Rotavirus C (RVC) is an enteric pathogen frequently found in pig holdings. It is known to cause mild to severe gastrointestinal symptoms especially in suckling and weaned piglets. As most of RVC strains cannot be propagated in cell culture, serological surveys and the development of autologous vaccines are hampered. In order to gain better insight into their diversity, genetic studies are therefore particularly useful for identifying RVC genotypes. In this study, the distribution of circulating RVC genotypes (G-types and P-types) was analysed in six countries in Central Europe. Our investigations revealed the occurrence of ten different G-types, 16 different P-types and 24 different G-P-combinations. The largest number of different genotypes was found in the regions with the highest pig densities. Overall, two clearly dominant genotypes both in the comparison of countries and federal states were identified: G6 and P21. Genotype P21 has so far only been detected in Europe. Focusing on coinfections, this study revealed the lowest coinfection rates within the most frequently detected two genotypes (G6 and P21). Overall, the study provides a unique dataset that raises further questions regarding the underlying reasons for the distribution of specific RVC strains and the notably low coinfection rates observed within certain genotypes.

Feline morbillivirus (FeMV) has been associated with renal pathology in cats; however, the specific pathological alterations caused by FeMV infection remain controversial. This study aimed to investigate histopathological changes, viral localization, and apoptotic activity in the kidneys of FeMV-infected cats. Kidney tissues from 150 deceased cats with suspected or confirmed chronic kidney disease (CKD) were screened for FeMV using conventional reverse-transcription PCR (cRT-PCR). Positive cases were genotyped and quantified for viral load using reverse-transcription digital PCR (RT-dPCR). A control group of nine FeMV-negative kidneys with CKD was included for comparison. Histological evaluation was conducted using hematoxylin and eosin (H E), periodic acid–Schiff (PAS), and Masson’s trichrome staining. Immunohistochemistry (IHC) and in situ hybridization (ISH) were employed to localize viral antigens and assess expression of apoptotic markers, including cleaved caspase-3 (cCasp3), B-cell lymphoma 2 (BCL-2), and BCL-2–associated X protein (BAX). FeMV RNA was detected in 6

The virus family Flaviviridae encompasses approximately 90 species within four genera: flavivirus, pegivirus, hepacivirus and pestivirus (Simmonds et al., 2017). Viruses of this family are represented as small enveloped single stranded RNA viruses with positive genome polarity of approximately 9–13 kb. The genus flavivirus consists of 53 ICTV-accepted species with most of them being arthropod-borne. Based on the transmission vectors involved, members of this genus are further classified into 'mosquito-borne' and 'tick-borne' viruses which are separated from species infecting 'insects-only' and from viruses for which no vector is known so far. Mature virions of this genus are spherical in shape and 50 nm in diameter (Fig. 1). The genomic RNA is enclosed by a small capsid protein (C) which is surrounded by a double lipid membrane including the two viral envelope proteins, designated M and E, in which E serves as the primary attachment protein for receptor-mediated endocytosis. In infected cells seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) are synthesized and play an important role for viral replication, assembly and immune evasion (reviewed in Barrows et al., 2018).

Domestic cats are the natural host of feline morbilliviruses (FeMV). Although other species can also be infected (such as dogs and opossums), no laboratory animal infection model is established so far. In vitro models for studying the molecular pathogenesis are therefore needed. For this purpose, propagation and titration of FeMV are key techniques. Unlike other morbilliviruses, such as canine distemper virus (CDV) or measles virus (MV), FeMV is a slow growing virus in cell culture and is difficult to titrate using classical plaque techniques. Here we describe methods for the efficient isolation of FeMV from natural sources (e.g., urine), the propagation of viral stocks, and their titration. In addition, we establish the generation of a three-dimensional infection model mimicking the feline tubular epithelium.

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern–central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38–4.40) for WNV, 5.6% (95% CI: 4.44–7.04) for TBEV, and 0.4% (95% CI: 0.14–0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern–central Germany, as long as they are not vaccinated against WNV.

