key: cord-0881002-xbe2wlna
authors: Laconi, Andrea; Listorti, Valeria; Franzo, Giovanni; Cecchinato, Mattia; Naylor, Clive; Lupini, Caterina; Catelli, Elena
title: Molecular characterization of whole genome sequence of infectious bronchitis virus 624I genotype confirms the close relationship with Q1 genotype
date: 2018-09-20
journal: Transbound Emerg Dis
DOI: 10.1111/tbed.13000
sha: 714e8e796950149e0c7fb7b80f162d957c030813
doc_id: 881002
cord_uid: xbe2wlna

Infectious Bronchitis virus (IBV) genotype Q1 was detected for the first time in China in 1996, and then spread worldwide. The first report of Q1 genotype in Italy occurred in 2011 and a deep molecular investigation of a Q1 isolated in Italy in 2013 has led to speculation regarding the origin of this genotype. Phylogenetic analysis of the S1 sequence of a Q1 Italian strain revealed a close relationship with sequences of the 624I strains circulating in Italy in the early 1990s and this led to the idea that 624I was an ancestor of the Q1 genotype. Despite the fact that most heterogeneity of IBVs occurs in the S1 gene, the sequence analysis of this gene alone was not sufficient to confirm or deny this hypothesis. In the present study, an Italian 624I (gammaCoV/AvCov/Ck/Italy/IP14425/96) was fully sequenced for the first time and compared to all available complete Q1 genome sequences. This analysis confirmed the genetic correlation between GammaCoV/AvCov/Ck/Italy/IP14425/96 and Q1 strains, suggesting a common origin between 624I and Q1 genotypes.

Infectious bronchitis (IB) is an avian disease distributed worldwide that represents one of the most persistent health problems of the commercial poultry industry (Cook, Jackwood, & Jones, 2012; de Wit, Cook, & van der Heijden, 2011) . It is caused by a Gammacoronavirus called Infectious Bronchitis Virus (IBV) and has a positive sense single-stranded 27.6 kb RNA genome (Jackwood & De Wit, 2013) . The IBV genome can evolve rapidly by mutation and recombination events, resulting in the emergence of new IBV variants which sometimes confer minimal or negligible cross-protection (de Wit et al., 2011) . A majority of such variants cause a transitory problem, which then disappear or remain confined into a specific geographical region. However, a few variants can persist and spread to new areas where they continue to cause disease (Jackwood, 2012; de Wit et al., 2011) .

Recently, genotypes of Asian origin have spread to cause worldwide disease and major economic losses (de Wit et al., 2011) . IBV Q1 is one such genotype. It was first detected in China in 1996 (Yu et al., 2001) and then reported in Asia, Middle East, Europe, and South America (Ababneh, Dalab, Alsaad, & Al-Zghoul, 2012; Huang, Lee, Cheng, & Wang, 2004; Jackwood, 2012; Marandino et al., 2015; Rimondi et al., 2009) . In Italy, the Q1 genotype was reported for the first time in 2011 after causing an outbreak of disease associated with respiratory signs, increased mortality, kidney lesions, and proventriculitis (Toffan et al., 2013) . Since then, the genotype has been continuously detected in Italy (Massi et al., 2015) . Phylogenetic analysis performed using full or partial S1 sequences showed a high identity (>99%) with Chinese Q1 isolates (Franzo et al., 2015; Massi et al., 2015; Toffan et al., 2013) . At a similar period, a high identity (94.1%) between those strains and strains belonging to the 624I genotype was observed (Franzo et al., 2015; Massi et al., 2015) , such that the recently proposed new IBV nomenclature based on the S1 sequence placed them in the same lineage (GI-16) (Valastro et al., 2016) . IBV 624I had been reported for the first time in Italy in 1993 (Capua, Gough, Mancini, Casaccia, & Weiss, 1994; Capua et al., 1999) , producing disease associated with kidney lesions and drop in egg production in breeders and layers (Capua, Grasso, Ferdinandi, Weiss, & Casaccia, 1996) . This genotype continued to be detected in Italy until 2004 and then reappeared from 2010 when it was again detected in a few broiler farms affected by respiratory disease located in different areas of Italy. In the following years, the number of detections increased (Massi, 2013) but since 2013 the 624I genotype has not been further detected (Massi et al., 2015) .

