Bálint, Ádám

Farsang, Attila

Homonnay, Zalán G.

Kiss, István

Kojer, Judit

Mató, Tamás

Palya, Vilmos

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Acta Veterinaria Hungarica 63 (3), pp. 382–388 (2015) DOI: 10.1556/4.2015.036  0236-6290/$ 20.00 © 2015 Akadémiai Kiadó, Budapest SURVEY INDICATES CIRCULATION OF 4/91 AND  QX-TYPE INFECTIOUS BRONCHITIS VIRUSES IN HUNGARY IN 2014 – SHORT COMMUNICATION István KISS1, Tamás MATÓ1, Zalán G. HOMONNAY1, Judit KOJER1, Attila FARSANG2, Ádám BÁLINT3 and Vilmos PALYA1* 1Scientific Support and Investigation Directorate, Ceva-Phylaxia Ltd.,  Szállás u. 5, H-1107 Budapest, Hungary; 2Directorate of Veterinary Medicinal Products,  National Food Chain Safety Office, Budapest, Hungary; 3Veterinary Diagnostic  Directorate, National Food Chain Safety Office, Budapest, Hungary (Received 23 January 2015; accepted 22 June 2015) Understanding the epidemiology and improving vaccinal protection against the highly variable chicken infectious bronchitis virus (IBV) requires the knowledge of circulating IBV serotypes/genotypes in defined geographic areas. Accordingly, the authors initiated a survey among the major poultry producers in Hungary in order to reveal the prevailing IBV serotypes in the country. Tracheal swabs and organ samples (caecal tonsils, kidneys, and trachea) were collected from broiler, layer, and meat-type breeder flocks, and were subjected to IBV de-tection by virus isolation and polymerase chain reaction (PCR). The IBV-positive samples were further characterised by nucleotide sequencing and phylogenetic analysis of a portion of the S1 IBV gene. Seventeen out of the 26 submitted sam-ples proved to be positive for IBV. Sequence analyses revealed ten 4/91 and six QX serotypes, and a single D274 type IB virus. One sample contained a mixture of QX and Massachusetts serotype viruses. Presumably most of the 4/91 and D274 type viruses were vaccine strains. The proportion of QX type viruses and their observed variation are in good agreement with the situation in a few other European countries. The detected viruses clustered largely according to their geo-graphic origin, with a few exceptions. If updated regularly, the preliminary ‘virus map’ will be useful for the adjustment of vaccination protocols. Key words: Infectious bronchitis virus, diversity, Hungary  Chicken infectious bronchitis (IB), recognised in the 1930s, causes various problems in the poultry industry worldwide. Disease manifestations include res-piratory and reproductive disorders, as well as kidney damages. The causative coronavirus (infectious bronchitis virus, IBV) is highly contagious, and its ge-nome shows great variations due to mutations and recombination events. Conse-quently, several genotypes and serotypes exist and evolve over time. Some of                                                  *Corresponding author; E-mail: vilmos.palya@ceva.com  CIRCULATION OF 4/91 AND QX-TYPE IBV IN HUNGARY 383 Acta Veterinaria Hungarica 63, 2015 these persist and spread, while others disappear within a short time (de Wit et al., 2011). This variation undermines vaccinal protection against IB due to the lack of cross-protection between the serotypes. Although homologous vaccine strains provide the best protection against challenge or natural infections, partial protec-tion can also be reached by appropriate combination of heterologous vaccines (Cook et al., 1996). Elaboration of proper vaccination protocols basically re-quires the knowledge of the prevailing serotypes in a given region/flock, and animal trials in order to determine vaccine efficacy and the protectotype of IBVs (de Wit et al., 2010). Because of the lack of such domestic data, Ceva-Phylaxia Co. Ltd. initiated a surveillance program to reveal prevalent IBV serotypes in Hungarian chicken flocks in 2014. The study was confined to partner companies of Ceva-Phylaxia and, therefore, has its inherent limitations regarding its scale. By providing archival IBV isolates from its own collection, the National Food Chain Safety Office (NFCSO) also supported this preliminary work.  