Research Note: An overview on distribution of fowl adenoviruses.

Fowl adenoviruses (FAdV), detected during routine diagnostic investigations from 38 countries (5 continents) over a decade, were partially sequenced and grouped by phylogenetic analysis. The partial polymerase gene nucleotide sequences of the 365 fowl adenovirus isolates resulted in the following species distribution: 11% FAdV-A; 3% FAdV-B; 2% FAdV-C; 34% FAdV-D; and 50% FAdV-E. Noticeably, only 79 of the detected strains could be associated with adenovirus-specific pathologic conditions: 62 (79%) with inclusion body hepatitis; 9 (11%) with gizzard erosion; and 8 (10%) with hepatitis hydropericardium syndrome. The remainder of the FAdV strains was detected as concomitant infection from other disease conditions almost exclusively in boilers of 27 to 42 d of age: the majority of them was FAdV-E followed by FAdV-D, and to a lesser extent of FAdV-A, B, and C, the latter ones have not been associated with any of the established adenovirus-caused syndromes in our collection. The highest ratio of coinfections was observed for FAdV-B (62%), while it was about 30% for the rest of the FAdV species. The most frequent coinfection, in connection with all FAdV species, was with the avian infectious bronchitis virus. The presented database will serve as the basis for comparative whole genome and cross-neutralization analysis of selected FAdV isolates.

Molecular Diversity and Genetic Relatedness of Candida albicans Isolates from Birds in Hungary.

The molecular epidemiology of Candida albicans infections in animals has been rarely studied. In this study, multilocus sequence typing was used to characterise the genetic diversity and population structure of 24 avian origin C. albicans isolates collected from different birds with candidiasis and compared to human isolates. Fourteen diploid sequence types (DSTs) including six new DSTs were determined. Cluster analysis revealed that isolates grouped into 8 clades. Bird isolates mainly belonged to minor clades and Clade 15 with DST 172 was the most common (11 isolates; 45.8%). The remaining isolates were clustered into Clade 7 (5 isolates; 20.8%), Clade 10 (4 isolates; 16.6%), Clade 8 (2 isolates; 8.3%), Clade 4 (1 isolate; 4.2%) and Clade 16 (1 isolate; 4.2%). Unweighted pair group method with arithmetic averages (UPGMA) and eBURST analyses showed that the genetic construction of avian origin C. albicans population is fairly diverse. Although species-specific lineages were not found, some degree of separation in the evolution of bird and human strains could be observed.

Coding-complete sequence of a vaccine-derived recombinant porcine reproductive and respiratory syndrome virus strain isolated in Hungary.

Porcine reproductive and respiratory syndrome virus 1 is a major cause of swine morbidity and mortality in various parts of the world, including Hungary. A national elimination programme to reduce the associated economic burden was initiated in Hungary in 2012. Using extensive laboratory surveillance, we identified and isolated an unusual PRRSV strain. The complete coding sequence of this isolate was determined and analyzed. The genome of this Hungarian PRRSV1 strain, HUN60077/16, is 15,081 nucleotides in length. Phylogenetic and recombination analysis showed a mosaic structure of the genome where a large fragment of ORF1b and the genomic region coding for ORF3 to ORF7 showed a very close genetic relationship to the vaccine virus Unistrain, while the ORF1a region, the 3' end of ORF1b, and the whole ORF2 were only distantly related to this or any other PRRSV1 strain whose genome sequence is available in the GenBank database. Genomic characterization of PRRSV strains is crucial when possible vaccine-associated cases are identified. This approach not only helps to identify genetic interactions between vaccine and wild-type PRRSV1 strains but may also be needed to prevent trust in commercial vaccines from being undermined.

Country-wide distribution of bluetongue virus with expanding host spectrum and evidence of vector competence in Hungary.

Following the introduction of bluetongue virus type 4 (BTV-4) in 2014, country-wide monitoring of bluetongue (BT) disease was performed to see whether the virus has become enzootic in Hungary. To analyse the epizootiology of BT, over 110,000 samples collected from domestic and wild ruminants were screened for the presence of BTV RNA and virus-specific antibodies using real-time RT-PCR assay and commercial ELISA kit, respectively. During laboratory analysis, specimens collected from 333 (0.8%) cattle, 79 (2.2%) sheep, 4 (0.9%) goats, and 1 (2.3%) mouflon were found to be positive by viral RNA-detection assay. In addition, antibody to BTV was detected in 5.5% (3158/57,250) of cattle, 10.1% (517/5120) of sheep, 40% (116/290) of goat, and 5.6% (16/284) of buffalo origin samples. The majority of positive samples originated from south-western counties; however, 18 out of 19 counties reported cases or antibody prevalence in the examined animals. Genome sequencing of a representative BTV-4 strain from 2015 was also performed. When comparing this strain with the isolate BTV4-HUN2014 detected only a year earlier in Hungary, mutations at 14 sites were identified within the amplified and sequenced genome. Our findings reinforce the need for continued surveillance of BT disease in Hungary. Keywords: reoviridae; orbivirus; cattle; sheep; goat; biting midge.

