Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1.

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5'-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Phylogeny of Hungarian EBLV-1 strains using whole-genome sequence data.

European bat lyssavirus 1 (EBLV-1) is a widespread lyssavirus across Europe, whose epizootic cycle is linked to a few bat species. Occasionally, EBLV-1 infection may occur in domestic animals and humans. EBLV-1 can be classified into two subtypes, where subtype EBLV-1a shows a wide geographic distribution between France and Russia whereas subtype EBLV-1b is distributed between Spain and Poland. In this study, we determined the genome sequence of two recent EBLV-1a strains detected in Hungary and analysed their adaptive evolution and phylodynamics. The data set that included 100 EBLV-1 genome sequences identified positive selection at selected sites in genes coding for viral proteins (N, codon 18; P, 141 and 155; G, 244 and 488; L, 168, 980, 1597 and 1754). A major genetic clade containing EBLV-1a isolates from Hungary, Slovakia, Denmark and Poland was estimated to have diverged during the 19th century whereas the divergence of the most recent ancestor of Hungarian and Slovakian isolates dates back to 1950 (time span, 1930 to 1970). Phylogeographic analysis of the EBLV-1a genomic sequences demonstrated strong evidence of viral dispersal from Poland to Hungary. This new information indicates that additional migratory flyways may help the virus spread, a finding that supplements the general theory on a west-to-east dispersal of EBLV-1a strains. Long-distance migrant bats may mediate the dispersal of EBLV-1 strains across Europe; however, structured surveillance and extended genome sequencing would be needed to better understand the epizootiology of EBLV-1 infections in Europe.

Time-course profiling of bovine alphaherpesvirus 1.1 transcriptome using multiplatform sequencing.

Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. Bovine alphaherpesvirus 1 (BoHV-1) is an important pathogen of cattle worldwide. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technologies MinION, and the LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps.

Complete genome analysis confirms that the pygmy marmoset adenovirus is a variant of the skunk adenovirus 1 - Short communication.

The complete genomic sequence along with phylogenetic analyses of an adenovirus (AdV), isolated from a dead captive pygmy marmoset (Callithrix pygmaea) from a Hungarian zoo is reported. Earlier, based on the phylogenetic analysis of the sequence of a PCR-amplified fragment from the DNA polymerase gene, the pygmy marmoset AdV (PMAdV) has been reported to cluster closest to certain chiropteran AdVs. In the following years similar AdVs were discovered in additional mammalian hosts, including a skunk (Mephitis mephitis), African pygmy hedgehogs (Atelerix albiventris), North American porcupines (Erethizon dorsatum) and grey fox (Urocyon cinereoargenteus). After the full genome analysis of the skunk adenovirus (SkAdV-1), a novel species Skunk mastadenovirus A (SkAdV-A) has been established. The AdVs, originating from the African pygmy hedgehogs, have been found to belong to virus species SkAdV-A. Partial gene sequences from the porcupine AdVs have also implied their very close genetic relatedness to SkAdV-A. The complete genomic sequence of PMAdV, examined in this study, was found to share 99.83% nucleotide identity with SkAdV-1, thus unequivocally represents a genomic variant of SkAdV-1. The observation that viruses classifiable as SkAdV-A are able to infect and cause diseases in several, distantly related mammals seems to deserve further studies to elucidate the infection biology of this intriguing AdV.

Low incidence of Schmallenberg virus infection in natural cases of abortion in domestic ruminants in Hungary.

An epizootic caused by a new orthobunyavirus called Schmallenberg virus (SBV) was recognised in European ruminants in 2011 and 2012. The re-emergence of the infection was reported in several countries in the subsequent years. Although the main clinical sign of SBV infection is abortion, the impact of SBV in natural cases of abortion in domestic ruminants had not been systematically examined before this study. The aim of the study was to investigate the role of SBV infection and to compare it to the importance of other causes of abortion by examining 537 natural cases of abortion that had occurred between 2011 and 2017 in Hungary. The cause of abortion was determined in 165 (31%) cases. An infectious cause was proved in 88 (16%) cases. SBV infection was found only in a total of four cases (0.8%) using real-time polymerase chain reaction. Three of them proved to be inapparent SBV infection, and one case was attributed to SBV-induced abortion by detecting non-purulent encephalitis and SBV nucleoprotein by immunohistochemistry in a brain tissue sample. According to the results, SBV played a minor role in natural cases of domestic ruminant abortion in Hungary during the 7-year period following the first SBV outbreak in 2011.

Isolation and complete genome sequence analysis of a novel ovine adenovirus type representing a possible new mastadenovirus species.