PURPOSE AND AIM:The presenting complaints, clinical signs, diagnostic evaluation, therapy, and outcome of 12 horses with clinically apparent West-Nile-Virus (WNV) infection are described. MATERIAL AND METHODS:Case series RESULTS: The adult horses (age 6-18 years, 7 mares, 5 geldings) from Saxony and Saxony-Anhalt were presented with various clinical histories between September 2018 and September 2020. All horses were presented in August or September and no horse was vaccinated against WNV. Fever as the most common general clinical sign was present in 8/12 horses. The most common neurological signs were muscle fasciculations (11/12 horses), ataxia (8/12 horses), hyperesthesia and head tilt (6/12 horses each). Diagnosis of WNV infection was confirmed by demonstrating IgM antibody and neutralizing antibody production in all horses; 2 euthanized horses also tested positive by PCR. Therapy was symptomatic and primarily included non-steroidal anti-inflammatories or dexamethasone as well as fluid therapy. Duration of hospitalization was 7.5 days on average. According to their owners, seven horses recovered completely, while information was missing for 2 horses. CONCLUSIONS AND CLINICAL RELEVANCE:In eastern-central Germany, WNV-encephalomyelitis must be considered a differential diagnosis for unvaccinated horses with acute neurologic disease occurring in summer and late summer. The reported clinical signs and the outcome of therapy are mostly congruent with reports from North America and other European countries.

West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study’s overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health- authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turn-out), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.

West Nile virus (WNV) and Usutu virus (USUV) are important flaviviruses circulating in Germany. While USUV was first reported more than 10 years ago, WNV has only reached the country in 2018. Wild birds are important amplifying hosts for both viruses. Therefore, we have been monitoring the bird population in different regions of Germany by a previously established network for many years. This report summarizes the results of molecular and/or serological methods of 2345 blood samples from birds of 22 different orders and over 2900 bird carcasses from 2019 and 2020. USUV RNA circulation was found in different regions of Germany, with emphasis on USUV lineages Europe 3 and Africa 3. Increased evidence of USUV lineage Europe 2 was detected in eastern Germany. WNV RNA was found only in birds from the eastern part of the country. The seroprevalence for USUV was between 3.11% and 7.20% in all three regions investigated, whereas the WNV seroprevalence spanned from 14.77% to 16.15% in eastern Germany, with a noticeable tendency for a westward and southward expansion in both years. Thus, wild bird monitoring for WNV and USUV can serve as an early warning system for a human exposure risk.

Usutu virus (USUV) is an emerging mosquito-borne arbovirus within the genus Flavivirus, family Flaviviridae. Similar to the closely related West Nile virus (WNV), USUV infections are capable of causing mass mortality in wild and captive birds, especially blackbirds. In the last few years, a massive spread of USUV was present in the avian population of Germany and other European countries. To date, no specific antiviral therapies are available. Nine different approved drugs were tested for their antiviral effects on the replication of USUV in vitro in a screening assay. Ivermectin was identified as a potent inhibitor of USUV replication in three cell types from different species, such as simian Vero CCL-81, human A549 and avian TME R. A 2- to 7-log10 reduction of the viral titer in the supernatant was detected at a non-cytotoxic concentration of 5 µM ivermectin dependent on the applied cell line. IC50 values of ivermectin against USUV lineage Africa 3 was found to be 0.55 µM in Vero CCL-81, 1.94 µM in A549 and 1.38 µM in TME-R cells. The antiviral efficacy was comparable between the USUV lineages Africa 2, Africa 3 and Europe 3. These findings show that ivermectin may be a candidate for further experimental and clinical studies addressing the treatment of USUV disease, especially in captive birds.

