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Circulation of QX-like infectious bronchitis virus in the Middle EastO. G. M. Amin, V. Valastro, A. Salviato, A. Drago, G. Cattoli, I. Monne

Infectious bronchitis virus (IBV) is a highly contagious viral dis-ease of chickens, caused by an aetiologic agent belonging to the genus Coronavirus. It mainly affects the upper and lower respira-tory tracts, and frequently causes damage to the kidneys and the reproductive system (Cavanagh and Naqi 2003). Although vacci-nation is commonly adopted, outbreaks have occurred worldwide in commercial poultry farms, with significant economic conse-quences due to a decrease in productive performances (Cavanagh 2005, 2007). The virus is characterised by a great genetic and path-ogenic variability. More than 50 different serotypes of IBV have been identified, and new variants continue to emerge (Bochkov and others 2006).

The genotype, designated as QX IBV, has spread from Asia, where it was described for the first time in 1996 in China (Wang and others 1998), to Europe (Beato and others 2005, Landman and others 2005, Valastro and others 2010), and recently to the southern part of the African continent (Zimbabwe) as well (Toffan and others 2011). This serotype has become the field strain predominant in the majority of poultry farms in many countries, and is associated with respiratory signs, nephritis and with the so-called ‘false layer syndrome’ (Worthington and others 2008, De Wit and others 2011). To date, no information on the QX-like strain circulation in the Middle East is available, nor has this virus been detected in Iraq, as also recently reviewed (De Wit and others 2011). In 2009, IB infec-tions in vaccinated broiler farms in Kurdistan-Iraq have been reported causing kidney lesions. In this case, based on genetic characterisation, a putative novel IB variant not related to QX-like strain was revealed (Mahmood and others 2011). In Iraq, the vaccination against IBV is performed with vaccines that contain live-attenuated or killed viruses belonging to the Massachusetts serotype. In spite of this, IBV infec-tion is considered endemic and widely spread both in vaccinated and unvaccinated poultry farms, generally associated with kidney damage and urolithiasis (Mahmood and others 2011).

The aim of this study is to provide information on the molecular characteristic and the phylogenetic relationship of the first QX- like strain reported in Kurdistan-Iraq, with other strains recently identified in Iraq and in other Eurasian countries.

O. G. M. Amin, DVMGeneral Directorate of Veterinary Services, Kurdistan Ministry of Agriculture, Kurdistan Regional Government, Erbil, IraqV. Valastro, PhDA. Salviato, biotechnologistA. Drago, biotechnologistG. Cattoli, DVMI. Monne, DVM

Research and Development Department, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy

E-mail for correspondence: vvalastro@izsvenezie.it

Provenance: not commissioned; externally peer reviewed

Accepted September 7, 2012

Veterinary Record (2012) doi: 10.1136/vr.100896

In December 2011, tracheal swabs were collected from a 35-day-old broiler breeder flock located in Erbil City (Kurdistan-Iraq). The flock had been vaccinated against IBV with commercial monovalent live-attenuated Mass-type vaccines, H120 and Ma5 strains, at 1 and 21 days of age, respectively. The birds were suffering from several clinical signs related to respiratory diseases, such as gasping, sneezing and ruffled feathers accompanied by severe conjunctivitis and increase of mortality rate (up to 19 per cent). Postmortem examination of dead birds revealed caseous plug in bronchial bifurcation and oedema in subcutaneous tissue.

In the laboratory, the viral RNA was extracted from 12 swabs (Nucleospin RNA II kit-Macherey-Nagel, Germany) and a real-time RT-PCR, targeting the conserved 5′-untranslated region of the viral genome (Callison and others 2006), was performed to screen the presence of IBV genomes. Six out of the 12 samples were found IBV-positive, and were processed by RT-PCR following the inhouse protocol (Valastro and others 2010). The set of primers which was used anneal to a conserved region of the S1 gene lying upstream of the segment, amplified by primers previously published and used for a RT nested-PCR (Jones and others 2005) which gave negative results on the samples included in this report. The application of the protocol allowed for the generation of a PCR product of approx-imately 300 base pairs, which was further processed for sequence analysis.