Pathological examination of a suckling male lamb showed severe viral pneumonia with suspected bacterial superinfection. Adenovirus was detected by immunohistochemical examination of the affected lung samples. Detection of the suspected adenovirus by PCR and subsequent isolation of the virus were successful. Using next-generation sequencing, the full genome of this ovine adenovirus was sequenced and analysed. A genome sequence comparison showed that it was a novel mastadenovirus type (named "ovine adenovirus 8") that did not belong to any of the established adenovirus species. The genome is 36,206 bp long, containing 93-bp inverted terminal repeats and 29 predicted genes, including the two genus-specific genes (encoding proteins V and IX). Ovine adenovirus 8 shows the closest relationship to ovine adenovirus 6. These two viruses seem to merit the establishment of a novel ovine mastadenovirus species for them, for which we proposed the name "Ovine mastadenovirus C".

Vaccine-associated rabies in red fox, Hungary.

Rabies vaccine strain was isolated from a red fox (Vulpes vulpes) with signs of neurological disorder during an oral vaccination campaign in 2015, Hungary. The whole genome sequence of the isolated strain shared >99.9% nucleotide sequence identity to the whole genomes of vaccines strains recently used in Hungarian oral vaccination campaigns. The neuroinvasive potential of rabies vaccines that leads to development of clinical manifestations is rarely seen among wild animals; however, the observed residual pathogenicity needs awareness of field experts and requires close monitoring of rabies cases in areas where elimination programs are implemented.

Cellular localisation of the proteins of region 3 of feline enteric coronavirus.

Feline enteric coronaviruses have three open reading frames (ORFs) in region 3 (3a, 3b, and 3c). All three ORFs were expressed with C-terminal eGFP and 3xFLAG tags in different cell lines and their localisation was determined. ORF 3a is predicted to contain DNA-binding and transcription activator domains, and it is localised in the nucleus and in the cytoplasm. ORF 3b is also predicted to contain DNA-binding and activator domains, and was found to localise in the mitochondrion. Besides that, in some of the non-infected and FIPV-infected cells nucleolar, perinuclear or nuclear membrane accumulation of the eGFP-tagged 3b was observed. The exact compartmental localisation of ORF 3c is yet to be determined. However, based on our co-localisation studies 3c does not seem to be localised in the ER-Golgi network, ERGIC or peroxisomes. The expression of 3c-eGFP is clearly cell type dependent, it is more stable in MARC 145 cells than in Fcwf-4 or CrFK cells, which might reflect in vivo stability differences of 3c in natural target cells (enterocytes vs. monocytes/macrophages).

Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

Demonstration of homologous recombination events in the evolution of bovine viral diarrhoea virus by in silico investigations.

Complete genome sequences of bovine viral diarrhoea virus types 1 and 2 (BVDV-1 and 2) deposited in the GenBank were submitted to bioinformatic analysis using a recombination-detecting software. The results indicate that recombination events are not rare in the case of BVDV, which frequently causes immunotolerance and, consequently, persistent infection in calves. The lack of specific immunity provides an ideal possibility for multiple infections by antigenically related but genetically different BVDV strains, and hence recombinations may occur. Among the 62 BVDV-1 genomes five recombinants and their possible parent strains, while among the 50 BVDV-2 genomes one simple recombinant and its parent strains were identified, which were supported by extremely strong probability values (P values varying between 1.26 × 10-4 and 1.58 × 10-310). Besides the newly identified recombinants, recombination events described previously were confirmed, but in some of these cases former information was completed with new data, or different parent(s) were suggested by the programme (RDP 4.46 BETA) used in this study.

Prevalence of bovine viral diarrhoea virus in cattle farms in Hungary.

A study was performed to survey the virological prevalence of bovine viral diarrhoea (BVD) virus (BVDV) in cattle herds in Hungary between 2008 and 2012. A total of 40,413 samples for BVDV detection and 24,547 samples for antibody testing were collected from 3,247 herds (570,524 animals), thus representing approximately 75% of the cattle population in Hungary. Retrospective Bayesian analysis demonstrated that (1) the herd-level true virus prevalence was 12.4%, (2) the mean individual (within-herd) true virus prevalence was 7.2% in the herds having at least one virus-positive animal and 0.89% for all investigated herds with a mean apparent prevalence of 1.15% for the same population. This is the first study about BVDV prevalence in Hungary.

Seroprevalence of bovine viral diarrhoea virus in Hungary - situation before launching an eradication campaign.

