Candidate 'Avian orthoreovirus B': An emerging waterfowl pathogen in Europe and Asia?

A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.

West Nile virus host-vector-pathogen interactions in a colonial raptor.

BACKGROUND: Avian host species have different roles in the amplification and maintenance of West Nile virus (WNV), therefore identifying key taxa is vital in understanding WNV epidemics. Here, we present a comprehensive case study conducted on red-footed falcons, where host-vector, vector-virus and host-virus interactions were simultaneously studied to evaluate host species contribution to WNV circulation qualitatively. RESULTS: Mosquitoes were trapped inside red-footed falcon nest-boxes by a method originally developed for the capture of blackflies and midges. We showed that this approach is also efficient for trapping mosquitoes and that the number of trapped vectors is a function of host attraction. Brood size and nestling age had a positive effect on the number of attracted Culex pipiens individuals while the blood-feeding success rate of both dominant Culex species (Culex pipiens and Culex modestus) markedly decreased after the nestlings reached 14 days of age. Using RT-PCR, we showed that WNV was present in these mosquitoes with 4.2% (CI: 0.9-7.5%) prevalence. We did not detect WNV in any of the nestling blood samples. However, a relatively high seroprevalence (25.4% CI: 18.8-33.2%) was detected with an enzyme-linked immunoabsorbent assay (ELISA). Using the ELISA OD ratios as a proxy to antibody titers, we showed that older seropositive nestlings have lower antibody levels than their younger conspecifics and that hatching order negatively influences antibody levels in broods with seropositive nestlings. CONCLUSIONS: Red-footed falcons in the studied system are exposed to a local sylvatic WNV circulation, and the risk of infection is higher for younger nestlings. However, the lack of individuals with viremia and the high WNV seroprevalence, indicate that either host has a very short viremic period or that a large percentage of nestlings in the population receive maternal antibodies. This latter assumption is supported by the age and hatching order dependence of antibody levels found for seropositive nestlings. Considering the temporal pattern in mosquito feeding success, maternal immunity may be effective in protecting progeny against WNV infection despite the short antibody half-life measured in various other species. We conclude that red-footed falcons seem to have low WNV host competence and are unlikely to be effective virus reservoirs in the studied region.

In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model.

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.

Monitoring of West Nile virus in mosquitoes between 2011-2012 in Hungary.

West Nile virus (WNV) is a widely distributed mosquito-borne flavivirus. WNV strains are classified into several genetic lineages on the basis of phylogenetic differences. Whereas lineage 1 viruses are distributed worldwide, lineage 2 WNV was first detected outside of Africa in Hungary in 2004. Since then, WNV-associated disease and mortality in animal and human hosts have been documented periodically in Hungary. After the first detection of WNV from a pool of Culex pipiens mosquitoes in 2010, samples were collated from several sources and tested in a 2-year monitoring program. Collection areas were located in the Southern Transdanubium, in northeastern Hungary, in eastern Hungary, and in southeastern Hungary. During the 2 years, 23,193 mosquitoes in 645 pools were screened for WNV virus presence with RT-PCR. Three pools were found positive for WNV in 2011 (one pool of Ochlerotatus annulipes collected in Fényeslitke in June, one pool of Coquillettidia richiardii collected in Debrecen, Fancsika-tó, in July, and one pool of Cx. pipiens captured near Red-Footed Falcon colonies at Kardoskút in September). The minimal infection rate (MIR=proportion of infected mosquitoes per 1000 mosquitoes) of all mosquito pools was 0.25, whereas the MIR of infected species was 2.03 for O. annulipes, 0.63 for C. richiardii, and 2.70 for C.x pipiens. Molecular data have demonstrated that the same lineage 2 WNV strain has circulated in wild birds, horses, humans, and mosquitoes in Hungary since 2004. Mosquito-based surveillance successfully complemented the ongoing, long-term passive surveillance system and it was useful for the early detection of WNV circulation.

Detection and characterization of a divergent avian reovirus strain from a broiler chicken with central nervous system disease.

Avian orthoreoviruses have been associated with a variety of diseases in chickens, including tenosynovitis, runting-stunting syndrome, hepatitis, myocarditis, osteoporosis, respiratory diseases, and central nervous system disease. The primary objective of our study was the molecular characterization of an avian reovirus strain, T1781, which was isolated from a broiler chicken with a central nervous system disorder in Hungary during 2012. The complete genome sequence was determined using a traditional sequencing method after cell culture adaptation of the strain. Sequence and phylogenetic analyses showed that T1781 shared only moderate nucleic acid sequence identity in several genes to previously analyzed reovirus strains from chickens, and each gene formed separate branches in the corresponding phylogenetic trees. The maximum nucleotide sequence identities of strain T1781 genes to reference avian reovirus strains ranged from 79 % to 90 %. Collectively, our analyses indicated that T1781 is a divergent chicken reovirus strain. The genetic background of this and other avian reoviruses associated with various disease manifestations needs further investigation.

Emergence and characterisation of pandemic H1N1 influenza viruses in Hungarian swine herds.

In 2010, two novel porcine H1N1 influenza viruses were isolated from pigs with influenza-like illness in Hungarian swine herds. Sequence and phylogenetic analysis of these strains revealed that they shared molecular features with the pandemic H1N1 influenza virus strains, which emerged globally during 2009. The PB2, HA and NA genes contained unique amino acid changes compared to the available new H1N1 influenza virus sequences of pig origin. Furthermore, the investigated strains could be separated with respect to parallel amino acid substitutions affecting the polymerase genes (PB2, PB1 and PA) and the nucleoprotein (NP) gene, supporting the proposed complementarities between these proteins, all required for the viral fitness. Molecular characterisation of two Hungarian human pandemic H1N1 isolates was also performed, so that we could compare contemporaneous strains of different host species origins. Shared molecular motifs in various genes of animal and human influenza strains suggested that the Hungarian porcine strains could have originated from humans through direct interspecies transmission. This study is among the few that support the natural human-to-pig transmission of the pandemic H1N1 influenza virus.

Genome sequence of a monoreassortant H1N1 swine influenza virus isolated from a pig in Hungary.

The genome of a porcine H1N1 influenza A strain is reported in this study. The strain proved to be a monoreassortant strain with a typical porcine N1 gene on the genetic backbone of the pandemic H1N1 influenza A virus strain. Monitoring of descendants of the pandemic 2009 H1N1 strain is needed because of concerns that more-virulent strains may emerge in forthcoming epidemic seasons.

Putative novel genotype of avian hepatitis E virus, Hungary, 2010.

To explore the genetic diversity of avian hepatitis E virus strains, we characterized the near-complete genome of a strain detected in 2010 in Hungary, uncovering moderate genome sequence similarity with reference strains. Public health implications related to consumption of eggs or meat contaminated by avian hepatitis E virus, or to poultry handling, require thorough investigation.