Serological screening in wild ruminants in Germany, 2021/2022: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus.

Wildlife animals may be susceptible to multiple infectious agents of public health or veterinary relevance, thereby potentially forming a reservoir that bears the constant risk of re-introduction into the human or livestock population. Here, we serologically investigated 493 wild ruminant samples collected in the 2021/2022 hunting season in Germany for the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and four viruses pathogenic to domestic ruminants, namely, the orthobunyavirus Schmallenberg virus (SBV), the reovirus bluetongue virus (BTV) and ruminant pestiviruses like bovine viral diarrhoea virus or border disease virus. The animal species comprised fallow deer, red deer, roe deer, mouflon and wisent. For coronavirus serology, additional 307 fallow, roe and red deer samples collected between 2017 and 2020 at three military training areas were included. While antibodies against SBV could be detected in about 13.6% of the samples collected in 2021/2022, only one fallow deer of unknown age tested positive for anti-BTV antibodies, and all samples reacted negative for antibodies against ruminant pestiviruses. In an ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2, 25 out of 493 (5.1%) samples collected in autumn and winter 2021/2022 scored positive. This sero-reactivity could not be confirmed by the highly specific virus neutralisation test, occurred also in 2017, 2018 and 2019, that is, prior to the human SARS-CoV-2 pandemic, and was likewise observed against the RBD of the related SARS-CoV-1. Therefore, the SARS-CoV-2 sero-reactivity was most likely induced by another hitherto unknown deer virus belonging to the subgenus Sarbecovirus of betacoronaviruses.

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.

Chronic wasting associated with Chlamydia pneumoniae in three ex situ breeding facilities for tropical frogs.

A number of different Chlamydia spp. have been detected in the class Amphibia with C. pneumoniae being the predominant species involved. Chlamydiae have been linked to mass mortality events, thereby representing significant pathogens that deserve attention with respect to worldwide amphibian decline. We here present six cases of chlamydiosis and asymptomatic chlamydial infections in different frog species from three ex situ amphibian conservation facilities. Clinical signs predominantly characterised by regurgitation, chronic wasting, lethargy and suspended breeding were associated with C. pneumoniae infection. Despite various treatment regimens, it was not possible to clear infections. However, intra vitam diagnostics succeeded from skin, faeces and urine for the first time.

Expanding the host range: infection of a reptilian host (Furcifer pardalis) by an atypical Brucella strain.

Atypical brucellae show deviant phenotypes and/or genotypes. Besides Brucella inopinata, B. microti and B. vulpis, atypical strains have been described infecting humans, rodents, amphibians and fish. They represent potential zoonotic agents. Here, we provide evidence that reptiles as the remaining poikilothermic vertebrate class also represent susceptible hosts for atypical Brucella.

Fetal infection with Schmallenberg virus - An experimental pathogenesis study in pregnant cows.

Since its first appearance in 2011, Schmallenberg virus (SBV) has been repeatedly detected in aborted ruminant foetuses or severely malformed newborns whose mothers were naturally infected during pregnancy. However, especially the knowledge about dynamics of foetal infection in cattle is still scarce. Therefore, a total of 36 pregnant heifers were experimentally infected during two animal trials with SBV between days 60 and 150 of gestation. The foetuses were collected between 10 and 35 days after infection and virologically and pathologically investigated. Overall, 33 heifers yielded normally developed, macroscopically inconspicuous foetuses, but abundant virus replication was evident at the maternal/foetal interface and viral genome was detectable in at least one organ system of 18 out of 35 foetuses. One heifer was found to be not pregnant at autopsy. One of the animals aborted at day 4 after infection, viral RNA was detectable in the lymphatic tissue of the dam, in the maternal and foetal placenta, and in organs and lymphatic tissue of the foetus. In another foetus, SBV typical malformations like torticollis and arthrogryposis were observed. The corresponding dam was infected at day 90 of pregnancy and viral genome was detectable in the cerebellum of the unborn. Interestingly, no common patterns of infected foetal organs or maternal/foetal placentas could be identified, and both, sites of virus replication and genome loads, varied to a high degree in the individual foetuses. It is therefore concluded, that SBV infects in many cases also the bovine foetus of naïve pregnant cattle, however, the experimentally observed low abortion/malformation rate is in concordance to the reported low rates in the field during the first outbreak wave following the introduction of SBV. This observation speaks for a natural resistance of most bovine foetuses even during the vulnerable phase of early pregnancy, which has to be further studied in the future.

Detection of canine adenovirus 1 in red foxes ( Vulpes vulpes) and raccoons ( Procyon lotor) in Germany with a TaqMan real-time PCR assay.

We developed a real-time (rt)PCR assay based on TaqMan probe technology for the specific detection of canine adenovirus 1 (CAdV-1). The assay is able to detect three 50% tissue culture infectious dose/mL in CAdV-1-containing cell culture supernatant. Viral genomes were not amplified of canine adenovirus 2 or of several bovine, porcine, and avian adenoviruses. In silico analysis provided no indication of amplification of other heterologous genomes. The sensitivity of the real-time assay exceeded that of a conventional gel-based CAdV-1 PCR by a factor of 100. Following the integration of the novel PCR into the Hessian wildlife-monitoring program, CAdV-1 DNA was detected in none of the tested raccoons ( n = 48) but in 11 of 97 foxes.

