High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Comparative examination of a rapid immunocytochemical test for the detection of highly pathogenic avian influenza virus in domestic birds in field outbreaks.

RESEARCH HIGHLIGHTS: Avian influenza virus (AIV) antigen detection was examined in field outbreaks.Bird brain smears were tested using immunocytochemistry (IC).IC results strongly correlated with real-time RT-PCR results.The IC method was rapid, specific, sensitive, and cost-effective in AIV field outbreaks.

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.

Eradication of PRRS from Hungarian Pig Herds between 2014 and 2022.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread infectious disease that is currently a major cause of economic losses in pig production. In Hungary, a National PRRS Eradication Program has been introduced to attain a more efficient, economic, and competitive international market position. The program has been also approved by the EU, but the resulting legal obligations have imposed a burden on Hungarian producers to comply with EU competition rules. The implementation of the program has been carried out by the veterinary authorities with the consent of, continuous support from and monitoring conducted by organisations within the pig sector as well as a scientific committee. The PRRS eradication program in Hungary was based on a regional territorial principle and was compulsory for all pig holdings within the regions. In Hungary, large fattening farms operate as all-in/all-out or continuous flow systems. Large-scale breeding herds are predominantly farrow-to-finish types. Although its significance has decreased in recent decades, 20% of the Hungarian pig population is still kept on small (backyard) farms (<100 animals). All PRRSV-infected large-scale farms had to develop a unit-adapted eradication plan, including external and internal biosecurity measures, vaccinations, etc. It was crucial to render each fattening unit free of the disease, as fattening units play a significant role in spreading the virus within the country. The eradication efforts mainly implemented were depopulation-repopulation methods, but on some farms a testing and removal method has been used. As the eradication progressed over the years, the introduction of infected fattening pigs was restricted. Thanks to these measures, Hungarian large-scale fattening farms became PRRSV-free by the end of 2018. The PRRSV-free status of small-scale herds was achieved by the end of 2015 and was maintained between 2016 and 2021. By 31 December 2021, all breeding pigs in large-scale farms in Hungary were free of wild-type PRRS virus. By 31 March 2022, the total pig population of the country, including all backyard farms and fattening units, achieved PRRSV-free status. The future goal is to ensure and maintain the PRRSV-free status of Hungary via strict import regulations of live animals combined with the continuous and thorough screening of incoming and resident herds for the presence of the virus.

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.

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.

The Impact of PRRS Eradication Program on the Production Parameters of the Hungarian Swine Sector.

BACKGROUND: The Hungarian national eradication program of PRRS was successfully completed between 2014 and 2022. There were doubts about the efficiency of the eradication program in Hungary from the beginning to the tune that it might only be carried out efficiently through depopulation-repopulation of the infected herds, which is a very costly procedure. In our study, we investigated the impact of the depopulation-repopulation procedure, which played a prominent role in the PRRS eradication program on the productivity of the Hungarian swine sector-namely, on the number of slaughter pigs per sow per year and the total live slaughter weight per sow per year. MATERIAL AND METHODS: Since 2014, we monitored the evolution of the PRRS eradication through the depopulation-repopulation approach on the large-scale breeding herds in Hungary. Most producers replaced their herds with animals that were free of PRRS and other infectious diseases (mycoplasmosis, actinobacillosis, swine dysentery, atrophic rhinitis, etc.). On this basis, we evaluated the change in the number of slaughter pigs per sow per year as a consequence of depopulation-repopulation of the herds being carried out. In the statistical analysis linear regression was used. CONCLUSIONS: The results of our study demonstrate that the PRRS eradication program with the herd depopulation-repopulation approach led to a considerable improvement of the productivity of Hungarian pig farming. This result also demonstrates that, independent of the PRRS eradication, it is still necessary to consider investments into the individual production units to increase efficiency, and to carry out herd depopulation-repopulation in cases where the current genetics limits improvements in productivity.

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.