Feline morbillivirus (FeMV) is a recently discovered virus belonging to the genus Morbillivirus of the virus family Paramyxoviridae. Often, the virus has been detected in urine of cats with a history of urinary disease and has a worldwide distribution. Currently, it is unclear which receptor the virus uses to enter the target cells. Furthermore, many aspects of FeMV biology in vivo, including tissue tropism, pathogenesis, and virus excretion in the natural host remain unclear. In this study we analyzed the replication of FeMV in various cell lines. Secondly, we tested if the presence of feline SLAMF1 (Signaling Lymphocytic Activation Molecule family 1/CD150, principal entry receptor for other members of the Morbillivirus genus) improved FeMV replication efficiency in vitro. Finally, to elucidate in vivo biology in cats, as a natural host for FeMV, we experimentally infected a group of cats and monitored clinical symptoms, viremia, and excretion of the virus during the course of 56 days. Our study showed that FeMV shares some features with other morbilliviruses like the use of the SLAMF1 receptor. For the first time, experimental infection of SPF cats showed that FeMV does not induce an acute clinical disease like other morbilliviruses but can induce lesions in the kidneys, including tubulointerstitial nephritis. Further investigations are needed to confirm the site and dynamics of replication of FeMV in the urinary tract and the longer-term impact of FeMV-induced lesions on the renal function. Whether FeMV infection can result in chronic kidney disease will require the monitoring of cats over a longer period.

The mosquito-borne Usutu virus (USUV) is a zoonotic flavivirus and an emerging pathogen. So far therapeutical options or vaccines are not available in human and veterinary medicine. The bioenergetic profile based on extracellular flux analysis revealed an USUV infection-associated significant increase in basal and stressed glycolysis on Vero and with a tendency for basal glycolysis on the avian cell line TME-R derived from Eurasian blackbirds. On both cell lines this was accompanied by a significant drop in the metabolic potential of glycolysis. Moreover, glycolysis contributed to production of virus progeny, as inhibition of glycolysis with 2-deoxy-D-glucose reduced virus yield on Vero by one log10 step. Additionally, the increase in glycolysis observed on Vero cells after USUV infection was lost after the addition of exogenous type I interferon (IFN) β. To further explore the contribution of the IFN response pathway to the impact of USUV on cellular metabolism, USUV infection was characterized on human A549 respiratory cells with a knockout of the type I IFN receptor, either solely or together with the receptor of type III IFN. Notably, only the double knockout of types I and III IFN receptor increased permissiveness to USUV and supported viral replication together with an alteration of the glycolytic activity, namely an increase in basal glycolysis to an extent that a further increase after injection of metabolic stressors during extracellular flux analysis was not noted. This study provides evidence for glycolysis as a possible target for therapeutic intervention of USUV replication. Moreover, presented data highlight type I and type III IFN system as a determinant for human host cell permissiveness and for the infection-associated impact on glycolysis.

Zusammenfassung Gegenstand und Ziel Die Vorstellungsgründe, klinische Symptomatik, Diagnostik, Therapie und Ergebnis der Therapie von 12 Pferden mit klinisch apparenter West-Nil-Virus (WNV)-Infektion werden beschrieben. Material und Methoden Fallserie Ergebnisse Die adulten Pferde (Alter 6–18 Jahre, 7 Stuten, 5 Wallache) aus Sachsen oder Sachsen-Anhalt wurden zwischen September 2018 und September 2020 mit unterschiedlichen Vorberichten vorgestellt. Alle Pferde wurden im August oder September vorgestellt und keines der Pferde war gegen das WNV geimpft. Fieber war das häufigste Allgemeinsymptom und trat bei 8/12 Pferden auf. An neurologischen Symptomen traten Muskelfaszikulationen (11/12 Pferde), Ataxie (8/12 Pferde) sowie Hyperästhesie und Kopfschiefhaltung (je 6/12 Pferde) am häufigsten auf. Bei allen Pferden wurde die Infektion mittels Nachweises von IgM sowie neutralisierenden Antikörpern gegen das WNV diagnostiziert, zwei euthanasierte Pferde waren zudem PCR-positiv. Die symptomatische Therapie beinhaltete vor allem nicht-steroidale Antiphlogistika oder Dexamethason sowie Infusionstherapie. Die Dauer des Klinikaufenthaltes betrug im Durchschnitt 7,5 Tage. Sieben Pferde erholten sich laut Besitzerangaben vollständig, für 2 Pferde war keine Information erhältlich. Schlussfolgerungen und klinische Relevanz Die WNV-Enzephalomyelitis muss in Mitteldeutschland seit 2018 als Differentialdiagnose von im Sommer und Spätsommer auftretenden akuten neurologischen Erkrankungen ungeimpfter Pferdes in Betracht gezogen werden. Die beschriebenen Symptome sowie das Ergebnis der Therapie sind weitgehend deckungsgleich mit Berichten aus Nordamerika und anderen europäischen Ländern.

