Ádám Bálint

Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area (Beringia)

Coronaviruses (CoVs) can cause mild to severe disease in humans and animals, their host range and environmental spread seem to have been largely underestimated, and they are currently being investigated for their potential medical relevance. Infectious bronchitis virus (IBV) belongs to gamma-coronaviruses and causes a costly respiratory viral disease in chickens. The role of wild birds in the epidemiology of IBV is poorly understood. In the present study, we examined 1,002 cloacal and faecal samples collected from 26 wild bird species in the Beringia area for the presence of CoVs, and then we performed statistical and phylogenetic analyses. We detected diverse CoVs by RT-PCR in wild birds in the Beringia area. Sequence analysis showed that the detected viruses are gamma-coronaviruses related to IBV. These findings suggest that wild birds are able to carry gamma-coronaviruses asymptomatically. We concluded that CoVs are widespread among wild birds in Beringia, and their geographic spread and frequency is higher than previously realised. Thus, Avian CoV can be efficiently disseminated over large distances and could be a genetic reservoir for future emerging pathogenic CoVs. Considering the great animal health and economic impact of IBV as well as the recent emergence of novel coronaviruses such as SARS-coronavirus, it is important to investigate the role of wildlife reservoirs in CoV infection biology and epidemiology.

Molecular Characterization of Feline Infectious Peritonitis Virus Strain DF-2 and Studies of the Role of ORF3abc in Viral Cell Tropism

ABSTRACT 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 log10 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.

Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus

Abstract A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV®). One TCID50 was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.

Characterisation of the first complete genome sequence of the roe deer (Capreolus capreolus) papillomavirus

The complete genomic DNA of a novel roe deer (Capreolus capreolus) papillomavirus (CcPV1) was amplified and sequenced from fibropapillomatous skin lesions of a Hungarian roe deer. Viral DNA was detected by a pair of degenerate primers and the remaining genomic sequence was amplified by a long-template high-fidelity PCR and sequenced. The CcPV1 genome was 8032 bp long and contained open reading frames (ORFs) typical for Delta-papillomaviruses (E6, E7, E1, E2, E4, E5, E9, L2, and L1) and a 799 bp long untranslated regulatory region (URR). Phylogenetic analysis based on the 3861 bp long concatenated sequence of the E1-E2-L2-L1 ORFs and on separate alignments of all major ORFs using both neighbour-joining and maximum parsimony methods placed CcPV1 on a distinct branch between Ovine papillomavirus 1 and the other deer papillomaviruses within the Delta-papillomavirus genus, although pairwise nucleotide alignments of L1 ORF sequences determined highest identities with European Elk Papillomavirus (71.2%) and Reindeer Papillomavirus (70.3%).

Pathobiology of highly pathogenic avian influenza virus (H5N1) infection in mute swans (Cygnus olor)

The results of pathological, virological and polymerase chain reaction examinations carried out on 35 mute swans (Cygnus olor) that succumbed to a highly pathogenic avian influenza virus (H5N1) infection during an outbreak in Southern Hungary are reported. The most frequently observed macroscopic lesions included: haemorrhages under the epicardium, in the proventricular and duodenal mucosa and pancreas; focal necrosis in the pancreas; myocardial degeneration; acute mucous enteritis; congestion of the spleen and lung, and the accumulation of sero-mucinous exudate in the body cavity. Histopathological lesions comprised: lymphocytic meningo-encephalomyelitis accompanied by gliosis and occasional perivascular haemorrhages; multi-focal myocardial necrosis with lympho-histiocytic infiltration; pancreatitis with focal necrosis; acute desquamative mucous enteritis; lung congestion and oedema; oedema of the tracheal mucosa and, in young birds, the atrophy of the bursa of Fabricius as a result of lymphocyte depletion and apoptosis. The observed lesions and the moderate to good body conditions were compatible with findings in acute highly pathogenic avian influenza infections of other bird species reported in the literature. Skin lesions and lesions typical for infections caused by strains of lower pathogenicity (low pathogenic avian influenza virus) such as emaciation or fibrinous changes in the reproductive and respiratory organs, sinuses and airsacs were not observed. The H5N1 subtype avian influenza virus was isolated in embryonated fowl eggs from all cases and it was identified by classical and molecular virological methods.