Bovine viral diarrhoea (BVD) is a viral disease appearing in various forms and causing high economic losses in the cattle stocks of Hungary. The aim of the present study was to determine the prevalence of bovine viral diarrhoea virus (BVDV) in Hungary through a monitoring survey carried out on samples collected in cattle-keeping units throughout the country. Since no such survey had been carried out in Hungary during the last thirty years, our study may serve as a basis for later monitoring investigations aimed at following the progress of an expected eradication campaign of BVD. The tests were carried out using an ELISA method, on a total of 1200 blood samples submitted from 54 cattle herds. The herds had not been vaccinated against BVDV before the sampling. Out of the 1200 samples, 521 proved to be positive (43.4%), 40 gave doubtful result (3.3%) and 639 were negative (53.3%). In some stocks the samples were collected from cows having completed several lactation periods, and therefore the seronegativity indicates the BVDV-free status of the given stock. Moreover, among the positive herds we found a few where the seropositivity rate was rather low (< 5%). According to the results of the survey, a rather high portion (about one third) of the cattle-keeping units of Hungary can be regarded as BVDV free, which ratio is much higher than had been expected on the basis of surveys carried out on a lower number of samples and in smaller regions of the country. Hence, the chances of an eradication campaign launched in the near future, or carried out parallel to the IBR eradication programme, are better than previously expected.

Effective multiple oral administration of reverse genetics engineered infectious bursal disease virus in mice in the presence of neutralizing antibodies.

BACKGROUND: Despite spectacular successes in hepatitis B and C therapies, severe hepatic impairment is still a major treatment problem. The clinically tested infectious bursal disease virus (IBDV) superinfection therapy promises an innovative, interferon-free solution to this great unmet need, provided that a consistent manufacturing process preventing mutations or reversions to virulent strains is obtained. METHODS: To address safety concerns, a tissue culture adapted IBDV vaccine strain V903/78 was cloned into cDNA plasmids ensuring reproducible production of a reverse engineered virus R903/78. The therapeutic drug candidate was characterized by immunocytochemistry assay, virus particle determination and immunoblot analysis. The biodistribution and potential immunogenicity of the IBDV agent was determined in mice, which is not a natural host of this virus, by quantitative detection of IBDV RNA by a quantitative reverse transcriptase-polymerase chain reaction and virus neutralization test, respectively. RESULTS: Several human cell lines supported IBDV propagation in the absence of visible cytopathic effect. The virus was stable from pH 8 to pH 6 and demonstrated significant resistance to low pH and also proved to be highly resistant to high temperatures. No pathological effects were observed in mice. Single and multiple oral administration of IBDV elicited antibodies with neutralizing activities in vitro. CONCLUSIONS: Repeat oral administration of R903/78 was successful despite the presence of neutralizing antibodies. Single oral and intravenous administration indicated that IBDV does not replicate in mammalian liver alleviating some safety related concerns. These data supports the development of an orally delivered anti-hepatitis B virus/ anti-hepatitis C virus viral agent for human use.

Emergence of multireassortant bluetongue virus serotype 4 in Hungary.

The genome sequence and the phylogenetic relationships of a serotype 4 bluetongue virus (BTV-4) emerged during 2014 in Hungary are described in this study. Genome segment 2 encoding the major neutralization antigen, VP2, shared moderate sequence similarity (nt, ⩽ 94.3%) with the corresponding gene of contemporary and historic homotypic bluetongue viruses, whereas genome segments S1, S4, S5, S7-S10 were typically more closely related to the cognate genes of heterotypic isolates. Importantly, in many gene phylogenies the Hungarian BTV-4 strain showed genetic relationship to BTV strains identified in outbreaks in the western Mediterranean basin. Our results indicate the identified Hungarian bluetongue virus strain evolved through reassortment involving multiple genome segments from various heterotypic bluetongue viruses.

Sarcocystis-infection of cattle in Hungary.

BACKGROUND: Reports on Sarcocystis-infection of cattle are outdated or lacking in many European countries, including those in the Central-Eastern part of the continent. Therefore, to assess the prevalence of Sarcocystis spp. among bovids in Hungary, a countrywide survey was initiated. In addition, fulminant deaths of four cattle, that showed clinical signs and post mortem lesions resembling acute sarcocystiosis ("Dalmeny disease"), were investigated. METHODS: During the countrywide survey individual heart and oesophagus samples were collected at slaughterhouses from 151 beef cattle and from 15 buffalo, kept in 31 places of Hungary. Analysis for Sarcocystis spp. was carried out with conventional PCRs for the 18S rDNA gene and gel electrophoresis, followed by sequencing of 36 strongly positive samples. Mortality cases were evaluated by histological, molecular, bacteriological and virological analyses of samples from various organs. RESULTS: Among slaughtered cattle the rate of Sarcocystis-infection was 66%. S. cruzi was identified as the most prevalent species in aurochs-like breed, and the zoonotic S. hominis in Hungarian grey cattle. Concerning the sudden deaths of cattle, Sarcocystis-infection could not be demonstrated in organs showing haemorrhages, but S. cruzi cysts were present in the muscles. In one case "S. sinensis" was molecularly identified in the blood (indicating sarcocystaemia). Results of analyses for bacterial/viral pathogens were negative. CONCLUSIONS: S. cruzi appears to be the most prevalent Sarcocystis sp. in cattle in Hungary, followed by the zoonotic S. hominis. However, the rate of infection with both species was shown to differ between cattle breeds. The suspected role of Sarcocystis spp. as causative agents of the fatal cases could not be confirmed.

