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.

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.

In search of the spectral composition of an effective light trap for the mushroom pest Lycoriella ingenua (Diptera: Sciaridae).

Certain fungus gnats, like Lycoriella ingenua are notorious pests in agriculture, especially in mushroom production. While larvae cause mainly direct crop damage, adults are vectors of several dangerous fungal pathogens. To promote the development of pesticide-free management methods, such as light trapping, we measured the spectral sensitivity of L. ingenua compound eyes with electroretinography and performed two different behavioural experiments to reveal the wavelength dependence of phototaxis in this species. The spectral sensitivity of the compound eyes is bimodal with peaks at 370 nm (UV) and 526 nm (green). Behavioural experiments showed that attraction to light as a function of wavelength depends on light intensity. In our first experiment, where the minimal photon flux (105-109 photons/cm2/s) needed for eliciting a phototactic response was determined wavelength by wavelength, phototaxis was strongest in the green spectral range (~526 nm). In the other behavioural experiment, where wavelength preference was tested under a higher but constant light intensity (~1013 photons/cm2/s), the highest attraction was elicited by UV wavelengths (398 nm). Our results suggest that both UV and green are important spectral regions for L. ingenua thus we recommend to use both UV (~370-398 nm) and green (~526 nm) for trapping these insects.

Investigations on spreading of PRRSV among swine herds by improved minimum spanning network analysis.

In Hungary, the economic losses caused by porcine reproductive and respiratory syndrome virus (PRRSV) led to the launching of a national PRRSV Eradication Program. An important element of the program was investigating the spread of PRRSV among swine herds and the possible ways of introduction by sequencing of the open reading frame 5 (ORF5) gene. However, the classical phylogenetic tree presentation cannot explain several genetic relationships clearly, while more precise visualization can be represented by network tree diagram. In this paper, we describe a practical and easy-to-follow enriched minimum spanning similarity network application for improved representation of phylogenetic relations among viral strains. This method eliminated the necessity of applying a predefined, arbitrary cut-off or computationally extensive algorithms. The network-based visualization allowed processing and visualizing large amount of data equally for the laboratory, private and official veterinarians, and helped identify the potential connections between different viral sequences that support data-driven decisions in the eradication program. By applying network analysis, previously unknown epidemiological connections between infected herds were identified, and virus spreading was analyzed within short period of time. In our study, we successfully built and applied network analysis tools in the course of the Hungarian PRRSV Eradication Program.

Fungal Volatiles as Olfactory Cues for Female Fungus Gnat, Lycoriella ingenua in the Avoidance of Mycelia Colonized Compost.

The chemical signatures emitted by fungal substrates are key components for mycophagous insects in the search for food source or for suitable oviposition sites. These volatiles are usually emitted by the fruiting bodies and mycelia. The volatiles attract fungivorous insects, like flowers attract pollinators; certain flowers mimic the shape of mushroom fruiting bodies and even produce a typical mushroom odor to exploit on fungus-insect mutualism. There are numerous insects which are mycophagous or eat fungi additionally, but only a few are considered a threat in agriculture. Lycoriella ingenua is one of the most serious pests in mushroom cultivation worldwide. Here we attempt to examine the role of environmental volatiles upon behavioral oviposition preference. In two-choice bioassays, fungus gnats preferred uncolonized compost compared to colonized compost but preferred colonized compost against nothing. However, when colonized compost was paired against distilled water, no significant choice was observed. The comparison of fresh casing material and mycelium colonized casing material resulted in no significant preference. From colonized compost headspace, three antennally active volatiles were isolated by gas chromatography coupled with electroantennography and subsequently identified with gas chromatography coupled mass spectrometry as 1-hepten-3-ol, 3-octanone and 1-octen-3-ol. In behavioral assays the addition of said synthetic volatiles to uncolonized compost separately and in combination to mimic colonized compost resulted in avoidance. We thus partially elucidate the role of fungal volatiles in the habitat seeking behavior of Lycoriella ingenua.

An emerging new fowl adenovirus genotype.