Molecular characterization of unusual bovine rotavirus A strains having high genetic relatedness with human rotavirus: evidence for zooanthroponotic transmission.

We report here the genomic characterization of two rare rotavirus A (RVAs) G1P[11] and G9P[X] strains detected in cattle calves from two different geographical locations in India during routine rotavirus surveillance. These strains possessed unusual G types (VP7 gene) on a bovine/artiodactyl genotype constellation, G1-P[11]-I2-Rx-Cx-Mx-Ax-N2-T6-E2-H3 (HR-B91) and G9-P[X]-I2-Rx-Cx-Mx-Ax-N2-T6-E2-H3 (WB-H2). This is the first report on molecular characterization of G9 in cattle, and second report on G1 in cattle. The VP7 gene of HR-B91 occupied lineage IIc within G1 while that of WB-H2 occupied IIIb within G9 genotype. The latter was found to be very diverse from other RVA strains of G9 genotype, and this may emerge as a new genotype in due course. The study provides evidence of zooanthroponotic transmission of human G1 and G9 RVA genes to calves. Of note, the G9 genotype was found to serve as the ancestral genotype for G1. Phylogenetic analysis of remaining gene segments revealed close relatedness to artiodactyl or artiodactyl-like human RVA strains. The findings of this study highlight the potential role of interspecies transmission and reassortment events in generating the rare rotavirus strains.

Resurgence of rabies in Hungary during 2013-2014: An attempt to track the origin of identified strains.

In 2013-2014, accumulation of rabies episodes raised concerns regarding ongoing elimination programme in Hungary. Nearly four dozen cases were identified over a 13-month period in the central region of the country far behind the immunization zones. Although the outbreak was successfully controlled, the origin of disease remained unknown. In this study, we sequenced the partial N and G genes from 47 Hungarian rabies virus (RV) strains isolated from the 2013-2014 outbreak. Sequencing and phylogenetic analysis of the N and G genes showed that the Hungarian RV isolates share high nucleotide similarity among each other (up to 100%). When analysing the N gene, comparable sequence similarity was seen between the outbreak strains and some historic Romanian RV strains. Unfortunately, in the lack of available sequence data from the Romanian RV strains, the genetic relationship within the G gene could not be determined. Phylogenetic analysis of Hungarian RV isolates detected in the past revealed that multiple independent RV lineages circulated in our country over the past 25 years. The parental strain of the 2013-2014 outbreak may have been imported independently perhaps from east through transborder movement of a reservoir animal. Next to the introduction, this imported RV strain seems to have spread clonally in the affected area. Our findings indicate that despite effective control measures that, overall, minimized the incidence of rabies over the past decade, field and laboratory monitoring needs to be continued to make rabies elimination programme in Hungary successful.

Introduction and prolonged circulation of G12 rotaviruses in Sicily.

Genotype G12 strains are now considered to be the sixth most prevalent human rotaviruses worldwide. In two Sicilian cities, Palermo and Messina, surveillance of rotavirus circulation performed since 1985 and 2009, respectively, did not detect G12 strains until 2012. From 2012 to 2014 rotavirus infection was detected in 29·7% of 1647 stool samples collected from children admitted for acute gastroenteritis to three Sicilian hospitals in Palermo, Messina and Ragusa. In 2012, G12P[8] was first detected in Palermo and then in Messina where it represented the second most frequent genotype (20% prevalence) after G1P[8]. Thereafter, G12 strains continued to circulate in Sicily, showing a marked prevalence in Ragusa (27·8%) in 2013 and in Palermo (21%) and Messina (16·6%) in 2014. All but one of the Sicilian G12 strains carried a P[8] VP4 genotype, whereas the single non-P[8] rotavirus strain was genotyped as G12P[9]. Phylogenetic analysis of the VP7 and VP4 sequences allowed distinction of several genetic lineages and separation of the G12P[8] strains into three cluster combinations. These findings indicate independent introductions of G12 rotavirus strains in Sicily in recent years.

Novel bocaparvoviruses in rabbits.