First report on Babesia cf. microti infection of red foxes (Vulpes vulpes) from Hungary.

BACKGROUND: To date, only one report of a small Babesia infection based on microscopic observation which caused babesiosis in two dogs in Hungary has been published. Babesiosis due to Babesia canis - which is endemic in the local dogs - has only been detected in captive grey wolves. No information is available on babesial/theilerial infections in red foxes in Hungary. The aim of the study was to screen red foxes in Hungary for babesial parasites by PCR and to compare their partial 18S rRNA gene sequences to those parasites of domestic dogs and wild canids from other countries. METHODS: Blood samples of 404 red foxes originating from 316 locations representing all 19 Hungarian counties were screened in Hungary for babesial parasites by PCR and the partial 18S rRNA gene sequences were compared to those parasites of domestic dogs and wild canids from other countries. RESULTS: Altogether 81 red foxes out of 404 (20.0%; 95% CI: 16.4-24.2%) shot in 74 locations and in 17 of the 19 Hungarian counties were found to be infected with Babesia cf. microti by PCR. CONCLUSIONS: This is the first report to demonstrate the occurrence of Babesia cf. microti in Hungary, and its widespread presence in the fox population throughout the country. Further studies are needed to identify the tick species involved in its transmission, and whether other mechanisms of transmission are involved in its spread in fox populations.

First molecular evidence of Hepatozoon canis infection in red foxes and golden jackals from Hungary.

BACKGROUND: Recently, Hepatozoon canis infection has been detected among shepherd, hunting and stray dogs in the southern part of Hungary, which is considered to be free of Rhipicephalus sanguineus sensu lato and close to the border with Croatia. The aim of this study was to acquire information on the possibility that red foxes and/or golden jackals could play a role in the appearance and spread of H. canis in Hungary. METHODS: A conventional PCR was used to amplify a 666 bp long fragment of the Hepatozoon 18S rRNA gene from blood samples collected from 334 foxes shot in 231 locations in 16 counties and 15 golden jackals shot in 9 locations in two southwestern counties close to Croatia. A second PCR assay was performed in some of the samples positive by the first PCR to amplify a larger segment (approximately 1500 bp) of the 18S rRNA gene of Hepatozoon spp. for further phylogenetic analysis. RESULTS: Hepatozoon infection was detected in canids shot in 30 locations and 9 counties. Altogether 26 foxes (8.0%, 95% CI: 5-11%) and 9 jackals (60%, 95% CI: 33-81%) were PCR positive. Hepatozoon canis sequences were obtained from 12 foxes and 7 jackals. DNA sequences from 16 animals were 99-100% similar to H. canis from Croatian foxes or dogs while two of the sequences were 99% similar to an Italian fox. Half (13/26) of the infected red foxes and all golden jackals were shot in the two southwestern counties. CONCLUSIONS: This is the first report on molecular evidence of H. canis in red foxes (Vulpes vulpes) and golden jackals (Canis aureus) from Hungary, which is considered free from the tick vector of H. canis, R. sanguineus. Although no R. sanguineus sensu lato had been found on infected or non-infected wild canids, the detection of authochnous canine hepatozoonosis in Hungary might imply that the range of R. sanguineus sensu lato has reached this country.

Novel mastadenovirus infection and clinical disease in a pygmy marmoset (Callithrix [Cebuella] pygmaea).

We describe the detection and successful isolation of a novel mastadenovirus from a pygmy marmoset (Callithrix [Cebuella] pygmaea) that died following an episode of severe respiratory signs. Pathologic/histopathologic examination revealed hydrothorax and catarrhal bronchopneumonia with pronounced desquamation of the bronchiolar epithelial cells, while in other airways a marked hyperplasia of the epithelial lining and numerous giant cells could be observed. We obtained partial sequence data from the adenoviral DNA-dependent DNA-polymerase gene of the isolated strain and analyses of this region showed the highest level of identity to the recently described bat adenoviruses (strains PPV1 and TJM) and the type 2 canine adenovirus. Similar results were gained by phylogenetic calculations indicating that this novel marmoset adenovirus is only distantly related to reference Old and New World primate adenoviruses and formed a monophyletic group with bat and canine adenoviruses and the equine adenovirus 1. Even though the source of the infection remained unknown, our results could imply the possibility of a cross-species transmission of the virus from an anonymous host to the pygmy marmoset.

Molecular characterization of feline infectious peritonitis virus strain DF-2 and studies of the role of ORF3abc in viral cell tropism.

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log(10) titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.