Continuous presence of genetically diverse rustrela virus lineages in yellow-necked field mouse reservoir populations in northeastern Germany.

Rustrela virus (RusV; species Rubivirus strelense, family Matonaviridae) was discovered in different zoo animal species affected by fatal encephalitis. Simultaneous RusV RNA detection in multiple yellow-necked field mice (Apodemus flavicollis) suggested this rodent as a reservoir of RusV. Here, we investigated 1,264 yellow-necked field mice and sympatric other small mammals from different regions in Germany for RusV RNA using an optimized reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocol and high-throughput sequencing. The investigation resulted in the detection of RusV RNA exclusively in 50 of 396 (12.6 per cent) yellow-necked field mice but absence in other sympatric species. RT-qPCR-determined tissue distribution of RusV RNA revealed the highest viral loads in the central nervous system, with other tissues being only very rarely affected. The histopathological evaluation did not reveal any hints of encephalitis in the brains of infected animals despite the detection of viral RNA in neurons by in situ hybridization (ISH). The positive association between the body mass of yellow-necked field mice and RusV RNA detection suggests a persistent infection. Phylogenetic analysis of partial E1 and full-genome sequences showed a high diversification with at least four RusV lineages (1A-1D) in northeastern Germany. Moreover, phylogenetic and isolation-by-distance analyses indicated evolutionary processes of RusV mostly in local reservoir populations. A comparison of complete genome sequences from all detected RusV lineages demonstrated a high level of amino acid and nucleotide sequence variability within a part of the p150 peptide of the non-structural polyprotein and its coding sequence, respectively. The location of this region within the RusV genome and its genetic properties were comparable to the hypervariable region of the rubella virus. The broad range of detected RusV spillover hosts in combination with its geographical distribution in northeastern Germany requires the assessment of its zoonotic potential and further analysis of encephalitis cases in mammals. Future studies have to prove a putative co-evolution scenario for RusV in the yellow-necked field mouse reservoir.

First Insights into the Occurrence of Circular Single-Stranded DNA Genomes in Asian and African Cattle.

Circular replicase-encoding single-stranded (CRESS) DNA viruses and other circular DNA agents are increasingly found in various samples and animals. A specific class of these agents-termed bovine meat and milk factors (BMMF)-has been supposed to act as a factor in indirect carcinogenesis in humans. Initial observations attributed the BMMF to European cattle breeds and foodstuffs produced thereof. In the present study, blood and fecal samples from African and Asian cattle were examined. BMMF molecules and genomoviruses were detected in all bovids under study. The majority (79%) of the 29 circular elements could be assigned to BMMF groups 1 and 2, whereas CRESS viruses of the family Genomoviridae accounted for the smaller part (21%). Two genomoviruses belong to the genus Gemykibivirus and one to the genus Gemykrogvirus. The remaining three might be considered as novel species within the genus Gemycircularvirus. The majority of all isolated molecules originated from fecal samples, whereas only three derived from blood. The results from this study expand our knowledge on the diversity and presence of circular DNA in different ruminants that serve for food production in many countries over the world.

Rustrela virus infection - An emerging neuropathogen of red-necked wallabies (Macropus rufogriseus).

The rustrela virus (RusV) was recently described as a novel pathogen in a circumscribed area of northern Germany close to the Baltic Sea. Up to now, the virus has been detected in cases of fatal non-suppurative meningoencephalitis in zoo animals of different species and a single wild carnivore as well as in apparently healthy yellow-necked field mice (Apodemus flavicollis). Data regarding the background of this previously undiscovered pathogen, including clinical presentation of the disease, host range and distribution of the virus, are still limited. Here, three euthanized red-necked wallabies (Macropus rufogriseus) from zoos of different areas in northeastern Germany were submitted for necropsy after presenting with apathy and therapeutically unresponsive neurological signs. A moderate to severe, non-suppurative meningoencephalitis was diagnosed in all three cases. RusV was consistently detected via RT-qPCR and RNA in situ hybridization in the brains of all wallabies. Other commonly known neuropathogens could not be detected.

