A novel gyrovirus in a common pheasant (Phasianus colchicus) with poult enteritis and mortality syndrome.

A novel gyrovirus was detected in an intestinal specimen of a common pheasant that died due to poult enteritis and mortality syndrome. The genome of the pheasant-associated gyrovirus (PAGyV) is 2353 nucleotides (nt) long and contains putative genes for the VP1, VP2, and VP3 proteins in an arrangement that is typical for gyroviruses. Gyrovirus-specific motifs were identified in both the coding region and the intergenic region of the PAGyV genome. The VP1 of PAGyV shares up to 67.6% pairwise nt sequence identity with reference sequences and forms a distinct branch in the phylogenetic tree. Thus, according to the recently described species demarcation criteria, PAGyV belongs to a novel species in the genus Gyrovirus, family Anelloviridae, for which we propose the name "Gyrovirus phaco 1".

Genome sequencing of a novel variant of fowl adenovirus B reveals mosaicism in the pattern of homologous recombination events.

We determined the genomic sequence of a Ukrainian strain of fowl adenovirus B (FAdV-B). The isolate (D2453/1) shared 97.2% to 98.4% nucleotide sequence identity with other viruses belonging to the species Fowl aviadenovirus B. Marked genetic divergence was seen in the hexon, fiber, and ORF19 genes, and phylogenetic analysis suggested that recombination events had occurred in these regions. Our analysis revealed mosaicism in the recombination patterns, a finding that has also been described in the genomes of strains of FAdV-D and FAdV-E. The shared recombination breakpoints, affecting the same genomic regions in viruses belonging to different species, suggest that similar selection mechanisms are acting on the key neutralization antigens and epitopes in viruses of different FAdV species.

Spatiotemporal Distribution of PRRSV-1 Clades in Hungary with a Focus on the Era of Disease Eradication.

Porcine reproductive and respiratory syndrome (PRRS) is the cause of the most severe economic losses in the pig industry worldwide. PRRSV is extremely diverse in Europe, which poses a significant challenge to disease control within a country or any region. With the combination of phylogenetic reconstruction and network analysis, we aimed to uncover the major routes of the dispersal of PRRSV clades within Hungary. In brief, by analyzing >2600 ORF5 sequences, we identified at least 12 clades (including 6 clades within lineage 1 and 3 clades within lineage 3) common in parts of Western Europe (including Denmark, Germany and the Netherlands) and identified 2 novel clades (designated X1 and X2). Of interest, some genetic clades unique to other central European countries, such as the Czech Republic and Poland, were not identified. The pattern of PRRSV clade distribution is consistent with the route of the pig trade among countries, showing that most of the identified clades were introduced from Western Europe when fatteners were transported to Hungary. As a result of rigorous implementation of the national eradication program, the swine population was declared officially free from PRRSV. This map of viral diversity and clade distribution will serve as valuable baseline information for the maintenance of PRRSV-free status in the post-eradication era.

Transmission Dynamics of Imported Vaccine-Origin PRRSV-2 within and between Commercial Swine Integrations in Hungary.

This study reports on the molecular epidemiology of Ingelvac-PRRS-MLV-associated cases in Hungary for the period 2020-2021. Field epidemiology investigations led the experts to conclude that imported pigs, which were shipped through transit stations in Denmark, introduced the vaccine virus. The movement of fatteners and the neglect of disease control measures contributed to the spread of the virus to PRRS-free pig holdings in the vicinity. Deep sequencing was performed to genetically characterize the genes coding for the virion antigens (i.e., ORF2 through ORF7). The study isolates exhibited a range of 0.1 to 1.8% nucleotide sequence divergence from the Ingelvac PRRS MLV and identified numerous polymorphic sites (up to 57 sites) along the amplified 3.2 kilo base pair genomic region. Our findings confirm that some PRRSV-2 vaccine strains can accumulate very high number of point mutations within a short period in immunologically naive pig herds.