The six nucleotide sequences obtained in this study were identi-cal to each other. One representative sequence (GenBank accession number JQ863360) was aligned and compared with publicly available QX-like strain sequences of viruses from several Eurasian countries, and also with IBV sequences representative of major IB serotypes. A maximum likelihood (ML) tree was estimated using the best-fit gen-eral time-reversible (GTR) + I + Γ4 model of nucleotide substitution using PAUP* (Wilgenbusch and Swofford 2003). A bootstrap resam-pling process (1000 replications) using the neighbour-joining method was used to assess the robustness of individual nodes on the phylog-eny, incorporating the ML substitution model described above.

The phylogenetic analysis showed that the Kurdistani-Iraqi virus was closely related (98.9 per cent) to QX-strains collected in China between 2009 and 2010. Lower nucleotide similar-ity (95.4 per cent) was revealed with the unique QX-type virus sequence from the Middle East deposited in GenBank and isolated in Israel in 2004 (IS/1201 isolate, accession number DQ400359). It is noteworthy that the QX-type virus identified in Erbil City is genetically unrelated (nucleotide similarity: 47.3 per cent, Fig 1) to the novel IBV variant (Sul/01/09 isolate) detected in Sulaimani about two years ago (Mahmood and others 2011), indicating the circulation in the same area of two different IBV field variants in vaccinated flocks. All these data taken together underline the need to revise the vaccination programme in order to prevent the occur-rence of new IBV outbreaks.

Although the lack of QX sequences from the region does not allow to infer on the origin of this virus in Iraq based on the nucleotide similarity existing between the Kurdistani-Iraqi and Chinese sequenc-es, it is possible that the QX-like strain’s introduction in Iraq origi-nated from the Far East rather than from Israel, as the virus identified in this country was genetically more correlated with the European QX-like strain. As occurred with the first introduction of the QX strain in Europe (Beato and others 2005, Worthington and others 2008), the Asian region seems to have also played a crucial role in the spreading of the QX variant in this Middle Eastern area. However, the reasons of this westward spreading of IBV variants are currently unknown, and neither of the two main means of dissemination, that is, through wild birds (De Wit and others 2011) or trade of poultry, can be excluded based on the data available. Further studies are needed to highlight the epidemiological link existing between these geographic areas, and to clarify migration patterns of this IBV variant.

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2 of 2 | Veterinary Record | 10.1136/vr.100896

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VALASTRO, V., MONNE, I., FASOLATO, M., CECCHETTIN, K., PARKER, D., TERREGINO, C. & CATTOLI, G. (2010) QX-type infectious bronchitis virus in com-mercial flocks in the UK. The Veterinary Record 167, 865–866

WANG, Y. D., WANG, Y. L., ZHANG, Z., FAN, G., JIANG, Y., LIU, X., DING, J. & WANG, S. (1998) Isolation and identification of glandular stomach type IBV (QX IBV) in chickens. Chinese Journal of Animal Quarantine 15, 1–3

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FIG 1: Maximum likelihood phylogenetic tree of the S1 gene segment. The sequence of the QX-like strain collected in Kurdistan-Iraq is in bold. The QX-like strain sequences identified in China between 2009 and 2010 and in Israel are highlighted with a black square and with a black circle, respectively. The numbers on each branch point represent bootstrap values. Accession numbers of the S1 gene sequences downloaded from GenBank are reported in the phylogenetic tree. Strain abbreviations: CH China, Ck chicken, IR Iran, IS Israel, IT Italy, K Korea, NL The Netherlands, SLO Slovenia, SP Spain, RF Russia, THA Thailand, UK United Kingdom.

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doi: 10.1136/vr.100896 published online October 7, 2012Veterinary Record

 O. G. M. Amin, V. Valastro, A. Salviato, et al. virus in the Middle EastCirculation of QX-like infectious bronchitis

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