Search for polyoma-, herpes-, and bornaviruses in squirrels of the family Sciuridae.

BACKGROUND: Squirrels (family Sciuridae) are globally distributed members of the order Rodentia with wildlife occurrence in indigenous and non-indigenous regions (as invasive species) and frequent presence in zoological gardens and other holdings. Multiple species introductions, strong inter-species competition as well as the recent discovery of a novel zoonotic bornavirus resulted in increased research interest on squirrel pathogens. Therefore we aimed to test a variety of squirrel species for representatives of three virus families. METHODS: Several species of the squirrel subfamilies Sciurinae, Callosciurinae and Xerinae were tested for the presence of polyomaviruses (PyVs; family Polyomaviridae) and herpesviruses (HVs; family Herpesviridae), using generic nested polymerase chain reaction (PCR) with specificity for the PyV VP1 gene and the HV DNA polymerase (DPOL) gene, respectively. Selected animals were tested for the presence of bornaviruses (family Bornaviridae), using both a broad-range orthobornavirus- and a variegated squirrel bornavirus 1 (VSBV-1)-specific reverse transcription-quantitative PCR (RT-qPCR). RESULTS: In addition to previously detected bornavirus RNA-positive squirrels no more animals tested positive in this study, but four novel PyVs, four novel betaherpesviruses (BHVs) and six novel gammaherpesviruses (GHVs) were identified. For three PyVs, complete genomes could be amplified with long-distance PCR (LD-PCR). Splice sites of the PyV genomes were predicted in silico for large T antigen, small T antigen, and VP2 coding sequences, and experimentally confirmed in Vero and NIH/3T3 cells. Attempts to extend the HV DPOL sequences in upstream direction resulted in contiguous sequences of around 3.3 kilobase pairs for one BHV and two GHVs. Phylogenetic analysis allocated the novel squirrel PyVs to the genera Alpha- and Betapolyomavirus, the BHVs to the genus Muromegalovirus, and the GHVs to the genera Rhadinovirus and Macavirus. CONCLUSIONS: This is the first report on molecular identification and sequence characterization of PyVs and HVs and the detection of bornavirus coinfections with PyVs or HVs in two squirrel species. Multiple detection of PyVs and HVs in certain squirrel species exclusively indicate their potential host association to a single squirrel species. The novel PyVs and HVs might serve for a better understanding of virus evolution in invading host species in the future.

Investigations into the presence of nidoviruses in pythons.

BACKGROUND: Pneumonia and stomatitis represent severe and often fatal diseases in different captive snakes. Apart from bacterial infections, paramyxo-, adeno-, reo- and arenaviruses cause these diseases. In 2014, new viruses emerged as the cause of pneumonia in pythons. In a few publications, nidoviruses have been reported in association with pneumonia in ball pythons and a tiger python. The viruses were found using new sequencing methods from the organ tissue of dead animals. METHODS: Severe pneumonia and stomatitis resulted in a high mortality rate in a captive breeding collection of green tree pythons. Unbiased deep sequencing lead to the detection of nidoviral sequences. A developed RT-qPCR was used to confirm the metagenome results and to determine the importance of this virus. A total of 1554 different boid snakes, including animals suffering from respiratory diseases as well as healthy controls, were screened for nidoviruses. Furthermore, in addition to two full-length sequences, partial sequences were generated from different snake species. RESULTS: The assembled full-length snake nidovirus genomes share only an overall genome sequence identity of less than 66.9% to other published snake nidoviruses and new partial sequences vary between 99.89 and 79.4%. Highest viral loads were detected in lung samples. The snake nidovirus was not only present in diseased animals, but also in snakes showing no typical clinical signs. CONCLUSIONS: Our findings further highlight the possible importance of snake nidoviruses in respiratory diseases and proof multiple circulating strains with varying disease potential. Nidovirus detection in clinical healthy individuals might represent testing during the incubation period or reconvalescence. Our investigations show new aspects of nidovirus infections in pythons. Nidoviruses should be included in routine diagnostic workup of diseased reptiles.

Camelids and Cattle Are Dead-End Hosts for Peste-des-Petits-Ruminants Virus.

Peste-des-petits-ruminants virus (PPRV) causes a severe respiratory disease in small ruminants. The possible impact of different atypical host species in the spread and planed worldwide eradication of PPRV remains to be clarified. Recent transmission trials with the virulent PPRV lineage IV (LIV)-strain Kurdistan/2011 revealed that pigs and wild boar are possible sources of PPRV-infection. We therefore investigated the role of cattle, llamas, alpacas, and dromedary camels in transmission trials using the Kurdistan/2011 strain for intranasal infection and integrated a literature review for a proper evaluation of their host traits and role in PPRV-transmission. Cattle and camelids developed no clinical signs, no viremia, shed no or only low PPRV-RNA loads in swab samples and did not transmit any PPRV to the contact animals. The distribution of PPRV-RNA or antigen in lymphoid organs was similar in cattle and camelids although generally lower compared to suids and small ruminants. In the typical small ruminant hosts, the tissue tropism, pathogenesis and disease expression after PPRV-infection is associated with infection of immune and epithelial cells via SLAM and nectin-4 receptors, respectively. We therefore suggest a different pathogenesis in cattle and camelids and both as dead-end hosts for PPRV.

Molecular identification and characterization of nonprimate hepaciviruses in equines.

Hepatitis C virus (HCV) is a positive-sense RNA virus belonging to the genus Hepacivirus, family Flaviviridae. Its genome has a length of 9.6 kb and encodes a single polyprotein flanked by two untranslated regions. HCV can cause liver cirrhosis and hepatocellular carcinoma, and approximately 2% of the world's population is chronically infected. The investigation of pathogenesis is complicated due to the lack of an animal model. The origin of this virus remains unclear, but in the last few years, relatives of HCV were initially identified in dogs and later in horses, rodents, bats and Old World monkeys. Non-primate hepacivirus (NPHV), which infects dogs and horses, is the closest relative to HCV. We established a pan-reactive "panHepaci"-RT-qPCR assay, which is able to detect human HCV as well as equine NPHV, and additionally, an equine-specific "equHepaci"-RT-qPCR for confirmation of positive results. Serum samples from 1158 clinically inconspicuous horses from Germany and several samples from other mammalian species were screened. We found 2.4% of the horses positive for hepacivirus RNA, and furthermore, the "panHepaci"-RT-qPCR assay also detected a hepacivirus in a donkey from Egypt. This virus had only 78% sequence identity in the E2 gene when compared to other known NPHVs. The established method could be useful for screening purposes, since it is likely that related hepaciviruses also occur in other species.

Occupation-Associated Fatal Limbic Encephalitis Caused by Variegated Squirrel Bornavirus 1, Germany, 2013.

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost's squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost's squirrel. Antibodies against bornaviruses were detected in the patient's cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.