Development of a farm-specific real-time quantitative RT-PCR assay for the detection and discrimination of wild-type porcine reproductive respiratory syndrome virus and the vaccine strain in a farm under eradication.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine causing severe economic losses worldwide, therefore intensive efforts are taken to eliminate PRRS virus (PRRSV) from infected herds for complete eradication. The most efficient, fastest but at the same time the most expensive eradication method is depopulation-repopulation. In order to reduce costs, a number of farms prefer to perform their eradication process with continuous production using modified live vaccine (MLV) immunisation. However, the commercial PRRSV RT-PCR kits do not have the capacity to discriminate infected from vaccinated animals. In this paper, we describe a simple discriminatory duplex TaqMan RT-PCR assay based on common forward and reverse primers, as well as two differently labelled MLV- and wild-type PRRSV-specific probes. The discriminatory PCR test we designed is a fast and efficacious method for processing large quantities of samples. The assay is cheap, flexible, easy to apply in different herds using different MLVs, but should be checked, and can be modified based on the sequence data obtained during the permanent monitoring examinations. Owing to its simplicity the test can serve as a significant complementary assay for PRRS control and elimination/eradication.

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.

Elimination of porcine reproductive and respiratory syndrome virus infection using an inactivated vaccine in combination with a roll-over method in a Hungarian large-scale pig herd.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses worldwide and only four countries in Europe are free from PRRSV. Complete depopulation-repopulation is the safest and fastest, but also the most expensive method for eradicating PRRSV from a population. Another possible way to eliminate an endemic PRRSV infection is to replace the infected breeding stock by gilts reared isolated and protected from PRRSV on an infected farm. With this method it is possible to maintain continuous production on the farm. The authors report the first successful elimination of PRRSV in a Hungarian large-scale pig farm by using an inactivated vaccine and performing segregated rearing of the offspring. CASE PRESENTATION: The study was performed on a PRRSV infected farm (Farm A) with 1475 sows. The clinical signs of reproductive failure had been eliminated previously by using an inactivated vaccine (Progressis®, Ceva). At the beginning of the elimination programme, gilts intended for breeding were vaccinated at 60 and 90-100 days of age. After that, gilts selected for breeding were vaccinated at 6 months of age, on the 60-70th day of pregnancy and at weaning. Approximately 1200 piglets from vaccinated sows were transported at 7 weeks of age to a closed, empty farm (Farm B) after being tested negative for PRRSV by a polymerase chain reaction (PCR) method, and then were reared here until 14 weeks of age. At this age, all pigs were tested by PRRS ELISA. Seronegative gilts (n = 901) were subsequently transported from Farm B to a third, closed and empty farm (Farm C), and (having reached the breeding age) they were inseminated here after a second negative serological test (ELISA). At the same time, Farm A was depopulated, cleaned and disinfected. All pregnant gilts were transported from Farm C to Farm A after being re-tested negative for antibodies against PRRSV. Follow-up serology tests were performed after farrowing and results yielded only seronegative animals. Based on the subsequent negative test results, the herd was declared PRRSV free by the competent authority. CONCLUSIONS: The presented farm was the first during the National PRRS Eradication Programme of Hungary to eradicate PRRSV successfully by vaccinating the sows with an inactivated vaccine and performing segregated rearing of the offspring. Production was almost continuous during the whole process of population replacement.

Genetic Variability of PRRSV Vaccine Strains Used in the National Eradication Programme, Hungary.

