In Vitro Modeling of Feline Morbillivirus Infections Using Primary Feline Kidney Cells.

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.

Borna disease in an adult free-ranging Eurasian beaver (Castor fiber albicus).

Borna disease (BD) associated with a peracute bacterial septicaemia with Escherichia coli was diagnosed in an adult female, naturally infected, free-ranging Eurasian beaver of the subspecies Castor fiber albicus, clinically characterized by weight loss, depression, weakness and gurgled peristaltic sounds. The beaver was euthanized humanely. Necropsy and light microscopy revealed a non-purulent meningoencephalitis with typical mononuclear perivascular cuffs and parenchymal infiltrates. The diagnosis of BD was confirmed by detection of viral antigen and RNA by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was sequenced and cluster analysis revealed a close relationship between endemic clusters in Saxony-Anhalt. This is the first report of naturally occurring BD in a free-ranging Eurasian beaver.

Horses as Sentinels for the Circulation of Flaviviruses in Eastern-Central Germany.

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.

An Explorative Study of the Causal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Meleagris gallopavo) Considering the Infectious Risk Factors.

A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health.

Case Series of Disseminated Xanthogranulomatosis in Red-crowned Parakeets (Cyanoramphus novaezelandiae) with Detection of Psittacine Adenovirus 2 (PsAdV-2).

Xanthogranulomatosis is a common dermatological disease in birds. This form of inflammation, possibly associated with lipometabolic disorders, can also be seen in visceral organs, which as yet has only rarely been described in avian medicine. In general, diseases related to impaired lipid metabolism are frequently reported in avian medicine, with hepatic steatosis and atherosclerosis being the most common. In human medicine, infectious agents-especially some strains of adenovirus-were implicated in contributing to lipometabolic disorders; this has also been described for chicken. Here, a case series of six Red-crowned Parakeets (Cyanoramphus novaezelandiae) is presented, all cases being characterized by psittacine adenovirus 2 (PsAdV-2) infection with or without disseminated xanthogranulomatosis. The affected individuals were examined alive by clinical examination. Total body radiographs were taken of two birds, haematology and blood biochemistry results were achieved in one bird. The birds either died immediately after clinical presentation or within two days, two individuals were euthanized due to worsening of their clinical condition. All birds underwent a post-mortem examination. While four birds were finally diagnosed with disseminated xanthogranulomatosis, all six individuals had large eosinophilic intranuclear inclusion bodies in the epithelial cells of the collecting ducts of the kidney and tested positive for PsAdV-2. Further examinations are needed to clarify to what extent PsAdV-2 might elicit lipometabolic disease in birds, or psittacines in general, and, in particular, the Red-crowned Parakeet.

Seroprevalence and Risk Factors for Equine West Nile Virus Infections in Eastern Germany, 2020.

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.

Spread of West Nile Virus and Usutu Virus in the German Bird Population, 2019-2020.

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.

Avian leukosis virus (ALV) is highly prevalent in fancy-chicken flocks in Saxony.

The current prevalence of avian leukosis virus (ALV) in fancy chickens in Germany is unknown. Therefore, 537 cloacal swabs from 50 purebred fancy-chicken flocks in Saxony were tested for the presence of the ALV p27 protein using a commercial antigen-capture ELISA. The detection rate was 28.7% at the individual-animal level and 56.0% at the flock level. Phylogenetic analysis of PCR products obtained from 22 different flocks revealed the highest similarity to ALV subtype K. When classifying breeds by their origin, ALV detection rates differed significantly. Evaluation of questionnaire data revealed no significant differences between ALV-positive and negative flocks regarding mortality.

The mycotoxin deoxynivalenol (DON) can deteriorate vaccination efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) at subtoxic levels.

BACKGROUND: Feedgrain contamination with mycotoxins, including deoxynivalenol (DON, "vomitoxin") is relatively frequently encountered. Pigs are particularly sensitive to the toxicity of DON. To assess the interplay between DON and porcine reproductive and respiratory syndrome virus (PRRSV), we performed an experimental DON exposure-PRRSV vaccination-challenge infection trial. Three-week-old piglets were divided into four groups. Groups I, II and III (10 animals/group) were vaccinated with a PRRSV modified live vaccine and 2 weeks later challenged with a heterologous field strain. While group I was not supplemented with DON, animals in groups II and III received DON for 4 weeks prior to challenge infection at levels that can be encountered in pig feed, employing a low-dose or high-dose regime (group II: 40 µg DON/kg body weight per day; group III: 80 µg DON/kg body weight per day, corresponding to approx. 1 or 2 mg DON/kg feed, respectively). Eight animals (group IV; unvaccinated, not DON exposed) served as control animals for the challenge infection. RESULTS: We assessed clinical signs, virus load in serum and various organs as well as antibody titres in the animals. All vaccinated animals mounted an efficient PRRSV-specific antibody response within 2 weeks, except for 20% of the animals receiving the higher DON dose. Upon virus challenge, the vaccinated animals in group I were protected from clinical signs. Vaccinated DON-exposed animals in group II and III were protected from clinical signs to a lesser extent. Clinical signs in group III receiving the higher dose of DON were as severe as in the (unvaccinated, not DON exposed) control group IV. The animals of group III also displayed lower antibody titres compared with the animals in group I and II. CONCLUSIONS: The experimental vaccination/challenge study therefore revealed that exposure of pigs to DON for a period of 4 weeks deteriorates the efficacy of vaccination against clinical signs of PRRS.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Mutated Measles Virus Matrix and Fusion Protein Influence Viral Titer In Vitro and Neuro-Invasion in Lewis Rat Brain Slice Cultures.