Four different sublineages of highly pathogenic avian influenza H5N1 introduced in Hungary in 2006-2007

Highly pathogenic avian influenza (HPAI) H5N1 viruses were introduced to Hungary during 2006-2007 in three separate waves. This study aimed at determining the full-length genomic coding regions of the index strains from these epizootics in order to: (i) understand the phylogenetic relationship to other European H5N1 isolates, (ii) elucidate the possible connection between the different outbreaks and (iii) determine the putative origin and way of introduction of the different virus variants. Molecular analysis of the HA gene of Hungarian HPAI isolates obtained from wild birds during the first introduction revealed two groups designated Hungarian1 (HUN1) and Hungarian2 (HUN2) within sublineage 2.2B and clade 2.2.1, respectively. Sequencing the whole coding region of the two index viruses A/mute swan/Hungary/3472/2006 and A/mute swan/4571/Hungary/2006 suggests the role of wild birds in the introduction of HUN1 and HUN2 viruses: the most similar isolates to HUN1 and HUN2 group were found in wild avian species in Croatia and Slovakia, respectively. The second introduction of HPAI H5N1 led to the largest epizootic in domestic waterfowl in Europe. The index strain of the epizootic A/goose/Hungary/14756/2006 clustered to sublineage 2.2.A1 forming the Hungarian3 (HUN3) group. A common ancestry of HUN3 isolates with Bavarian strains is suggested as the most likely scenario of origin. Hungarian4 (HUN4) viruses isolated from the third introduction clustered with isolate A/turkey/United Kingdom/750/2007 forming a sublineage 2.2.A2. The origin and way of introduction of HUN4 viruses is still obscure, thus further genetic, phylogenetic, ecological and epidemiological data are required in order to elucidate it.

Unique genomic organization of a novel Avipoxvirus detected in turkey (Meleagris gallopavo)

Avipoxviruses are emerging pathogens affecting over 200 bird species worldwide. Genetic characterization of avipoxviruses is performed by analysis of genomic regions encoding the 4b and DNA polymerase. Whole genome sequence data are limited to a few avipoxvirus isolates. Based on phylogenetic analysis three major genetic clades are distinguished. In this study we report a novel avipoxvirus strain causing skin lesions in domestic turkey. The virus was identified in Hungary during 2011 in a flock of turkey vaccinated against avipoxvirus infection. The genome of the isolated strain, TKPV-HU1124/2011, was uniquely short (∼188.5kbp) and was predicted to encode reduced number of proteins. Phylogenetic analysis of the genes encoding the 4b and DNA polymerase separated TKPV-HU1124/2011 from other turkey origin avipoxviruses and classified it into a new genetic clade. This study permits new insight into the genetic and genomic heterogeneity of avipoxviruses and pinpoints the importance of strain diversity in vaccine efficacy.

Viral Sequence Insertions and a Novel Cellular Insertion in the NS2 Gene of Cytopathic Isolates of Bovine Viral Diarrhea Virus as Potential Cytopathogenicity Markers