Altogether 26 broiler, layer and breeder flocks were sampled regardless of the presence or lack of respiratory signs at the time of sampling, though IB asso-ciated signs were observed at each farm from time to time. Five birds per flock were sampled by means of collecting either tracheal swabs or organ specimens (caecal tonsil, kidney and trachea) from freshly collected carcasses and processing them pooled. The origin of the samples and related basic data are shown in Table 1. Six QX-type IBV samples were also provided by the NFCSO as purified RNA. These had been collected in the previous years, from the following locations: Kiskunmajsa (broiler, two farms), Szentpéterúr (broiler), Imrehegy (broiler), Szigetszentmiklós (layer), Bana (broiler). The presence of IBV was tested by virus isolation attempts in specific pathogen free (SPF) embryonated chicken eggs, looking for the typical lesions of IBV infection, i.e. stunting and curling of the embryos, and simultaneously by PCR (Capua et al., 1999). PCR products were submitted to nucleotide sequencing. The subsequent phylogenetic analysis was based on the 763–1129 nucleotide positions of the S1 gene (excluding the primers used for sequencing), using the Neighbour-Joining algorithm and the Kimura 2-parameter method, conducted in MEGA5 (Tamura et al., 2011). None of the farms reported clinical signs or noticeable losses of produc-tion at the time of sampling. Eighteen submissions proved to be positive by PCR for IBV out of the sampled 26 ones, but one yielded a weak product, which was not suitable for sequencing (the positive results are shown in Table 1). The se-quence analysis revealed ten 4/91, six QX-like, and one D274 type IBV. Out of the PCR positive samples, virus isolation was successful for IBV in 13 cases.     Acta Veterinaria Hungarica 63, 2015 384 KISS et al.Table 1 List and data of submissions included in the study. Superscript figures differentiate farms in the same region Origin County Type  of bird Age Applied IBV vaccine strains Positive sample IBV  test  result Concomitant pathogen detected GenBank accession number Nyírmada Sz-Sz-B B no info no info CT – –  Monyoród Baranya B 5 weeks D0: H120; D6–8: 4/91 CT 4/91 reovirus KP851921Lippó Baranya B 5 weeks D0: H120; D6–8: 4/92 CT 4/91 – KP851920Pápa Veszprém B 4 weeks D0: H120, D274 CT – –  Balotaszállás Bács-Kiskun B 3 weeks no info CT 4/91 reovirus KP851935Szakáld B-A-Z B 4 weeks D0: H120, D274 CT QX –  Mezőkövesd1 B-A-Z B 4 weeks D0: H120, D274 CT – –  Mezőkövesd2a  B-A-Z BB 22 weeks D0: H120, D274; D14: 4/91; D42: Ma5; D70: H120, D274; wk 18: inactivated M41, D274  CT  4/91  –  KP851923Mezőkövesd2b  B-A-Z BB 55 weeks D0: H120, D274; D14: 4/91; D42: Ma5; D70: H120, D274; wk 18: inactivated M41, D274  CT  QX  –   Mezőkövesd3 B-A-Z B 24 days D0: H120, D274 CT – reovirus  Jakabszállás Bács-Kiskun B 23 days D0: H120 CT QX – KP851927Jakabszállás Bács-Kiskun B 28 days D0: H120 CT QX/H120– KP851928KP851934Rém Bács-Kiskun BB 41 weeks D0: H120; D9: 4/91; D21: Ma5; wk 10: H120, D274 CT, K, T D274 – KP851933Pusztazámor Pest B 24 days D0: Ma5; D14: 4/91 CT – –  Kisfalud Gy-M-S LB 10 weeks D0: H102, D274 CT, K – –  Kisfalud Gy-M-S L 45 weeks D0: H120; D21: Ma5; D49: 4/91 CT QX – KP851929Taktaharkány B-A-Z L  D21: 1/96; w 6. & w 9: H120 CT 1/96 – KP851931Pásztó Nógrád B 21 days no vaccination against IB CT,K 4/91 reovirus KP851925Sényő Sz-Sz-B free-ranging B63 days D0: H120; D11: 4/91 CT 4/91 – KP851926Rétközberencs Sz-Sz-B B 19 days D0: H120 CT – –  Fegyvernek J-N-Sz B 23 days D0: H120 CT – –  Dombóvár Tolna B 4/6 weeks D0: H120 CT – reovirus  Ráckeve Pest L 38 weeks 3 wks: 4/91; 9 wks: H120 CT, K QX – KP851930Őriszentpéter Vas B 41 days D0: H120, D274 CT, T 4/91 – KP851922Balmazújváros Hajdú-Bihar B 38 days D0: B48; D11: 1/96 CT 1/96 – KP851932Nagyfüged Heves B 8 weeks D0 H120; D11: 1/96 CT 4/91 reovirus KP851924Sz-Sz-B = Szabolcs-Szatmár-Bereg; B-A-Z = Borsod-Abaúj-Zemplén; Gy-M-S = Győr-Moson-Sopron; J-N-Sz = Jász-Nagykun-Szolnok; B = broiler; L = layer;  BB = broiler breeder; LB = layer breeder; CT = caecal tonsil; T = trachea; K = kidney; n = negative; n.a. = not applicable; wk = week; wks = weeks  CIRCULATION OF 4/91 AND QX-TYPE IBV IN HUNGARY 385 Acta Veterinaria Hungarica 63, 2015 The group of 4/91 serotype IBVs seemed rather homogeneous. The ampli-fied fragment did not allow the discrimination between vaccine and field strains. In a broiler and in a layer flock a 1/96 strain of vaccine origin was detected sev-eral weeks after vaccination. In spite of the widespread use of vaccines containing Massachusetts-type strains (de Wit et al., 2011), this serotype was not detected except in one sample containing a mixture of H120 vaccine strain and a QX-like virus in one case (28 days old broilers vaccinated with H120 at day-old age). QX IBV had been first described in China in 2004 in connection with dif-ferent forms of renal lesions of chickens (Liu and Kong, 2004). The QX-type IBVs have spread all over Europe and become a predominant strain (Terregino et al., 2008; de Wit et al., 2011), although so-called European QX strains are quite rarely associated with kidney problems (Worthington et al., 2008). In Hungary it was first reported in 2008 (Benyeda et al., 2009). The present survey demonstrated the presence of QX serotype viruses in broiler, breeder and layer flocks after various vaccination protocols, even after using all registered serotypes in a broiler breeder flock (see Table 1, line Mező-kövesd2b). The database was supplemented with the sequence data of the NFCSO, including strains from the past few years (Fig. 1). The QX-like viruses formed geography-based groups, i.e. Jakabszállás and Kiskunmajsa. At the same time, high nucleotide identity in the amplified frag-ment was found between geographically moderately (Hajdúnánás–Mezőnyárád, approx. 