Bocaparvovirus is a newly established genus within the family Parvoviridae and has been identified as a possible cause of enteric, respiratory, reproductive/neonatal and neurological disease in humans and several animal species. In this study, metagenomic analysis was used to identify and characterise a novel bocaparvovirus in the faeces of rabbits with enteric disease. To assess the prevalence of the novel virus, rectal swabs and faecal samples obtained from rabbits with and without diarrhoea were screened with a specific PCR assay. The complete genome sequence of the novel parvovirus was reconstructed. The virus was distantly related to other bocaparvoviruses; the three ORFs shared 53%, 53% and 50% nucleotide identity, respectively, to homologous genes of porcine bocaparvoviruses. The virus was detected in 8/29 (28%) and 16/95 (17%) samples of rabbits with and without diarrhoea, respectively. Sequencing of the capsid protein fragment targeted by the diagnostic PCR identified two distinct bocaparvovirus populations/sub-types, with 91.7-94.5% nucleotide identity to each other. Including these novel parvoviruses in diagnostic algorithms of rabbit diseases might help inform their potential pathogenic role and impact on rabbit production and the virological profiles of laboratory rabbits.

Full-genome sequencing of a Hungarian canine G3P[3] Rotavirus A strain reveals high genetic relatedness with a historic Italian human strain.

A canine Rotavirus A strain was identified in the fecal specimen of a young dog during 2012 in Hungary. The strain RVA/Dog-wt/HUN/135/2012/G3P[3] shared complete genotype constellation (G3-P[3]-I3-R3-C3-M3-A15-N2-T3-E3-H6) and high genome sequence similarity (nt, 98.8 %) with a historic human strain, RVA/Human-tc/ITA/PA260-97/1997/G3P[3]. This study provides evidence for the canine origin of the unusual NSP1 genotype, A15, and reinforces the hypothesis of direct interspecies transmission of canine rotaviruses to humans.

West Nile virus surveillance in mosquitoes, April to October 2013, Vojvodina province, Serbia: implications for the 2014 season.

After the West Nile virus (WNV) outbreak in 2012, we collected mosquito samples from Vojvodina province, Serbia, in 2013. We found high WNV infection rate in two species, Culex pipiens and Anopheles maculipennis. Phylogenetic analysis showed that Serbian WNV strains from 2013 were most closely related to Italian and Greek strains isolated in 2012 and 2010, respectively. Public health authorities should be aware of a potentially increased risk of WNV activity during the 2014 season.

Novel European lineages of bat astroviruses identified in Hungary.

KEYWORDS: bat, astroviruses, coronaviruses, snRT-PCR, Hungary.

Analysis of early strains of the norovirus pandemic variant GII.4 Sydney 2012 identifies mutations in adaptive sites of the capsid protein.

Global surveillance for norovirus identified in 2012 the emergence of a novel pandemic GII.4 variant, termed Sydney 2012. In Italy, the novel pandemic variant was identified as early as November 2011 but became predominant only in the winter season 2012-2013. Upon sequencing and comparison with strains of global origin, the early Sydney 2012 strains were found to differ from those spreading in 2012-2013 in the capsid (ORF2) putative epitopes B, C and D, segregating into a distinct phylogenetic clade. At least three residues (333, 340 and 393, in epitopes B, C and D, respectively) of the VP1 varied among Sydney 2012 strains of different clades. These findings suggest that the spread of the pandemic variant in Italy during the winter season 2012-2013 was due to the introduction of strains distinct from those circulating at low frequency in the former winter season and that similar strains were also circulating elsewhere worldwide.

One year survey of human rotavirus strains suggests the emergence of genotype G12 in Cameroon.

In this study the emergence of rotavirus A genotype G12 in children <5 years of age is reported from Cameroon during 2010/2011. A total of 135 human stool samples were P and G genotyped by reverse transcriptase PCR. Six different rotavirus VP7 genotypes were detected, including G1, G2, G3, G8, G9, and G12 in combinations with P[4], P[6] and P[8] VP4 genotypes. Genotype G12 predominated in combination with P[8] (54.1%) and P[6] (10.4%) genotypes followed by G1P[6] (8.2%), G3P[6] (6.7%), G2P[4] (5.9%), G8P[6] (3.7%), G2P[6] (0.7%), G3P[8] (0.7%), and G9P[8] (0.7%). Genotype P[6] strains in combination with various G-types represented a substantial proportion (N=44, 32.6%) of the genotyped strains. Partially typed strains included G12P[NT] (2.2%); G3P[NT] (0.7%); G(NT)P[6] (1.5%); and G(NT)P[8] (0.7%). Mixed infections were found in five specimens (3.7%) in several combinations including G1+ G12P[6], G2+ G3P[6] + P[8], G3+ G8P[6], G3 + G12P[6] + P[8], and G12P[6] +P[8]. The approximately 10% relative frequency of G12P[6] strains detected in this study suggests that this strain is emerging in Cameroon and should be monitored carefully as rotavirus vaccine is implemented in this country, as it shares neither G- nor P-type specificity with strains in the RotaTeq® and Rotarix® vaccines. These findings are consistent with other recent reports of the global spread and increasing epidemiologic importance of G12 and P[6] strains.