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.

Seroprevalence of Infection with Feline Morbilliviruses Is Associated with FLUTD and Increased Blood Creatinine Concentrations in Domestic Cats.

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.

High seroprevalence of feline morbilliviruses in free-roaming domestic cats in Chile.

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.

Identification of Novel Feline Paramyxoviruses in Guignas (Leopardus guigna) from Chile.

The family of paramyxoviruses has received growing attention as several new species have been identified recently, notably two different clusters in domestic cats, designated as feline morbillivirus (FeMV) and feline paramyxovirus (FPaV). Their phylogenetic origin and whether wild felids also harbor these viruses are currently unknown. Kidney samples from 35 guignas (Leopardus guigna), a wild felid from Chile, were investigated for paramyxoviruses using consensus-RT-PCR. In addition, thirteen serum samples of guignas were screened for the presence of FeMV-specific antibodies by an immunofluorescence assay (IFA). Viral RNA was detected in 31% of the kidney samples. Phylogenetic analyses revealed two well-supported clusters, related to isolates from domestic cats, rodents and bats. No significant histopathology changes were recorded in infected guignas. Serology identified two samples which were positive for FeMV-specific antibodies. Our study highlights the diversity of paramyxovirus infections in felids with special emphasis on guignas from Chile.

Field Trial Vaccination against Cowpox in Two Alpaca Herds.

In Europe, cowpox virus (CPXV) infection in South American camelids occurs as a so-called spill-over infection. Although infected animals generally have a mild form of the disease and survive, cases of fatal generalised CPXV infection have also been described. Prevention by prophylactic vaccination is the only way to protect animals from disease. In the present study, modified vaccinia virus Ankara (MVA) vaccine, which has been successfully used in many animal species, was used in a prime-boost vaccination regimen in two alpaca herds with a history of CPXV infection. The focus of the study was the prevention of further clinical cases, and to determine the safety and immunogenicity of the MVA vaccine in alpacas. The MVA vaccine was well tolerated and safe in the 94 animals vaccinated. An indirect immunofluorescence assay (IFA) using MVA as an antigen showed that the seroprevalence of antibody after booster vaccination was 81.3% in herd I and 91.7% in herd II. Detectable antibody titres declined to 15.6% in herd I and 45.8% in herd II over a 12-month period after booster vaccination. Animals could be divided into four groups based on individual antibody titres determined over one year: Group 1 consisted of 19.3% of animals that were seropositive until the end of the trial period; Group 2 consisted of 58.0% of animals that were seropositive after booster vaccination, but seronegative one year later; Group 3 consisted of 14.7% of animals that were not seropositive at any time point; and Group 4 consisted of 7.9% of animals that were seropositive after initial immunisation, seronegative six months later, but seropositive or intermediate in IFA one year after immunisation, likely because of natural exposure. In new-born crias born to MVA-vaccinated mares, specific maternal antibodies were detected in 50.0% of animals up to 14 weeks of age. Our results confirm that MVA vaccination is a feasible tool for the prevention of CPXV disease in alpacas. Long-term studies are needed to verify future vaccination regimen in CPXV affected herds.

Prevalence of feline immunodeficiency virus and feline leukaemia virus in domestic cats in Hungary.

OBJECTIVES: Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting cats worldwide. The objectives of the study were to estimate the prevalence of these retroviruses in domestic cats in Hungary and to characterise the phylogenetic relationships of FIV strains. METHODS: A total of 335 anticoagulated whole-blood samples obtained from both a healthy and ill cat population were examined for the presence of FIV and FeLV with two methods: ELISA and PCR. Statistical analysis was carried out to analyse the data obtained. Sequencing and phylogenetic analysis of partial polymerase (pol) gene sequences was performed to describe circulating FIV subtypes. RESULTS: Statistical analysis showed 11.8% and 9.9% true prevalence of FeLV and FIV, respectively, with ELISA. The apparent prevalence calculated from the PCR results were 17.3% for FeLV and 13.1% for FIV. Phylogenetic analysis of partial pol gene sequences obtained from 22 FIV strains showed that all observed Hungarian strains belonged to FIV subtype B. The strains were grouped into several monophyletic subgroups reflecting the geographic locations of the origin of the samples. The overall mean genetic similarity between the analysed strains was 98.2%. CONCLUSIONS AND RELEVANCE: We report the first thorough overview of the prevalence of FeLV and FIV in Hungary, which is relatively high, and give insight into the genetic diversity of Hungarian strains of FIV.