60 km), and considerably (Bana–Medgyesbodzás – from 2012, approx. 280 km; Jakabszállás–Ráckeve–Szentpéterúr, approx. 200 km) distant strains of QX-like viruses, suggesting epizootiological link(s) between these farms (Fig. 2). Noticeably, the QX-like strain of Imrehegy origin, collected in 2011 by the NFCSO, separated from the rest of the QX-like group and showed high iden-tity with A2-type strains from Xindadi and Xinnong provinces (Ji et al., 2011), regarded as nephropathogenic variants of IBV. Therefore, this strain deserves further analysis. During the isolation process, embryo pathology characteristic of reovi-ruses (haemorrhagic embryos with greenish foci on their liver) was observed in six cases, and the presence of reoviruses was confirmed by virus isolation in cell culture and by PCR (Bruhn et al., 2005), either from IBV-negative (two cases) or 4/91-positive flocks (four cases), in the absence of characteristic reovirus-associated clinical signs. This study provided a snapshot on the prevailing IBV strains in Hungary and also showed that caecal tonsils are rather useful samples for IBV monitoring purposes. Like the situation in several European countries, we also found that 4/91 and QX-like viruses are the most prevalent. While the latter certainly repre-sent field viruses, the detected 4/91 and D274 strains can most probably be con-sidered vaccine viruses, based on the vaccination history of the respective flocks and the spreading capacity of IBV (Cook and Mockett, 1995). 386 KISS et al. Acta Veterinaria Hungarica 63, 2015  Fig. 1. Phylogenetic tree of the detected IBV strains. The bootstrap test (500 replicates) confidence levels ≥ 50% are shown next to the branches. Sequences originating from the survey are in bold let-ters, NFCSO samples are shown in italics  CIRCULATION OF 4/91 AND QX-TYPE IBV IN HUNGARY 387 Acta Veterinaria Hungarica 63, 2015  Fig. 2. Geographic distribution of the farms submitting the samples. The detected serotypes are identified with polygonal symbols  Reoviruses are frequently isolated from clinically normal chickens and the outcome of reovirus infection depends on several factors including age and im-mune status of the bird, route of exposure, virus pathotype, and interactions with other pathogens. Certain reovirus strains are even immunosuppressive and, thus, may enhance the severity of concomitant infections (Engström et al., 1988; Jones, 2013). While not sought for specifically, our results confirmed the ubiqui-tous nature of reoviruses in meat-type chickens (Jones, 2013), apparently in the lack of clinical disease. The applied vaccination protocols appeared satisfactory for preventing the clinical manifestations of IB, but the spread of QX-like variants was obvious. This draws the attention to more targeted vaccination regimes against this serotype. The developing ‘virus map’, supplemented with epizootiological informa-tion, will be useful for monitoring the efficacy of vaccination protocols. It will also show the prevalence and spread of IBV variants, thus serving the interest of farmers, vaccine companies, diagnostic laboratories and scientists involved in IBV research.  Acknowledgements The authors acknowledge the invaluable contribution of veterinarians from the Domestic Sales Department of Ceva-Phylaxia: Mihály Albert, Zsolt Punyi, István Szigeti, István Pálfy and András Medveczki, and the technical help received from mem-bers of the Scientific Support Directorate. Attila Farsang’s contribution was kindly sup-ported by the ‘János Bolyai’ Research Fellowship of the Hungarian Academy of Science (BO/125/13). The authors thank Tímea Tatár-Kis for the critical reading of the manu-script.  QX (9)  4/91 (8)  1/96 (2)  D274 (1)  QX/H120 (1)  Other/No value (6) 388 KISS et al. 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Survey indicates circulation of 793/B and QX-type infectious bronchitis viruses in Hungary in 2014

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