More than 20 years after the first outbreaks, the phylogenetic picture of PRRSV is still incomplete and full of gaps, especially in regards of PRRSV 1. Due to the exceptional diversity observed at the eastern borders of Europe and the low number of available sequences from Central Eastern European countries, the authors collected and analyzed both recent as well as already submitted sequences comparing them to a large backbone set of available ORF5 sequences representing the full spectrum of PRRSV 1 Subtype 1 diversity to conduct a systematic phylogenetic analysis and reclassification elucidating the diversity of the virus in these countries. Moreover, further analyses of the EUROSTAT data regarding the live pig movement trends revealed their influence of virus diversity and evolution. The results indicate that besides the effect of local, isolated divergent evolution and the use of modified live vaccines, the most important factor influencing a given country’s virus diversity is the transboundary movement of live, infected animals.

Porcine respirovirus 1, also kown as Porcine parainfluenza virus 1 (PPIV‐1) was first identified in Hong Kong in 2013, later in the USA and most recently in Chile. Here we report the first detection of PPIV‐1 outside these three regions. We screened 22 farms in Hungary by testing 15 nasal swab samples obtained from 3‐week‐old piglets (3 randomly chosen piglets from 5 litters in each farm. Only one farm was found to be positive. We subsequently sampled the positive farm by taking cross‐sectional 20 nasal swab samples from 2, 4, 6 and 8‐week‐old piglets. Virus detection by qRT‐PCR showed that although all investigated age groups were positive to PPIV‐1, a higher number of infected animals and higher viral loads were found among 4‐week‐old animals. Based on the phylogenetic analyses of partial F and L genes, the 3 Hungarian strains are genetically closely related to the very first PPIV‐1 strain identified in Hong Kong in 2013, whereas the overall genetic difference compared to the recently described North American isolates was around 10%.

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin–biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30–50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections.

Porcine respirovirus 1 (PRV1) is also known as porcine parainfluenza virus 1 (PPIV1). The prevalence and the role of PRV1 infections for pig health is largely unknown. In order to assess the PRV1 prevalence in Poland, nasal swabs and oral fluids collected from pigs from 30 farms were examined with RT real-time PCR. Additionally, IAV and PRRSV infection statuses of PRV1-positive samples were examined. The results showed that the virus is highly prevalent (76.7% farms positive) and different patterns of PRV1 circulation in herds with mild–moderate respiratory disease were observed. Co-infections with IAV and PRRSV were infrequent and detected in 8 (23.5%) and 4 (11.8%) out of 34 PRV1-positive nasal swab pools from diseased pens, respectively. In one pen PRV1, IAV, and PRRSV were detected at the same time. Interestingly, PRV1 mean Ct value in samples with co-infections was significantly lower (29.8 ± 3.1) than in samples with a single PRV1 infection (32.5 ± 3.6) (p < 0.05), which suggested higher virus replication in these populations. On the other hand, the virus detection in pig populations exhibiting respiratory clinical signs, negative for PRRSV and IAV, suggests that PRV1 should be involved in differential diagnosis of respiratory problems.

Porcine circovirus type 3 (PCV3) is an emerging pathogen that has been reported worldwide in all ages of healthy and clinically ill pigs. The presence of this virus in Hungary has been confirmed in a commercial farm experiencing reproductive failures, but there were no data on the circulation of PCV3 in the country. Here we report the prevalence and the genetic diversity of PCV3 in Hungarian herds. To estimate the prevalence, 1855 serum samples, 176 oral fluid and 97 processing fluid samples were collected in a systematic, cross-sectional method from 20 large scale swineherds and tested by real-time qPCR. PCV3 was present in at least one type of diagnostic matrix in 19 out of the 20 (95%) pig farms. The highest detection rates were observed in the processing fluid samples (61%), but 41% of the oral fluid and 23% of the serum samples were positive. The virus was found in all age groups, and slightly more adult animals were infected than growing pigs, but the viral burden was lower amongst them. Phylogenetic analysis of nine complete genomes, obtained from either the sampled herds or organ samples of PCV3-positive carcasses, showed high nucleotide identity between the detected sequences, which all belonged to the PCV3a genotype. Our results indicate that PCV3 is widespread in Hungary, but in most cases, the virus seems to circulate subclinically, infecting all age groups and production phases without the presence of apparent clinical disease.