Identification of novel reassortant mammalian orthoreoviruses from bats in Slovenia.

BACKGROUND: Recently, mammalian orthoreoviruses (MRVs) were detected for the first time in European bats, and the closely related strain SI-MRV01 was isolated from a child with severe diarrhoea in Slovenia. Genetically similar strains have also been reported from other mammals, which reveals their wide host distribution. The aim of this study was to retrospectively investigate the occurrence and genetic diversity of MRVs in bats in Slovenia, from samples obtained throughout the country in 2008 to 2010, and in 2012 and to investigate the occurrence of the novel SI-MRV01 MRV variant in Slovenian bats. RESULTS: The detection of MRVs in bat guano was based on broad-range RT-PCR and specific bat MRV real-time RT-PCR. Subsequently, MRV isolates were obtained from cell culture propagation, with detailed molecular characterisation through whole-genome sequencing. Overall, bat MRVs were detected in 1.9% to 3.8% of bats in 2008, 2009 and 2012. However, in 2010 the prevalence was 33.0%, which defined an outbreak of the single SI-MRV01 strain. Here, we report on the identification of five MRV isolates of different serotypes that are designated as SI-MRV02, SI-MRV03, SI-MRV04, SI-MRV05 and SI-MRV06. There is high genetic variability between these characterised isolates, with evident genome reassortment seen across their genome segments. CONCLUSIONS: In conclusion, we have confirmed the presence of the SI-MRV01 strain in a Slovenian bat population. Moreover, according to genetic characterisation of S1 genome segment, all three MRV serotypes were present in the bat population. In this study, five independent MRV isolates were obtained and detailed whole genome analysis revealed high diversity between them. This study generates new information about the epidemiology and molecular characteristics of emerging bat MRV variants, and provides important molecular data for further studies of their pathogenesis and evolution.

Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia.

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5-100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia.

In November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were confirmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, amplified from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.

Post-epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia.

Schmallenberg virus (SBV) is a vector-borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA-positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re-emergence of SBV in Europe is predicted to occur when population-level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV-positive wild ruminants suggest that these species might be possible sources for the re-emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017-2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real-time RT-PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.

Nearly Complete Genome Sequences of Two Bovine Viral Diarrhea Virus Isolates, Subtype 1f Strain SLO/1170/2000 and Subtype 1d Strain SLO/2416/2002.

Bovine viral diarrhea virus (BVDV) subtypes 1f and 1d were isolated for the first time in Slovenia in 1999 and detected later in a majority of BVDV-infected cattle herds. Here, we report the first nearly complete genome sequences of noncytopathogenic BVDV-1f strain SLO/1170/2000 and cytopathogenic BVDV-1d strain SLO/2416/2002, isolated in Slovenia.

Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission.

A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (-29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23-fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS.

Vaccine-induced rabies in a red fox (Vulpes vulpes): isolation of vaccine virus in brain tissue and salivary glands.

Oral vaccination campaigns to eliminate fox rabies were initiated in Slovenia in 1995. In May 2012, a young fox (Vulpes vulpes) with typical rabies signs was captured. Its brain and salivary gland tissues were found to contain vaccine strain SAD B19. The Basic Logical Alignment Search Tool alignment of 589 nucleotides determined from the N gene of the virus isolated from the brain and salivary glands of the affected fox was 100% identical to the GenBank reference SAD B19 strain. Sequence analysis of the N and M genes (4,351 nucleotides) showed two nucleotide modifications at position 1335 (N gene) and 3114 (M gene) in the KC522613 isolate identified in the fox compared to SAD B19.