A recent retrospective study carried out on 123 IBV strains isolated in Italy between 1963 and 1989 revealed that 624I genotype had not only circulated long before its first reporting in 1993, but that in fact it has been one of the major IBV genotypes circulating in the Country at that time (Taddei et al., 2012) . Evidence of the presence of this genotype has also been found in Slovenia, where several 624I strains were isolated between 1991 and 1999 (Krapez, Slavec, Barlic-Maganja, & Rojs, 2010) , in Poland and South Africa (Capua et al., 1999) and eventually in Russia where 624I genotype was reported in 2002 (Bochkov, Batchenko, Shcherbakova, Borisov, & Drygin, 2006) .

The high identity observed between Q1 and 624I genotypes raises questions regarding their possible related origins. IBV 624I has been hypothesized to be an ancestor of the Q1 genotype (Franzo et al., 2015; Massi, 2013) , but unfortunately the unavailability of any 624I full genome sequence did not allow such final conclusions to be drawn.

In the present study, an IBV 624I was fully sequenced and phylogenetic analysis was performed both using a dataset based on available IBV full-length genome sequences and, due to the larger number of published sequences, a dataset based on full S1 gene. In addition, recombination analysis was carried out using the complete IBV 624I and Q1 strains.

IBV 624I strain was isolated in 1996 during a disease outbreak in chicken farms located in Northern Italy. The virus was isolated in specific pathogen-free (SPF) chicken eggs and the 3th passage was propagated in SPF chicken embryo tracheal organ cultures (TOC).

After isolation, the virus underwent serological analysis, resulting as belonging to the 624I serotype. In this study, this virus is named gammaCoV/AvCov/Ck/Italy/IP14425/96.

The RNA was extracted from the supernatant of infected TOC using Qiamp viral RNA mini kit (Qiagen, Hilden, Germany) following the manufacture's protocol. Viral RNA was firstly retro-transcribed using Super Script III enzyme (Invitrogen, Carlsbad, CA, USA), then amplified using Ranger enzyme (Bioline, London, UK) according to the manufacturer's instructions. Retrotranscription, amplification, and sequencing were carried out using primers previously designed for IBV full genome sequencing (Franzo et al., 2015; Listorti et al., 2017) . Where primers did not work due to sequence differences, new primers were designed based on the newly determined sequences flanking those genome regions (Table 1 ). Sequencing was performed by Source BioScience (Nottingham, UK). Each genome fragment was sequenced twice. Where gammaCoV/AvCov/Ck/Italy/ IP14425/96a sequence differed from those of Q1 strains, the locations were sequenced again, starting with a new retrotranscription of the region.

3′ end of the genome was determined using a 3′RACE protocol previously described (Laconi et al., 2016) . Briefly, RT was performed with a primer containing 20 Ts followed by an adaptor sequence at its 5′ terminus. This was amplified by PCR using two primers, one within the end of the genome and one matching the adaptor (Table 1) . These PCR products were sequenced towards the polyA tail.

Chromatograms were analysed using the program Chromas (http:// technelysium.com.au/wp/chromas/) and sequences aligned using BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) against the genome of IBV strains gammaCoV/Ck/Italy/I2022/13 (KP780179) and CK/CH/LDL/97I (JX195177). Open Reading Frame (ORF) prediction was carried out using ORFfinder program (https://www.ncbi.nlm.nih. gov/orffinder/).

A dataset containing 313 complete genome sequences (nt) of IBV was downloaded from ViPr, an open source bioinformatics database and analysis resource for virology research. To minimize the computational load, cd-hit-est test of the CD-HIT Suite (Li & Godzik, 2006) was used to cluster sequences that shared over 98% identity and a prototype sequence within each cluster was selected. After clustering, 187 representative sequences remained. Sequences were aligned using ClustaW and phylogenetic analysis was carried out with MEGA7 software (Kumar, Stecher, & Tamura, 2016) using Maximum Likelihood method with Tamura-Nei substitution model and 1,000 bootstrap replicates to assess the robustness of the branches.

A dataset containing all available complete or nearly complete S1 gene sequences (at least 1,000 bp) was downloaded from ViPr. After clustering, 320 sequences remained. The sequences were aligned using ClustalW method, and a phylogenetic tree was constructed using the Maximum Likelihood method with Kimura 2 parameter substitution model and 1,000 bootstrap replicates to assign confidence level to the branches in MEGA 7 software. Based on these results, a subset of sequences clustering with gammaCoV/AvCov/Ck/ Italy/IP14425/96 was selected and expanded to its original number of taxa. Sequences belonging to this dataset were aligned with Clus-talW method, and a phylogenetic tree was reconstructed using the parameters previously described.