In this work, we examined the diversity of fowl adenovirus (FAdV) types occurring in Hungary. From diseased chicken flocks in Eastern Hungary, 29 FAdV strains were isolated between 2011 and 2015. We performed molecular typing of the isolates based on their partial hexon sequences. The results showed that representatives from every FAdV species from A to E are present in Hungary, but compared to the findings from our previous survey, a lower number of different FAdV types were detected. Inclusion body hepatitis was always associated with FAdV-2 or -8b, gizzard erosion was caused in almost every case by FAdV-1. Numerous strains belonging to species FAdV-B were found. The complete genome sequence of a candidate new genotype strain, showing the highest divergence from the reference FAdV-5, was determined using next generation sequencing. In order to provide results compatible with the serology-based type classification, multiple genomic regions, including the major antigenic determinants, of the new isolate (strain 40440-M/2015) were compared to their counterparts in the prototype FAdV-5 (strain 340) from species FAdV-B, at both nucleotide and amino acid sequence levels. In different comparative analyses, the two strains were always found to have larger divergence between each other than any two of the most closely related FAdV serotypes. This new emerging FAdV genotype is already present in Hungary and Austria, though its exact pathological role requires further investigations. The introduction of a novel FAdV (geno)type for the classification of these strains is further supported.

Vaccine-associated rabies in red fox, Hungary.

Rabies vaccine strain was isolated from a red fox (Vulpes vulpes) with signs of neurological disorder during an oral vaccination campaign in 2015, Hungary. The whole genome sequence of the isolated strain shared >99.9% nucleotide sequence identity to the whole genomes of vaccines strains recently used in Hungarian oral vaccination campaigns. The neuroinvasive potential of rabies vaccines that leads to development of clinical manifestations is rarely seen among wild animals; however, the observed residual pathogenicity needs awareness of field experts and requires close monitoring of rabies cases in areas where elimination programs are implemented.

Lineage diversification, homo- and heterologous reassortment and recombination shape the evolution of chicken orthoreoviruses.

The near complete genome sequences of ten field avian orthoreovirus (ARV) strains collected from young chicken between 2002 and 2011 in Hungary have been determined in order to explore the genetic diversity and evolutionary mechanisms affecting ARVs in this region. Sequence analyses and phylogenetic calculations revealed similar geographic distribution and distinct evolution in case of eight studied strains that were closely related to the recently described Hungarian strain T1781. The remaining two strains showed the highest similarity with the US origin AVS-B. The topology of the phylogenetic trees of certain segments was affected by several potential homologous reassortment events between strains of Hungarian, Chinese and US origin. Analyzing the µB gene a possible heterologous reassortment event was identified in three Hungarian strains. Recombination events were detected in as much as a dozen cases implying that beside point mutations and reassorment this mechanism also plays an important role in the diversification of ARVs. All these mechanisms in concert may explain the reduced effectiveness of immunization using commercial vaccine strains.

Genome Sequences of Three Turkey Orthoreovirus Strains Isolated in Hungary.

We have investigated the genomic properties of three turkey reovirus strains-19831M09, D1246, and D1104-isolated in Hungary in 2009. Sequence identity values and phylogenetic calculations indicated genetic conservativeness among the studied Hungarian strains and a close relationship with strains isolated in the United States.

Full-length genome sequence analysis of a Hungarian porcine reproductive and respiratory syndrome virus isolated from a pig with severe respiratory disease.

Here, we report the isolation of a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) strain from a clinical outbreak of severe respiratory problems and high fever. Next-generation sequencing was used to determine the complete genome sequence of the isolate (9625/2012). The virus belongs to a new branch within subtype 1, clade D, and shows the highest similarity to PRRSV Olot/1991 and to the Amervac vaccine strain. Mutation analysis of 9625/2012 revealed no evidence of recombination but did show a high proportion of amino acid substitutions in the putative neutralizing epitopes, suggesting an important role of selective immune pressure in the evolution of PRRSV 9625/2012.

Molecular typing of fowl adenoviruses, isolated in Hungary recently, reveals high diversity.