Revisiting Rustrela Virus: New Cases of Encephalitis and a Solution to the Capsid Enigma.

Rustrela virus (RusV; species Rubivirus strelense) is a recently discovered relative of rubella virus (RuV) that has been detected in cases of encephalitis in diverse mammals. Here, we diagnosed two additional cases of fatal RusV-associated meningoencephalitis in a South American coati (Nasua nasua) and a Eurasian or European otter (Lutra lutra) that were detected in a zoological garden with history of prior RusV infections. Both animals showed abnormal movement or unusual behavior and their brains tested positive for RusV using specific reverse transcription quantitative PCR (RT-qPCR) and RNA in situ hybridization. As previous sequencing of the RusV genome proved to be very challenging, we employed a sophisticated target-specific capture enrichment with specifically designed RNA baits to generate complete RusV genome sequences from both detected encephalitic animals and apparently healthy wild yellow-necked field mice (Apodemus flavicollis). Furthermore, the technique was used to revise three previously published RusV genomes from two encephalitic animals and a wild yellow-necked field mouse. When comparing the newly generated RusV sequences to the previously published RusV genomes, we identified a previously undetected stretch of 309 nucleotides predicted to represent the intergenic region and the sequence encoding the N terminus of the capsid protein. This indicated that the original RusV sequence was likely incomplete due to misassembly of the genome at a region with an exceptionally high G+C content of >80 mol%. The new sequence data indicate that RusV has an overall genome length of 9,631 nucleotides with the longest intergenic region (290 nucleotides) and capsid protein-encoding sequence (331 codons) within the genus Rubivirus. IMPORTANCE The detection of rustrela virus (RusV)-associated encephalitis in two carnivoran mammal species further extends the knowledge on susceptible species. Furthermore, we provide clinical and pathological data for the two new RusV cases, which were until now limited to the initial description of this fatal encephalitis. Using a sophisticated enrichment method prior to sequencing of the viral genome, we markedly improved the virus-to-background sequence ratio compared to that of standard procedures. Consequently, we were able to resolve and update the intergenic region and the coding region for the N terminus of the capsid protein of the initial RusV genome sequence. The updated putative capsid protein now resembles those of rubella and ruhugu virus in size and harbors a predicted RNA-binding domain that had not been identified in the initial RusV genome version. The newly determined complete RusV genomes strongly improve our knowledge of the genome structure of this novel rubivirus.

Nevus lipomatosus cutaneus superficialis (Hoffmann-Zurhelle) in a chimpanzee (Pan troglodytes).

BACKGROUND: A 32-year-old, male chimpanzee (Pan troglodytes) kept in a zoo developed a focally extensive, proliferative, cerebriform, dermal mass at the left inner thigh extending to the inguinal region. After surgical removal, the mass recurred and extended progressively over a period of 5 years. METHODS AND RESULTS: At necropsy, a 20 x 20 cm large, well defined, papular and partly verrucous, rubbery mass composed of multiple large, soft nodules measuring up to 4 cm in diameter was observed in the left thigh and inguinal region. Histological examination revealed a multifocal expansion of the dermis by mature adipocytes that were arranged in small islands to large lobular aggregates. Dermal proliferations of adipocytes were almost completely separated from the subcutaneous adipose tissue. CONCLUSIONS: This is the first report of a unique lesion that resembles human Nevus lipomatosus cutaneus superficialis in a chimpanzee and is different from lipoma or liposarcoma.

Tp53 expressing squamous cell carcinoma of the tonsil in a captive polar wolf (Canis lupus arctos).

The gross and histopathologic findings for a primary tonsillar squamous cell carcinoma in a captive 11-yr-old male polar wolf (Canis lupus arctos) are described. The carcinoma had metastasized to regional lymph nodes of the pharynx, the precardial mediastinum, and the lungs. Tumor suppressor protein TP53 was detected by immunohistochemistry in the nuclei of poorly differentiated, cytokeratin-positive cells of the primary neoplasm and the metastases. Canine oral papillomavirus DNA was not detectable by polymerase chain reaction (PCR).