Human G9P[8] rotavirus strains circulating in Cameroon, 1999-2000: Genetic relationships with other G9 strains and detection of a new G9 subtype.

Group A rotaviruses (RV-A) are the leading cause of viral gastroenteritis in children worldwide and genotype G9P[8] is one of the five most common genotypes detected in humans. In order to gain insight into the degree of genetic variability of G9P[8] strains circulating in Cameroon, stool samples were collected during the 1999-2000 rotavirus season in two different geographic regions in Cameroon (Southwest and Western Regions). By RT-PCR, 15 G9P[8] strains (15/89=16.8%) were identified whose genomic configurations was subsequently determined by complete or partial gene sequencing. In general, all Cameroonian G9 strains clustered into current globally-spread sublineages of the VP7 gene and displayed 86.6-100% nucleotide identity amongst themselves and 81.2-99.5% nucleotide identity with global G9 strains. The full genome classification of all Cameroonian strains was G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 but phylogenetic analysis of each gene revealed that the strains were spread across 4 or more distinct lineages. An unusual strain, RVA/Human-wt/CMR/6788/1999/G9P[8], which shared the genomic constellation of other Cameroonian G9P[8] strains, contained a novel G9 subtype which diverged significantly (18.8% nucleotide and 19% amino acid distance) from previously described G9 strains. Nucleotide and amino acid alignments revealed that the 3' end of this gene is highly divergent from other G9 VP7 genes suggesting that it arose through extensive accumulation of point mutations. The results of this study demonstrate that diverse G9 strains circulated in Cameroon during 1999-2000.

Genetic heterogeneity and recombination in human type 2 astroviruses.

Novel lineages of human astrovirus (HAstV) types 2, 2c, and 2d have been identified. Upon sequencing of the 3' end of the genome, the type 2c and 2d HAstVs were found to be open reading frame 1b (ORF1b)-ORF2 recombinant, with ORF1b being derived from type 3 and type 1 HAstVs, respectively. An ORF2 interlineage recombinant strain, 2c/2b, was also identified.

Enteric disease in dogs naturally infected by a novel canine astrovirus.

Infection by a novel canine astrovirus was associated with gastroenteritis in two dogs. The virus displayed 70.3 to 73.9% amino acid identity to other canine astroviruses in the full-length capsid. Specific antibodies were detected in the convalescent-phase sera of the dogs, indicating seroconversion. Also, the virus appeared weakly related antigenically to the prototype canine astrovirus isolate ITA/2008/Bari.

Human infection with novel G3P[25] rotavirus strain in Taiwan.

Genotype P[25] rotaviruses are rare and to date have been reported to occur only in a few countries of mainland Asia. Here we report the molecular characterization of a novel human rotavirus genotype combination, G3P[25], detected in a 17-month-old child hospitalized due to severe gastroenteritis during 2009 in central Taiwan. Sequencing and phylogenetic analysis of the VP4 gene demonstrated a distinct origin from other strains bearing the P[25] VP4 gene, whereas the VP7, VP6 and NSP4 gene phylogenies identified common origins with cognate genes of other, presumed human-porcine reassortment Taiwanese strains. These results suggest that interactions between human and animal strains appear to contribute to the generation of genetic and antigenic diversity of rotavirus strains, with potential public health importance in Taiwan.

Monitoring of group A rotaviruses in wild-living birds in Hungary.

Rotavirus is a common pathogen causing gastroenteritis in humans and domesticated animals. The incidence of rotavirus in wild-living animals, particularly in avian species, has not been systematically investigated. In this study 1220 fecal samples and cloacal swabs collected from wild-living birds during 2008 in Hungary were tested for the presence of group A rotaviruses by a VP6 gene-specific reverse-transcription-polymerase-chain-reaction assay. Of the 1220 samples, 276 and 944 were processed as individual and pooled specimens, respectively. Rotavirus was identified in two pooled pheasant (Phasianus colchicus) samples and two individual reed bunting samples (Emberiza schoeniclus). These data indicated a very low prevalence of group A rotaviruses (0.3%) in our sample set. Nonetheless, the present study, together with existing literature data, implies that rotavirus infections occur in a wide spectrum of feral bird species. These findings are exciting and suggest that pursuing rotavirus monitoring is needed to uncover avian rotavirus strain diversity and understand rotavirus ecology in nature.