abnormalities in ruminants worldwide. Although they were initially isolated from ruminants and Culicoides biting midges about five decades ago in Nigeria, there is no current information on their prevalence and geographical distribution despite reports of abortions and congenital malformations in the country's ruminant population. Here, apparently healthy cattle and sheep obtained from eight states in the three major vegetation zones of Nigeria were screened for the presence of specific neutralizing antibodies against Schmallenberg virus (SBV), Simbu virus (SIMV) and Shamonda virus (SHAV). RESULTS: Using a cross-sectional design, 490 cattle and 165 sheep sera were collected between 2012 and 2014 and tested by a commercial SBV ELISA kit which enables the detection of antibodies against various Simbu serogroup viruses. The seropositivity rates for cattle and sheep were 91.2% and 65.4%, respectively. In cattle, there was no association between ELISA seropositivity and vegetation zone. However, the prevalence of anti-Simbu serogroup antibodies was significantly higher in Ebonyi State compared to other states in the rainforest vegetation zone. The seroprevalence was significantly higher in sheep obtained from live animal markets compared to farms (OR = 5.8). Testing of 20 selected ELISA-positive sera by serum neutralisation test showed that all were positive for one or more of SBV, SIMV and SHAV with the highest titres obtained for SHAV. Antibodies to SBV or a closely related virus were detected in the Sudan savannah and rainforest zones, anti-SIMV antibodies were detected only in the rainforest zone, while anti-SHAV antibodies were found in the three vegetation zones. CONCLUSION: The findings of this study reveal that following the early isolation of Simbu serogroup viruses in Nigeria in the 1960s, members of this virus group are still circulating in the country. Specifically, SBV, SIMV and SHAV or closely related viruses infect cattle and sheep across the three vegetation zones of Nigeria suggesting that insect vector activity is extensive in the country. The exact vegetation zone where the animals became exposed to the viruses could, however, not be determined in this study.

Development of within-herd immunity and long-term persistence of antibodies against Schmallenberg virus in naturally infected cattle.

BACKGROUND: In 2011, the teratogenic, insect-transmitted Schmallenberg virus (SBV) emerged at the German/Dutch border region and subsequently spread rapidly throughout the European continent. In cattle, one of the major target species of SBV, first antibodies are detectable between one and three weeks after infection, but the duration of humoral immunity is unknown. To assess the course of immunity in individual animals and the development of the within-herd seroprevalence, cattle kept in a German farm with a herd size of about 300 lactating animals were annually blood sampled between December 2011 and December 2017 and tested for the presence of SBV-specific antibodies. RESULTS: During the monitored period, the within-herd seroprevalence declined from 74.92% in 2011 to 39.93% in 2015 and, thereafter, slightly increased to 49.53% in 2016 and 48.44% in 2017. From the animals that were tested in 2014 and 2015 for the first time (between 24 and 35 months of age) only 14.77% and 7.45%, respectively, scored positive. Thereafter, the seropositivity rate of this age group rose markedly to 58.04% in 2016 and 48.10% in 2017 indicating a circulation of SBV. Twenty-three individual animals were consistently sampled once per year between 2011 and 2017 after the respective insect vector season, 17 of them tested positive at the first sampling. Fourteen animals were still seropositive in December 2017, while three cattle (17.65%) became seronegative. CONCLUSIONS: The regular re-emergence of SBV in Central Europe is a result of decreasing herd immunity caused by the replacement of animals by seronegative youngstock rather than of a drop of antibody levels in previously infected individual animals. The consequences of the overall decline in herd seroprevalence may be increasing virus circulation and more cases of fetal malformation caused by infection of naïve dams during gestation.