Sheep and goats as indicator animals for the circulation of CCHFV in the environment.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus, which causes a serious illness with case-fatality rates of up to 80% in humans. CCHFV is endemic in many countries of Africa, Asia and Southeastern Europe. Next to the countries with endemic areas, the distribution of CCHFV is unknown in Southeastern Europe. As the antibody prevalence in animals is a good indicator for the presence or absence of the virus in a region, seroepidemiological studies can be used for the definition of risk areas for CCHFV. The aim of the present study was to reveal which ruminant species is best suited as indicator for the detection of a CCHFV circulation in an area. Therefore, the prevalence rates in sheep, goats and cattle in different regions of Albania and Former Yugoslav Republic of Macedonia were investigated. As there are no commercial tests available for the detection of CCHFV-specific antibodies in animals, two commercial tests for testing human sera were adapted for the investigation of sera from sheep and goats, and new in-house ELISAs were developed. The investigation of serum samples with these highly sensitive and specific assays (94-100%) resulted in an overall prevalence rate of 23% for Albania and of 49% for Former Yugoslav Republic of Macedonia. Significant lower seroprevalence rates for CCHFV were found in cattle than in small ruminants in given areas. These results indicate that small ruminants are more suitable indicator animals for CCHFV infections and should therefore be tested preferentially, when risk areas are to be identified.

Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge.

UNLABELLED: Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were tested in vitro and in vivo in type I interferon receptor knockout mice (IFNAR(-/-)) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR(-/-) mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses. IMPORTANCE: SBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

Single immunization with an inactivated vaccine protects sheep from Schmallenberg virus infection.

The arthropod-borne Schmallenberg virus (SBV), family Orthobunyaviridae, emerged in Europe in 2011. SBV is associated with a mild disease in adult ruminants but fetal malformation after an infection during a critical phase of pregnancy. A number of inactivated vaccines have been developed; their efficacy after two injections was demonstrated. To make the vaccination of sheep more efficient and economic the effect of a single immunization with one of these vaccines was investigated in the present study. Five vaccinated sheep and five additional control sheep were inoculated with SBV three weeks after vaccination and the results of a competitive ELISA, a standard microneutralization test and an SBV-specific real-time RT-PCR confirmed vaccine efficacy by demonstrating complete inhibition of viral replication in immunized animals.

Evaluating the protective efficacy of a trivalent vaccine containing Akabane virus, Aino virus and Chuzan virus against Schmallenberg virus infection.

Schmallenberg virus (SBV), an arthropod borne pathogen, spread rapidly throughout the majority of Europe since 2011. It can cause a febrile disease, milk drop, diarrhea, and fetal malformation in ruminants. SBV, a member of the Simbu serogroup within the genus Orthobunyavirus, is closely related to Akabane virus (AKAV) and Aino virus (AINOV) among others. In the present study, 4 Holstein-Friesian calves were immunized twice four weeks apart with a multivalent, inactivated vaccine against AKAV and AINOV. Another 4 calves were kept as unvaccinated controls. All animals were clinically, serologically and virologically examined before and after challenge infection with SBV. AKAV- and AINOV-specific neutralizing antibodies were detected one week before challenge infection, while SBV-specific antibodies were detectable only thereafter. SBV genome was detected in all vaccinated animals and 3 out of 4 controls in serum samples taken after challenge infection. In conclusion, the investigated vaccine was not able to prevent an SBV-infection. Thus, vaccines for other related Simbu serogroup viruses can not substitute SBV-specific vaccines as an instrument for disease control.

Inactivated Schmallenberg virus prototype vaccines.

Schmallenberg virus (SBV), a novel Orthobunyavirus, is an insect-transmitted pathogen and was first described in Europe in 2011. SBV causes a mild transient disease in adult ruminants, but severe foetal malformation and stillbirth were observed after an infection of naive cows and ewes, which is responsible for considerable economic losses. The virus is now widely distributed in Europe, and no vaccines were available to stop transmission and spread. In the present study, 16 calves and 25 sheep, the major target species of SBV infection, were vaccinated twice 3 weeks apart with one of 5 newly developed, inactivated vaccine candidates. Six calves and 5 sheep were kept as unvaccinated controls. All animals were clinically, serologically and virologically examined before and after challenge infection. Immunisation with the inactivated preparations resulted in a neutralising antibody response three weeks after the second vaccination without any side effects. The number of animals that seroconverted in each group and the strength of the antibody response were dependent on the cell line used for virus growth and on the viral titre prior to inactivation. Four vaccine prototypes completely prevented RNAemia after challenge infection, a fifth candidate reduced RNAemia considerably. Although further evaluations e.g. regarding duration of immunity will be necessary, the newly developed vaccines are promising candidates for the prevention of SBV-infection and could be a valuable tool in SBV control strategies.