Presence of past recombination events for strain gammaCoV/AvCov/ The ORF analysis predicted 13 ORFs and revealed the following 

Sequence of the IBV strain gammaCoV/AvCov/Ck/Italy/IP14425/96 was submitted to the GenBank database and the following accession number was assigned: MG021194.

The phylogenetic analysis of the 187 representative full IBV genome sequences demonstrated that gammaCoV/AvCov/Ck/Italy/ IP14425/96 clustered together with Q1 strains gammaCoV/Ck/Italy/ I2022/13, CK/CH/LDL/97I and UY/09/CA/01, occupying a basal position in the specific cluster (Figure 1 ). In the same clade, the Uruguayan strain UY/11/CA/18 (MF421320), was also present, previously ascribed to the SAI genotype (Lineage G-11) (Figure 1 ) (Marandino et al., 2015) . 

Phylogenetic analysis using a dataset characterized by 320 representative full S1 sequences showed that gammaCoV/AvCov/Ck/Italy/ IP14425/96 clustered together with strains previously identified as 624/I genotype isolated in Italy in the late '80s and early '90s and strains belonging to Q1 genotype, isolated in Italy, in China, in Tai 

Recombination analysis was performed to assess the possible recom- 

The genome of the IBV strain gammaCoV/AvCov/Ck/Italy/IP14425/ 96 isolated in Italy in 1996 was fully sequenced and this represents the first report of a full genome sequencing of a virus belonging to the 624I genotype. The isolate shows a genome organization slightly different when compared to the genome organization of most IBVs previously reported (5′UTR-1a-1b-S-3a-3b-E-M-5a-5b-N-3′UTR) (Cavanagh, 2005) , since ORF analysis showed the presence of F I G U R E 2 (a) Phylogenetic tree based on Maximum Likelihood method with Kimura-2 model constructed using 320 S1 gene complete sequences. For easiness of representation, strain reported in the present study and closely related strains, are marked in red. (b) Expanded phylogenetic tree based on Maximum Likelihood method with Kimura-2 model constructed using S1 complete sequences closely related to gammaCoV/AvCov/Ck/Italy/IP14425/96 [Colour figure can be viewed at wileyonlinelibrary.com] accessory genes 4b, 4c, and 6b already reported for TCoV and other

IBVs (Abolnik, 2015; Hewson, Ignjatovic, Browning, Devlin, & Noormohammadi, 2011 Kint, Dickhout et al., 2015; Kint, Fernandez-Gutierrez et al., 2015 . A similar function might be hypothesized also for genes 4b, 4c, and 6b, especially in the light that the 4b homologous gene

in the MERS-CoV has been reported as an antagonist of type I interferon response (Yang et al., 2013) and that the 6b homologue in SARS-CoV was shown to be able to induce apoptosis (Ye, Wong, Li, & Xie, 2008) . More studies need to be done to improve the knowledge on these three accessory genes, in particular whether they are peculiar of certain genotypes and whether their expression influences the pathogenicity or the tropism of the virus.

Phylogenetic analyses performed using two different datasets, one built with IBV complete genome sequences and one built with IBV complete or nearly complete S1 gene sequences, showed that 624I and Q1 genotypes clustered together. Our findings, strongly F I G U R E 3 RDP screenshots displaying the possible recombination events associated with CK/CH/LDL/97I (a), gammaCoV/Ck/Italy/I2022/13 (b), and gammaCoV/AvCov/Ck/Italy/P14425/96 (c) Each panel displays the pairwise identities among the possible recombinant and its putative parents. Crossover sites indicating sharp changes in pairwise identity are pointed out by arrows. (a) Comparisons among the putative recombinant CK/CH/LDL/97I show sharp changes in the pairwise identity within the 1a gene, H120 vaccine strain FJ888351 has been identified as putative parental strains for that genomic region. (b) Comparison among the putative recombinant gammaCoV/Ck/Italy/I2022/13 shows sharp changes in the pairwise identity within the 1a gene; strain gammaCoV/AvCov/Ck/Italy/P14425I/96 has been identified as major parental strain for that region. (c) Comparison among the putative recombinant gammaCoV/AvCov/Ck/Italy/P14425/96 shows sharp changes in the pairwise identity within the 1a gene; strain gammaCoV/Ck/Italy/I2022/13 has been identified as major parental strain for that genomic region [Colour figure can be viewed at wileyonlinelibrary.com] suggest a common origin between the two genotypes. The basal location of the 624I strain in both the phylogenetic trees, coupled with the epidemiological data available, suggests that this genotype might have played a role in the emergence of Chinese Q1. This model requires long-distance intercontinental dispersion of the 624I genotype and this possibility is supported by its ability to circulate for extended periods within the same country (Taddei et al., 2012) and beyond a geographical area (Capua et al., 1999; Krapez et al., 2010 (Bochkov et al., 2006) . Further investigations are needed since, despite the IBV worldwide dissemination has been observed and accepted for other genotypes (Franzo et al., 2017) , the mechanism behind this evidence is not fully understood.