Sampling Strategies in PRRS Elimination in Hungary: An Observational Study Involving Four Farrow-to-Finish Swine Herds.

PRRS elimination strategies often rely on depopulation-repopulation. However, this approach is accompanied by a long-term loss of production. With adequate control measures, such as well-designed immunization programs and technological changes along with prevalence-based laboratory testing, the virus-free status of the most vulnerable age groups in swine herds can be achieved. The most common reason for acquiring PRRSV at large farrow-to-finish swine farm units is that the previously settled fattening pigs serve as a source of infection for the newly reared PRRS-free animals. Following such unwanted events, PRRSV may persist in an affected establishment for several years. In this observational study, we selected four farrow-to-finish type swine herds. We implemented different laboratory testing protocols to find the most optimal solution for a successful PRRS elimination program. To aid our objectives, we used a DIVA PCR technique. The PRRS DIVA PCR assay is a fast, reliable method to identify sows shedding farm-specific PRRSV strain(s). As a result of elimination efforts at the sentinel pig herds, we found that reliable detection of wild-type PRRSV shedding among sows requires sampling at least three weaned piglets per litter. The strict adherence to this sampling protocol, the systematic use of laboratory methods that quickly detect the presence of wild virulent virus in the herd during the rearing period and the culling of DIVA PCR positive litters and their sows decreased the presence of the resident virus markedly. These procedures at Hungarian farrow-to-finish type farms successfully inhibited the wild-type PRRSV infection of different age groups. The results of this study demonstrate that applying this methodology together with strict biosecurity measures enabled us to reach PRRS-vaccinated-free status in large, farrow-to-finish herds within two years.

Genomic Epidemiology and Evolution of Fowl Adenovirus 1.

Fowl adenovirus 1 (FAdV-1) is the main cause of gizzard erosion in chickens. Whole genome sequencing and sequence analyses of 32 FAdV-1 strains from a global collection provided evidence that multiple recombination events have occurred along the entire genome. In gene-wise phylogenies, only the adenoviral pol gene formed a tree topology that corresponded to whole genome-based phylogeny. Virus genetic features that were clearly connected to gizzard erosion were not identified in our analyses. However, some genome variants tended to be more frequently identified from birds with gizzard erosion and strains isolated from healthy birds or birds with non-specific pathologies tended to form common clusters in multiple gene phylogenies. Our data show that the genetic diversity is greater, and the evolutionary mechanisms are more complex within FAdV-1 than previously thought. The implications of these findings for viral pathogenesis and epidemiology await further investigation.

Deep Sequencing of Porcine Reproductive and Respiratory Syndrome Virus ORF7: A Promising Tool for Diagnostics and Epidemiologic Surveillance.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major concern worldwide. Control of PRRSV is a challenging task due to various factors, including the viral diversity and variability. In this study, we evaluated an amplicon library preparation protocol targeting the ORF7 region of both PRRSV species, Betaarterivirus suid 1 and Betaarterivirus suid 2. We designed tailed primers for a two-step PCR procedure that generates ORF7-specific amplicon libraries suitable for use on Illumina sequencers. We tested the method with serum samples containing common laboratory strains and with pooled serum samples (n = 15) collected from different pig farms during 2019-2021 in Hungary. Testing spiked serum samples showed that the newly designed method is highly sensitive and detects the viral RNA even at low copy numbers (corresponding to approx. Ct 35). The ORF7 sequences were easily assembled even from clinical samples. Two different sequence variants were identified in five samples, and the Porcilis MLV vaccine strain was identified as the minor variant in four samples. An in-depth analysis of the deep sequencing results revealed numerous polymorphic sites along the ORF7 gene in a total of eight samples, and some sites (positions 12, 165, 219, 225, 315, 345, and 351) were found to be common in several clinical specimens. We conclude that amplicon deep sequencing of a highly conserved region of the PRRSV genome could support both laboratory diagnosis and epidemiologic surveillance of the disease.