Cytopathogenicity of bovine viral diarrhea virus (BVDV) has been shown to correlate with the presence of insertions of cellular sequences, duplication of viral sequences with or without insertions, deletions, and point mutations in the genomes of cytopathogenic (cp) strains. In the present study we have investigated cytopathogenicity markers in the genomes of six cp BVDV isolates. The viruses were selected as representatives of various forms of BVDV infection, in some cases presumably induced by vaccination with a live attenuated vaccine. The complete NS2-3 coding region of the six isolates and of the vaccine virus were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. In the genomes of four isolates (H6379, H6712, H8427 and H-BVD MD) and of the vaccine virus, a 45-nucleotide viral insertion was found at nucleotide position 4355, encompassing nucleotides 8402–8446, that encode 15 amino acids of the NS4B/NS5A junction region in a normal BVDV genome. Isolate H3887 had a 21-nucleotide insertion of non-viral origin, also located at nucleotide position 4355. This insertion has a high homology with a gene coding for murine interferon-induced guanylate-binding protein 1, and represents the first non-viral insertion identified at this position of the NS2 coding region. Isolate H3142 carries a 42-nucleotide insertion at position 4361, identical to a part of the NS5B gene mapping to position 11078–11119. Additionally, this isolate also has a deletion of three nucleotides (positions 4448–4450). The role of the 45-nucleotide insertion in expression of NS3 was investigated using the vaccine virus. The NS2-3 gene of this virus, and that of a generated insertion-negative variant were cloned in the mammalian expression vector pCI, and expressed in bovine turbinate cells. Western blot analysis revealed that the insertion contributed to a partial cleavage of NS2-3 generating NS3, the marker protein for cytopathogenicity in BVDV. The genome rearrangements found in these isolates occurred preferentially at position 4355, suggesting that this part of the genome could represent a potential hot spot for recombination events in ncp BVDV. The molecular mechanism underlying this phenomenon, however, remains to be elucidated.

Recombinant feline coronaviruses as vaccine candidates confer protection in SPF but not in conventional cats

Abstract Feline infectious peritonitis virus (FIPV) is a major pathogen of Felidae. Despite the extensive efforts taken in the past decades, development of the “ideal” live attenuated FIPV vaccine was not successful yet. In the present study, we provide data of immunisation experiments with a recombinant FCoV pair differing only in the truncation (PBFIPV-DF-2) or completion (PBFIPV-DF-2-R3i) of their ORF3abc regions. In our previous in vivo studies, these viruses proved to show the characters of low virulent or avirulent FCoV phenotypes, respectively. Therefore, we hypothesised the ability of these viruses, as possible vaccine candidates, in conferring protection in specific pathogen free (SPF) Domestic Shorthair as well as in conventional purebred British Shorthair cats. In SPF cats, after two oronasal and two intramuscular vaccinations with two weeks intervals, both vaccine candidates provided 100% protection against lethal homologous challenge with the highly virulent FIPV DF-2 strain. In contrast, the conventional purebred British Shorthair cats did not develop protection when they were immunised with the same vaccination regimes. In these groups 100% of the PBFIPV-DF-2-R3i immunised animals developed antibody-dependent enhancement (ADE). Prolonged survival was observed in 40% of the animals, while 60% showed fulminant disease course. Genetic and more probably immunological differences between the SPF and non-SPF purebred kittens can explain the different outcome of the vaccination experiment. Our data highlight the diverse immune responses between SPF and conventional cats and suggest a decisive role of previous infection by heterologous causative agents in the outcome of the vaccination against FIP.

Comparison of two H1N2 swine influenza A viruses from disease outbreaks in pigs in Sweden during 2009 and 2010.

The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. In the present study, two relatively uncommon swine influenza virus (SIV) H1N2 subtypes, isolated in Sweden in 2009 and 2010, were compared regarding their molecular composition and biological characteristics. The differences regarding markers purportedly related to pathogenicity, host adaptation or replication efficiency. They included a truncated PB1-F2 protein in the earlier isolate but a full length version in the more recent one; differences in the number of haemagglutinin glycosylation sites, including a characteristic human one; and a nuclear export protein with altered export signal. Of particular interest, the NS1 amino acid sequence of swine H1N2-2009 and 2010 has a ‘unique or very unusual’ PDZ binding domain (RPKV) at the C-terminal of the protein, a motif that has been implicated as a virulence marker. Concerning biological properties, these viruses reached lower titre and showed reduced cytopathogenicity in MDCK cells compared with an avian-like H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the same lineage as the 2009 and 2010 isolates. The findings should contribute to better understanding of factors related to the survival/extinction of this uncommon reassortant variant.