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.

Safety and Efficacy Profile of Live, Intermediate Plus Vaccine Against Infectious Bursal Disease Based on Strain G6.

Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease of young chickens that causes considerable economic loss in the poultry industry worldwide. Vaccination with live attenuated vaccines is still the most important method used for the control and prevention of IBD in chickens. Here we present the results of in vitro characterization, as well as efficacy and safety testing of a live, intermediate plus vaccine against IBD based on strain G6. Strain characterization confirmed that G6 strain is an intermediate plus strain, showing a high degree of homology with the existing vaccine strains of the same virulence. Safety studies showed that chickens can be vaccinated from 10 days of age. Onset and duration of immunity in specific pathogen free and maternally derived antibodies (MDA) chickens was proven to be 14 and 35 days after vaccination, respectively. When immunizing MDA-positive chickens, vaccine is capable of breakthrough at a titer of ≤500 ELISA units. The field trial conducted on commercial broilers showed a 95% protection against vvIBDV challenge. Stability of the freeze-dried vaccine after reconstitution was confirmed over a period of 3 h. Overall, IBD G6 vaccine has shown good safety and efficacy profile in accordance with European Pharmacopoeia requirements.

First report of porcine parainfluenza virus 1 (species Porcine respirovirus 1) in Europe.

Porcine respirovirus 1, also known 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%.

Body temperature and motion: Evaluation of an online monitoring system in pigs challenged with Porcine Reproductive & Respiratory Syndrome Virus.

Highly contagious and emerging diseases cause significant losses in the pig producing industry worldwide. Rapid and exact acquisition of real-time data, like body temperature and animal movement from the production facilities would enable early disease detection and facilitate adequate response. In this study, carried out within the European Union research project RAPIDIA FIELD, we tested an online monitoring system on pigs experimentally infected with the East European subtype 3 Porcine Reproductive & Respiratory Syndrome Virus (PRRSV) strain Lena. We linked data from different body temperature measurement methods and the real-time movement of the pigs. The results showed a negative correlation between body temperature and movement of the animals. The correlation was similar with both body temperature obtaining methods, rectal and thermal sensing microchip, suggesting some advantages of body temperature measurement with transponders compared with invasive and laborious rectal measuring. We also found a significant difference between motion values before and after the challenge with a virulent PRRSV strain. The decrease in motion values was noticeable before any clinical sign was recorded. Based on our results the online monitoring system could represent a practical tool in registering early warning signs of health status alterations, both in experimental and commercial production settings.

Vaccination of broilers against Newcastle disease in the presence of maternally derived antibodies.

OBJECTIVE: The aim of this study was to evaluate the efficacy of a live attenuated vaccine against Newcastle disease in broilers with different levels of maternally derived antibodies (MDA). While vaccination remains the single most important means for controlling Newcastle disease, presence of MDA may interfere with the vaccination of young birds and decrease the efficacy of the vaccine. MATERIALS AND METHODS: Day-old chicks with variable levels of MDA (negative, low and high) were vaccinated with a live attenuated vaccine against Newcastle disease. Three most commonly used inoculation routes were compared; oculonasal, spray and oral (drinking water). Onset and duration of immunity were measured by serology and challenge with virulent virus. RESULTS: Immune response in vaccinated MDA-positive birds was delayed in comparison with SPF controls. Protection was well established already at 14 days post vaccination in SPF birds while in MDA-positive birds it was 1-2 weeks delayed and was lower throughout the study. Non-vaccinated MDA-positive birds lost passive protection completely at 3-4 weeks of age and were significantly more susceptible to challenge than vaccinated hatch mates at all test points. The protection rate increased in vaccinated birds towards the end of the experiment and reached 70-100 % at the last test points (35-42 days of age). Correlation of haemagglutination inhibition (HI) titre vs. protection rate revealed the importance of cellular and local immunity as most of the vaccinated birds with low HI titre were protected, contrary to their unvaccinated hatch mates with the same HI titre. Oculonasal route seems to provide slightly better protection than the other two routes. CONCLUSIONS AND CLINICAL RELEVANCE: Although immune protection in vaccinated MDA-positive birds may be decreased or delayed, vaccination still provides high protection against ND challenge in comparison with the unvaccinated hatch mates. The degree of interference seems to be proportional to the level of MDA. Vaccination schedules therefore need to be designed according to the immune status of the flock.