The recombination analysis showed that the Chinese strain CK/ CH/LDL/97I underwent recombination with a H120 vaccine strain, which has been previously demonstrated to be involved in recombination events leading to reversion to virulence and the emerge of new genotypes in China (Zhang et al., 2010) . The identification of such a recombination event within the 1a gene might explain the relatively high genetic diversity between 624I strain gammaCoV/ AvCov/Ck/Italy/IP14425/96 and Q1 Chinese strain CK/CH/LDL/97I, two viruses isolated only one year apart.

The absence of such recombination in the genome of Q1 Italian strain gammaCoV/Ck/Italy/I2022/13 suggests that not all Q1 strains emerged as a result of a recombination event with a H120

strain. The absence of a recombination event in the 1a gene of Q1 Italian strain gammaCoV/Ck/Italy/I2022/13 might indicate that Chinese and Italian Q1 strains are the result of independent evolutions from the 624I genotype. However, the huge differences in field conditions and therefore genetic pressures between the two countries, together with the phylogenetic results based on S1 gene sequences in which Q1 Italian strains cluster together with Chinese and Taiwanese strains, make this hypothesis highly unlikely.

Taken as a whole, the data presented in this study suggest the 624I genotype to be the ancestor of Q1. 

:FJ807933:1-1632|Organism:Infectious bronchitis virus|Strain Name:EJ95|Protein Name:S1 glycoprotein|Gene Symbol:S1 gb:JQ920388:1-3510|Organism:Infectious bronchitis virus|Strain Name:8067|Protein Name:spike protein|Gene Symbol:S gb:FJ807944:1-1632|Organism:Infectious bronchitis virus|Strain Name:K620/02|Protein Name:S1 glycoprotein|Gene Symbol:S1 gb:FJ807932:1-1632|Organism:Infectious bronchitis virus|Strain Name:B4|Protein Name:S1 glycoprotein|Gene Symbol:S1 gb:KC577388:1-1632|Organism:Infectious bronchitis virus|Strain Name:48SD-96VI|Protein Name:spike glycoprotein S1|Gene Symbol:S1 gb:KX668222:1-3510|Organism:Infectious bronchitis virus|Strain Name:S78/14|Protein Name:spike protein|Gene Symbol:s gb:KX529736:1-1614|Organism:Infectious bronchitis virus|Strain Name:DMV/1639|Protein Name:spike protein|Gene Symbol:S1 gb:KM087780:1-1664|Organism:Infectious bronchitis virus|Strain Name:GA-13/14255/14|Protein Name:spike protein|Gene Symbol:S1 gb:KM660630:1-1620|Organism:Infectious bronchitis virus|Strain Name:GA/10215/2010|Protein Name:spike protein|Gene Symbol:S1 gb:KM660635:1-1662|Organism:Infectious bronchitis virus|Strain Name:GA/13384/2013|Protein Name:spike protein|Gene Symbol:S1 gb:KM660634:1-1647|Organism:Infectious bronchitis virus|Strain Name:GA/12341/2012|Protein Name:spike protein|Gene Symbol:S1 gb:GU734804:1-1640|Organism:Infectious bronchitis virus|Strain Name:GA08-GA08-08 pass16|Protein Name:spike protein|Gene Symbol:S1 gb:KC478646:1-1632|Organism:Infectious bronchitis virus|Strain Name:Ck/CH/Shaanxi/2009/W09|Protein Name:spike glycoprotein 1|Gene Symbol:S1 gb:DQ490205:1-3510|Organism:Infectious bronchitis virus|Strain Name:Armidale|Protein