Feline morbilliviruses (FeMV) are fairly newly discovered paramyxoviruses found in cats. The first description indicated an association with widely distributed chronic kidney disease (CKD) in the host species. In various studies, a global prevalence and a further genotype, designated FeMV-2, and the involvement of other organ systems in infected individuals were shown. Using an immunofluorescence assay, we detected an overall seroprevalence of FeMV in almost half of the cats investigated (n = 380), with a significantly increased proportion in younger animals. In comparison to European Shorthair cats, the rate of seropositivity is higher in pedigree cats. Regardless of the breed, FeMV infection was associated with increased blood creatinine concentrations, suggesting an association with CKD. Further analysis indicated that this association was the strongest in animals having high IFA titers against FeMV-2. In addition, a significant association between FeMV-positive status and the prevalence of feline lower urinary tract disease (FLUTD, or idiopathic cystitis) was detected. This association was dominated by cats having antibodies against FeMV-1 only. To further evaluate the positive correlation between FeMV seroprevalence and CKD as well as FLUTD, consideration of additional clinical characteristics and laboratory parameters is warranted, and controlled infection studies with both FeMV genotypes are necessary. Clinicians should, however, be aware of a possible link between renal and lower urinary tract disease and FeMV infections.

Objective Pigeon rotavirus A (RVA) isolates of genotype G18P[17] are causing disease outbreaks and fatalities in pigeon lofts in Australia, Germany, Belgium, Denmark and USA since 2016. Most disease outbreaks have been reported from juvenile pigeons (Columba livia forma domestica). However, reports on RVA-associated disease outbreaks in fancy pigeons in connection with fancy pigeon shows in Germany are rare. Material and methods Overall 18 pigeons (16 fancy pigeons and one racing pigeon from 9 pigeon fanciers, as well as one feral pigeon from a rescue center) were sent in for routine diagnostic necropsy including histopathologic, parasitologic and microbiologic examinations. Molecular biologic examinations for detection of RVA, circovirus, Usutu virus, West Nile virus and Chlamydia psittaci were also carried out on all pigeons. An accompanying questionnaire filled in by the senders was used to generate basic information on the affected pigeon lofts. Results Disease outbreaks in juvenile and adult pigeons were reported 7-14 days after fancy pigeon shows. One fancier who had previously vaccinated his pigeons with an autogenous pigeon RVA vaccine, noted no morbidity and mortality among his pigeons and thus sent in a healthy pigeon for diagnostic purposes. Reported clinical signs in the other pigeons were regurgitation, green slimy diarrhea, anorexia, apathy and death after 24 hours. Hepatic necrosis and detection of pigeon RVA isolates of genotype G18P[17] confirmed disease outbreaks caused by pigeon RVA in all pigeons, except for the vaccinated pigeon. Besides pigeon circovirus, which was detected in 15 of 18 pigeons, all other pathogens were singular findings. Conclusion and clinical relevance In disease outbreaks following fancy pigeon shows in juvenile and adult pigeons diagnostics should include pigeon RVA of genotype G18P[17].

Measles virus (MV) can cause severe acute diseases as well as long-lasting clinical deteriorations due to viral-induced immunosuppression and neuronal manifestation. How the virus enters the brain and manages to persist in neuronal tissue is not fully understood. Various mutations in the viral genes were found in MV strains isolated from patient brains. In this study, reverse genetics was used to introduce mutations in the fusion, matrix and polymerase genes of MV. The generated virus clones were characterized in cell culture and used to infect rat brain slice cultures. A mutation in the carboxy-terminal domain of the matrix protein (R293Q) promoted the production of progeny virions. This effect was observed in Vero cells irrespective of the expression of the signaling lymphocyte activation molecule (SLAM). Furthermore, a mutation in the fusion protein (I225M) induced syncytia formation on Vero cells in the absence of SLAM and promoted viral spread throughout the rat brain slices. In this study, a solid ex vivo model was established to elucidate the MV mutations contributing to neural manifestation.