Molecular typing of 26 recent fowl adenovirus (FAdV) isolates obtained in Eastern Hungary between 2006 and 2011 was performed. The viruses were isolated on primary chicken embryo liver cell cultures from carcasses of chickens, sent for diagnostic investigation. The isolates were subjected to a PCR to amplify a fragment from the hexon gene. Sequence analysis of the amplicons revealed the presence of all the five FAdV species in the country, among them FAdV-B, which has been barely found in Hungary before. The three strains classified as FAdV-B might represent a novel type within this species, as their nucleotide sequence identity to strain 340 (type FAdV-5) is below the commonly accepted intratype limit. Most of the strains (63%) were classified into species FAdV-E (12 strains) and FAdV-D (7 strains) in the study. Four of the isolates proved to be the mixture of two adenovirus strains belonging to two different FAdV types, in three from these four cases, these two types even belonged to two different species. Analysing the pathological findings bolsters certain established connections between some FAdV types and disease conditions. Gizzard erosion was found in connection with FAdV-1 strains only, and inclusion body hepatitis with FAdV-D and -E strains.

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.

Evolution of porcine kobuvirus infection, Hungary.

Porcine kobuvirus was first identified in early 2007 in Hungary. Originally thought to be confined to the intestine, almost 2 years later the virus was found in the blood of clinically healthy pigs on the same farm. Porcine kobuvirus may be widely distributed on pig farms worldwide.

Incidence, diversity, and molecular epidemiology of sapoviruses in swine across Europe.

Porcine sapovirus is an enteric calicivirus in domestic pigs that belongs to the family Caliciviridae. Some porcine sapoviruses are genetically related to human caliciviruses, which has raised public health concerns over animal reservoirs and the potential cross-species transmission of sapoviruses. We report on the incidence, genetic diversity, and molecular epidemiology of sapoviruses detected in domestic pigs in a comprehensive study conducted in six European countries (Denmark, Finland, Hungary, Italy, Slovenia, and Spain) between 2004 and 2007. A total of 1,050 swine fecal samples from 88 pig farms were collected and tested by reverse transcription-PCR for sapoviruses, and positive findings were confirmed by sequencing. Sapoviruses were detected in 80 (7.6%) samples collected on 39 (44.3%) farms and in every country. The highest prevalence was seen among piglets aged 2 to 8 weeks, and there was no significant difference in the proportion of sapovirus-positive findings for healthy animals and animals with diarrhea in Spain and Denmark (the only countries where both healthy animals and animals with diarrhea were tested). On the basis of the sequence of the RNA polymerase region, highly heterogeneous populations of viruses representing six different genogroups (genogroups III, VI, VII, and VIII, including potential new genogroups IX and X) were identified, with a predominance of genogroup GIII (50.6%). Genogroup VIII, found in five of the six countries, had the highest degree of homology (up to 66% at the amino acid level) to human sapovirus strains. Sapoviruses are commonly circulating and endemic agents in swine herds throughout Europe. Highly heterogeneous and potential new genogroups of sapoviruses were found in pigs; however, no "human-like" sapoviruses were detected.

Molecular analysis and characterization of swine and human influenza viruses isolated in Hungary in 2006­2007.

In order to provide additional information to the epidemiological situation in Middle Europe and open further possibilities to investigate the transmission of influenza viruses between species, the viral genomes of three influenza A virus isolates (one human and two swine) collected from North-East Hungary in 2006­2007 have been fully sequenced and characterized. The sequence analysis reveals strong geographical relationships between the internal genes of the two swine viruses; the human isolate shows strict conservation to recent H1N1 strains, while the swine strains demonstrate and reflect a mixed avian­human origin, a characteristic of European swine influenza viruses. No evidence of interspecies interaction has been found among the studied isolates.

Genetic diversity of porcine reproductive and respiratory syndrome virus strains circulating in Hungarian swine herds.

Analysis of 37 ORF5 sequences of Hungarian porcine reproductive and respiratory syndrome virus (PRRSV) strains revealed that most of them (35) belonged to the European genotype, forming distinct subgroups, reflecting the exceptional diversity of Eastern European strains. Twelve vaccine-like strains were also found in non-vaccinated animals. Two strains belonged to the American genotype showing 90-91% nucleotide identity to the "Quebec" Canadian reference strain. The analysis of the putative ectodomains and their N-linked glycosylation sites of the vaccine strain and its variants suggested selective pressure on the first ectodomain, by a consistent amino acid change on epitope B and by loosing a glycosylation site in the otherwise conserved N-46 position.