Name:spike protein|Gene Symbol:S gb:DQ490214:1-3495|Organism:Infectious bronchitis virus|Strain Name:V1-71|Protein Name:spike protein|Gene Symbol:S gb:DQ490216:1-3504|Organism:Infectious bronchitis virus|Strain Name:V2-71|Protein Name:spike protein|Gene Symbol:S gb:DQ490209:1-3483|Organism:Infectious bronchitis virus|Strain Name:Q1-73|Protein Name:spike protein|Gene Symbol:S gb:EU930437:1-1200|Organism:Infectious bronchitis virus|Strain Name:SDJY0701|Protein Name:truncated S1 glycoprotein|Gene Symbol:S1 gb:JN983807:1-3504|Organism:Infectious bronchitis virus|Strain Name:VicS-del|Protein Name:spike protein|Gene Symbol:S gb:JX018209:1-1715|Organism:Infectious bronchitis virus|Strain Name:013|Protein Name:spike protein|Gene Symbol:S1 gb:DQ490208:1-3501|Organism:Infectious bronchitis virus|Strain Name:N2-75|Protein Name:spike protein|Gene Symbol:S gb:KT886447:1-1096|Organism:Infectious bronchitis virus|Strain Name:gammaCoV/Ck/Poland/162/1997|Protein Name:spike protein|Gene Symbol:S1 gb:KT886445:1-1442|Organism:Infectious bronchitis virus|Strain Name:gammaCoV/Ck/Poland/81/1989|Protein Name:spike protein|Gene Symbol:S1 gb:JQ739363:1-1672|Organism:Infectious bronchitis virus|Strain Name:ck/CH/LSD/110712|Protein Name:S1 protein|Gene Symbol:S1 gb:DQ458217:1-3510|Organism:Infectious bronchitis virus|Strain Name:AL/4614/98|Protein Name:spike glycoprotein|Gene Symbol:S gb:KC692237:1-1632|Organism:Infectious bronchitis virus|Strain Name:CK CH GD LY12|Protein Name:S1 glycoprotein|Gene Symbol:s1 gb:KU311691:1-1631|Organism:Infectious bronchitis virus|Strain Name:CA/3099/12|Protein Name:spike glycoprotein|Gene Symbol:S1 gb:JF774063:1-1677|Organism:Infectious bronchitis virus|Strain Name:IBV/CAL99 variant/07|Protein Name:spike protein|Gene Symbol:S1 gb:GQ229245:1-1629|Organism:Infectious bronchitis virus|Strain Name:3381/06|Protein Name:spike glycoprotein S1 subunit|Gene Symbol:S1 gb:JQ964061:1-1632|Organism:Infectious bronchitis virus|Strain Name:L165|Protein Name:spike glycoprotein S1|Gene Symbol:S1 gb:KC577394:1-1629|Organism:Infectious bronchitis virus|Strain Name:56GX-98I|Protein Name:spike glycoprotein S1|Gene Symbol

Infectious bronchitis virus|Strain Name:ck/CH/LDL/150434-III|Protein Name:spike protein|Gene Symbol:S gb:GQ229232:1-3498|Organism:Infectious bronchitis virus|Strain Name:3382/06|Protein Name:spike protein|Gene Symbol:S gb:EU822336:1-3498|Organism:Infectious bronchitis virus|Strain Name:3468/07|Protein Name:spike protein|Gene Symbol:S gb:JQ739314:1-1807|Organism:Infectious bronchitis virus|Strain Name:ck/CH/LHLJ/111043|Protein Name:S1 protein|Gene Symbol:S1 gb:KP868572:20113-23613|Organism:Infectious bronchitis virus|Strain Name:ck/CH/LHLJ/111043|Protein Name:S protein|Gene Symbol:S gb:KU364611:1-1620|Organism:Infectious bronchitis virus|Strain Name:cK/CH/SCMS/150527|Protein Name:spike glycoprotein|Gene Symbol

EU714029|SC021202|China|NA KF663560|ck/CH/IBWF/2007|China|NA

AY692454|Beaudette Vero cell adapted|NA|NA NC 001451|Beaudette|NA|NA

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