The mosquito-borne flavivirus Usutu virus (USUV) is responsible for countless deaths in both resident populations and birds kept in outdoor aviaries. Since 2001, USUV outbreaks have attracted increased attention due to the rapid geographical spread of the virus and its close relationship to West Nile virus (WNV), an emerging pathogen in humans and animals. Similar to WNV, the USUV enzootic transmission cycle predominantly involves Culex spp. as vectors, whereas birds serve as amplifying reservoir hosts. In Europe, USUV-associated disease outbreaks in birds are almost exclusively described during late spring and early autumn (early April to late October). Contagiousness of virus particles excreted by infected birds has not yet been proven, so that the role of non-vector-borne transmission, as it is known for the closely related WNV, remains unclear. Here we report the diagnosis of USUV infection in 15 of 24 birds from mortality outbreaks that occurred during the cold season between late October 2018 and early April 2019, in eight different aviaries located in Germany. Detection of USUV was performed using standardized molecular biological methods and immunohistochemistry for verification of the infection. USUV infection in a parrot species, a tropical finch and two estrildid finches are reported for the first time. Further research on the occurrence of USUV infection during the cold season is key to understanding the dynamics of viral transmission as well as for a profound health risk assessment for aviary birds as well as humans.

The family of Coronaviridae comprises numerous virus species, some of which play an outstanding role in veterinary medicine. In addition to infections of the respiratory and/or the gastrointestinal tract, which are usually mild, severe systemic infections and even fatal outcomes can occur. Wild and zoo animals are just as affected by coronavirus infections as their domesticated counterparts. In many cases, however, other virus species or virus variants are found in wild animals, which so far have not been characterized in detail. Knowledge of the virus reservoirs, the transmission mechanisms, the virus evolution and the underlying pathomechanisms is a basic requirement for combating relevant infections in pets and farm animals. In the area of coronaviruses in zoo and wild animals, there is still a considerable need for research in order to be able to answer pestering questions regarding the zoonotic potential and possible cross-species transmissions between wild and domesticated animals.

One year after the first autochthonous transmission of West Nile virus (WNV) to birds and horses in Germany, an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher compared to 2018 (12 birds, two horses), resulting in the observation of the first WNV epidemy in Germany: 76 cases in birds, 36 in horses and five confirmed mosquito-borne, autochthonous human cases. We demonstrated that Germany experienced several WNV introduction events and that strains of a distinct group (Eastern German WNV clade), which was introduced to Germany as a single introduction event, dominated mosquito, birds, horse and human-related virus variants in 2018 and 2019. Virus strains in this clade are characterized by a specific-Lys2114Arg mutation, which might lead to an increase in viral fitness. Extraordinary high temperatures in 2018/2019 allowed a low extrinsic incubation period (EIP), which drove the epizootic emergence and, in the end, most likely triggered the 2019 epidemic. Spatiotemporal EIP values correlated with the geographical WNV incidence. This study highlights the risk of a further spread in Germany in the next years with additional human WNV infections. Thus, surveillance of birds is essential to provide an early epidemic warning and thus, initiate targeted control measures.

Feline morbillivirus infections have gained increased attention due to repeated reports of their association with urinary tract disease in cats. In the present study, 112 serum samples from free-roaming domestic cats in Chile were tested for antibodies against feline morbillivirus genotypes 1 and 2 (FeMV-1 and FeMV-2) using an indirect immunofluorescence assay. In total, 63% of the animals showed antibodies against one or both FeMV genotypes. Antibodies directed exclusively against FeMV-2 were significantly more prevalent in male cats. The correlation of sex and FeMV-2 infection might give insight into potential routes of transmission. We provide, for the first time, serological data on FeMV in Chile.

A total of 289 cloacal swabs from pigeons from 29 different breeders in Germany were collected. In addition, samples from pigeons exhibited at shows were collected. The detailed health status of the pigeon flocks was recorded. Samples were analysed for the presence of the recently discovered pigeon rotavirus and pigeon circovirus. Pigeon rotavirus was found in 10.3% and pigeon circoviruses was found in 65.5% of sampled pigeon lofts. The study revealed a strong relationship between the attendance of shows and the occurrence of different clinical signs. The higher prevalence of pigeon rotavirus in exhibited animals indicates that exhibitions are a risk factor for the transmission of this pathogen.