Porcine reproductive and respiratory syndrome (PRRS) is a globally spread, highly infectious viral disease. Live, attenuated vaccines against PRRS virus (PRRSV) decrease virus excretion and evoke protective immunity reducing the economic damage caused by the disease. In a longitudinal molecular epidemiological study accompanying ongoing national eradication programme we evaluated the suitability of PRRSV ORF5 and ORF7 sequences to identify possible field strains of vaccine-origin. In total, 2342 ORF5 sequences and 478 ORF7 sequences were analysed. Vaccine strains were identified by sequence identity values and phylogenetic network analysis. Strains that shared greater than 98% nucleotide identity within ORF5 and/or ORF7 were considered to have originated from vaccine. A total of 882 (37.6%) ORF5 and 88 (18.4%) ORF7 sequences met these criteria. In detail, 618, 179 and 35 ORF5 and 51, 29 and 8 ORF7 sequences were related to Porcilis PRRS vaccine, Unistrain PRRS vaccine, and ReproCyc PRRS EU vaccine, respectively. Data showed that the Porcilis vaccine was genetically more stable. Whereas, the variability of the Unistrain and the ReproCyc strains was significantly higher. Given that ORF7 shares, in some instances, complete identity between a particular vaccine strain and some historic variants of field PRRSV strains, care must be taken when evaluating vaccine relatedness of a field isolate based on the ORF7. On the contrary, ORF5 sequences were more suitable to predict the vaccine origin making a distinction more robustly between field and vaccine strains. We conclude that ORF5 based molecular epidemiological studies support more efficiently the ongoing PRRS eradication programmes. The conclusions presented in this large-scale PRRS molecular epidemiological study provides a framework for future eradication programmes planned in other 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.

Recombination Events Shape the Genomic Evolution of Infectious Bronchitis Virus in Europe.

Infectious bronchitis of chicken is a high morbidity and mortality viral disease affecting the poultry industry worldwide; therefore, a better understanding of this pathogen is of utmost importance. The primary aim of this study was to obtain a deeper insight into the genomic diversity of field infectious bronchitis virus (IBV) strains using phylogenetic and recombination analysis. We sequenced the genome of 20 randomly selected strains from seven European countries. After sequencing, we created a genome sequence data set that contained 36 European origin field isolates and 33 vaccine strains. When analyzing these 69 IBV genome sequences, we identified 215 recombination events highlighting that some strains had multiple recombination breaking points. Recombination hot spots were identified mostly in the regions coding for non-structural proteins, and multiple recombination hot spots were identified in the nsp2, nsp3, nsp8, and nsp12 coding regions. Recombination occurred among different IBV genotypes and involved both field and vaccine IBV strains. Ninety percent of field strains and nearly half of vaccine strains showed evidence of recombination. Despite the low number and the scattered geographical and temporal origin of whole-genome sequence data collected from European Gammacoronaviruses, this study underlines the importance of recombination as a major evolutionary mechanism of IBVs.

Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary.

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain "neglected" genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species "Bopivirus B" in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine-caprine interspecies transmission of certain bopiviruses.

Multiple SARS-CoV-2 Introductions Shaped the Early Outbreak in Central Eastern Europe: Comparing Hungarian Data to a Worldwide Sequence Data-Matrix.

Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019, the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of 21 April 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here, we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.

PRRS eradication from swine farms in five regions of Hungary.

Porcine reproductive and respiratory syndrome (PRRS) causes significant losses to the swine industry worldwide, which leads to launching eradication programmes. The PRRS eradication programme in Hungary is based on the territorial principle, and it is obligatory for each swine farm irrespective of the number of animals kept there. Hungary has an exceptionally large herd size in large-scale pig farms. Large fattening farms operate as all-in/all-out or continuous flow systems. The large-scale breeding herds are predominantly farrow-to-finish types. In large-scale breeding farms, PRRS eradication was carried out by the depopulation-repopulation method in 33 farms, of which 23 received state compensation, 18 farm units either finished production or changed to producing fatteners only. Two farms used the test and removal method for eradication. One farm was classified as 'vaccinated free'. At this farm the breeding animals are vaccinated continuously but there is no vaccination of the progeny at any age, and the PRRS-free status of the farm is strictly controlled and monitored. By 31 December 2019, all pigs in five euroregions of Hungary had become free from PRRS virus, while the PRRS eradication process is still ongoing in the remaining two regions.