Generation of simian rotavirus reassortants with diverse VP4 genes using reverse genetics.

Species A rotaviruses (RVAs) are a major cause of gastroenteritis in animals and humans. Their genome consists of 11 segments of dsRNA, and reassortment events between animal and human strains can contribute to the high genetic diversity of RVAs. We used a plasmid-based reverse genetics system to investigate the reassortment potential of the genome segment encoding the viral outer capsid protein VP4, which is a major antigenic determinant, mediates viral entry and plays an important role in host cell tropism. We rescued reassortant viruses containing VP4 from porcine, bovine, bat, pheasant or chicken RVA strains in the backbone of simian strain SA11. The VP4 reassortants could be stably passaged in MA-104 cells and induced cytopathic effects. However, analysis of growth kinetics revealed marked differences in replication efficiency. Our results show that the VP4-encoding genome segment has a high reassortment potential, even between virus strains from highly divergent species. This can result in replication-competent reassortants with new genomic, growth and antigenic features.

Molecular and serological survey of carnivore pathogens in free-roaming domestic cats of rural communities in southern Chile.

Owned, free-roaming domestic cats are abundant in the Chilean countryside, having high probability of contact with wildlife and potentially participating as reservoirs of zoonotic pathogens. In the present study, 131 cats from two remote study areas (Valdivia and Chiloe Island) in southern Chile were analyzed for infection/exposure to eight pathogens. Serum samples from 112 cats were tested for antigens against feline leukemia virus (FeLV antigen-ELISA) and antibodies against feline immunodeficiency virus (FIV-ELISA) and canine distemper virus (CDV-serum neutralization), yielded occurrence of 8.9, 1.7 and 0.8% respectively. The presence of DNA of five vector-borne pathogens, piroplasmids, Ehrlichia spp., Anaplasma spp., Rickettsia spp. and Bartonella spp. was investigated in thirty cats. Overall observed occurrence was 6.6% (2/30) for both Anaplasma platys, and B. henselae, and 3.3% (1/30) for both Bartonella sp. and Theileria equi. Observed occurrence for all vector-borne pathogens in Valdivia area was significantly higher than in Chiloe Island (5/15 vs 0/15; P=0.04). Our results represent the first description of exposure to CDV and DNA detection of T. equi and A. platys in domestic cats in Chile. The results highlight the importance of performing pathogen screening in owned, free-roaming rural cats to evaluate their potential role as reservoirs of infection and vectors for disease transmission to wildlife.

Evidence for West Nile Virus and Usutu Virus Infections in Wild and Resident Birds in Germany, 2017 and 2018.

Wild birds play an important role as reservoir hosts and vectors for zoonotic arboviruses and foster their spread. Usutu virus (USUV) has been circulating endemically in Germany since 2011, while West Nile virus (WNV) was first diagnosed in several bird species and horses in 2018. In 2017 and 2018, we screened 1709 live wild and zoo birds with real-time polymerase chain reaction and serological assays. Moreover, organ samples from bird carcasses submitted in 2017 were investigated. Overall, 57 blood samples of the live birds (2017 and 2018), and 100 organ samples of dead birds (2017) were positive for USUV-RNA, while no WNV-RNA-positive sample was found. Phylogenetic analysis revealed the first detection of USUV lineage Europe 2 in Germany and the spread of USUV lineages Europe 3 and Africa 3 towards Northern Germany. USUV antibody prevalence rates were high in Eastern Germany in both years. On the contrary, in Northern Germany, high seroprevalence rates were first detected in 2018, with the first emergence of USUV in this region. Interestingly, high WNV-specific neutralizing antibody titers were observed in resident and short-distance migratory birds in Eastern Germany in 2018, indicating the first signs of a local WNV circulation.

A New Genotype of Feline Morbillivirus Infects Primary Cells of the Lung, Kidney, Brain and Peripheral Blood.

Paramyxoviruses comprise a large number of diverse viruses which in part give rise to severe diseases in affected hosts. A new genotype of feline morbillivirus, tentatively named feline morbillivirus genotype 2 (FeMV-GT2), was isolated from urine of cats with urinary tract diseases. Whole genome sequencing showed about 78% nucleotide homology to known feline morbilliviruses. The virus was isolated in permanent cell lines of feline and simian origin. To investigate the cell tropism of FeMV-GT2 feline primary epithelial cells from the kidney, the urinary bladder and the lung, peripheral blood mononuclear cells (PBMC), as well as organotypic brain slice cultures were used for infection experiments. We demonstrate that FeMV-GT2 is able to infect renal and pulmonary epithelial cells, primary cells from the cerebrum and cerebellum, as well as immune cells in the blood, especially CD4⁺ T cells, CD20⁺ B cells and monocytes. The cats used for virus isolation shed FeMV-GT2 continuously for several months despite the presence of neutralizing antibodies in the blood. Our results point towards the necessity of increased awareness for this virus when clinical signs of the aforementioned organs are encountered in cats which cannot be explained by other etiologies.