Genome stability assessment of PRRS vaccine strain with new ARTIC-style sequencing protocol.

A tiling amplicon sequencing protocol was developed to analyse the genome sequence stability of the modified live PRRSV vaccine strain, Porcilis MLV. The backbone of the ARTIC-style protocol was formed by 34 individual primer pairs, which were divided into two primer pools. Primer pairs were designed to amplify 532 to 588 bp fragments of the corresponding genomic region. The amplicons are suitable for sequencing on Illumina DNA sequencers with available 600-cycle sequencing kits. The concentration of primer pairs in the pools was optimized to obtain a balanced sequencing depth along the genome. Deep sequencing data of three vaccine batches were also analysed. All three vaccine batches were very similar to each other, although they also showed single nucleotide variations (SNVs) affecting less than 1 % of the genome. In the three vaccine strains, 113 to 122 SNV sites were identified; at these sites, the minority variants represented a frequency range of 1 to 48.7 percent. Additionally, the strains within the batches contained well-known length polymorphisms; the genomes of these minority deletion mutants were 135 to 222 bp shorter than the variant with the complete genome. Our results show the usefulness of ARTIC-style protocols in the evaluation of the genomic stability of PRRS MLV strains.

Corrigendum: Genetic diversity of imported PRRSV-2 strains, 2005-2020, Hungary.

[This corrects the article DOI: 10.3389/fvets.2022.986850.].

Genetic diversity of imported PRRSV-2 strains, 2005-2020, Hungary.

Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) remains sporadic in Europe. In this study, we investigated the molecular epidemiology of PRRSV-2 infections encompassing 15 years in Hungary. Partial (423 bp long) ORF5 sequences (n = 44) from 20 Hungarian pig herds were analyzed. The study strains fell into two genetic lineages, L1 and L5, being L5 strains more prevalent (88.6 vs. 11.4%). Pairwise sequence identities within Hungarian representative PRRSV-2 strains ranged between 84.7 to 100% (nucleotide, nt) and 85 to 100% (amino acid, aa). When compared with reference strains, identity values fell between 87 and 100% (L1, nt 87-91%, aa 87-93%, reference strain IAF-exp91; L5, nt 87-100%, aa 88-100%, reference strain Ingelvac MLV). Epidemiologic examination implied that the majority of L5 strains were imported repeatedly from other European countries where Ingelvac MLV was approved for routine use. The emergence of L1 strains was thought to be associated with a single introduction and subsequent dissemination between pig farms of a large integrator. Results presented here contribute to a better understanding of the epizootiology of PRRSV-2 infections and shed light on the genetic diversity of viral strains in non-endemic countries.

Genomic Epidemiology and Evolution of Duck Hepatitis A Virus.

Duck hepatitis A virus (DHAV), an avian picornavirus, causes high-mortality acute disease in ducklings. Among the three serotypes, DHAV-1 is globally distributed, whereas DHAV-2 and DHAV-3 serotypes are chiefly restricted to Southeast Asia. In this study, we analyzed the genomic evolution of DHAV-1 strains using extant GenBank records and genomic sequences of 10 DHAV-1 strains originating from a large disease outbreak in 2004-2005, in Hungary. Recombination analysis revealed intragenotype recombination within DHAV-1 as well as intergenotype recombination events involving DHAV-1 and DHAV-3 strains. The intergenotype recombination occurred in the VP0 region. Diversifying selection seems to act at sites of certain genomic regions. Calculations estimated slightly lower rates of evolution of DHAV-1 (mean rates for individual protein coding regions, 5.6286 × 10-4 to 1.1147 × 10-3 substitutions per site per year) compared to other picornaviruses. The observed evolutionary mechanisms indicate that whole-genome-based analysis of DHAV strains is needed to better understand the emergence of novel strains and their geographical dispersal.