key: cord-0921873-f3v8wi9h
authors: Ammayappan, Arun; Upadhyay, Chitra; Gelb, Jack; Vakharia, Vikram N
title: Complete genomic sequence analysis of infectious bronchitis virus Ark DPI strain and its evolution by recombination
date: 2008-12-22
journal: Virol J
DOI: 10.1186/1743-422x-5-157
sha: e4d4fde9df50047757fa69030407e861748c0eb3
doc_id: 921873
cord_uid: f3v8wi9h

An infectious bronchitis virus Arkansas DPI (Ark DPI) virulent strain was sequenced, analyzed and compared with many different IBV strains and coronaviruses. The genome of Ark DPI consists of 27,620 nucleotides, excluding poly (A) tail, and comprises ten open reading frames. Comparative sequence analysis of Ark DPI with other IBV strains shows striking similarity to the Conn, Gray, JMK, and Ark 99, which were circulating during that time period. Furthermore, comparison of the Ark genome with other coronaviruses demonstrates a close relationship to turkey coronavirus. Among non-structural genes, the 5'untranslated region (UTR), 3C-like proteinase (3CL(pro)) and the polymerase (RdRp) sequences are 100% identical to the Gray strain. Among structural genes, S1 has 97% identity with Ark 99; S2 has 100% identity with JMK and 96% to Conn; 3b 99%, and 3C to N is 100% identical to Conn strain. Possible recombination sites were found at the intergenic region of spike gene, 3'end of S1 and 3a gene. Independent recombination events may have occurred in the entire genome of Ark DPI, involving four different IBV strains, suggesting that genomic RNA recombination may occur in any part of the genome at number of sites. Hence, we speculate that the Ark DPI strain originated from the Conn strain, but diverged and evolved independently by point mutations and recombination between field strains.

Avian infectious bronchitis virus (IBV) is a pathogen of domestic chickens that causes acute, highly contagious respiratory disease [1] . IBV is a member of the Coronaviridae, order Nidovirales [2] and its genome consists of a 27.6 kb single stranded positive-sense RNA molecule that encodes for four structural proteins; the spike (S) glycoprotein, the small envelope (E) protein, the membrane (M) glycoprotein, and the nucleocapsid (N) protein [3, 4] .

Six subgenomic mRNAs are transcribed from the IBV genome in virus-infected cells. The mRNA 1 contains two large overlapping open reading frames, encoding two polyproteins 1a and 1b [5] , among which 1b is produced as 1ab polyprotein by ribosomal frame-shifting mechanism [6] . Many serotypes have been described for IBV, probably due to the frequent point mutations that occur in RNA viruses and also due to recombination events demonstrated for IBV [7] [8] [9] . For this reason, the characterization of virus isolates existing in the field is very important. The Ark DPI strain was first isolated from Delmarva Peninsula broiler flock [10, 11] and it is currently being used as a vaccine in the USA and Europe. In this study, we characterized the entire genome of virulent Ark DPI strain (embryo passage 11) and compared it with other IBV strains and coronaviruses from all over the world.

The Ark DPI virus was inoculated into 9-day-old SPF chicken eggs and allantoic fluid was collected 72 h post inoculation. The fluid was clarified by low speed centrifugation and clear supernatant was stored at -80°C. Genomic RNA was extracted from virus-infected allantoic fluid with Qiagen RNAeasy kit, following the manufacturer's instructions, and stored at -80°C until further use. Oligonucleotides were designed based on consensus sequence of the following IBV strains: Cal 99 [Gen-Bank:AY514485], Mass 41 [GenBank:AY851295] and BJ [GenBank:AY319651]. Overlapping primers were designed in a manner such that each pair of primer covered approximately two kb of genome. The RT-PCR was carried out as described earlier [12] and the RT-PCR products were cloned into pCR2.1 TOPO TA vector (Invitrogen, CA). Plasmid DNA from various clones was sequenced by dideoxy chain termination method, using an automated DNA sequencer (Applied Biosystems, CA). Three independent clones were sequenced for each amplicon to exclude errors that can occur from RT and PCR reactions. The assembly of contiguous sequences and multiple sequence alignments were performed with the GeneDoc software [13] . The pair-wise nucleotide identity and comparative sequence analyses were conducted using Vector NTI Advance 10 software (Invitrogen, CA) and BLAST search, NCBI. Phylogenetic analyses were conducted using the MEGA4 program [14] .

The GenBank accession number for the Ark DPI sequence is EU418976. The complete genomes of following strains are obtained from GenBank: TCoVMG10, NC_010800; Beaudette, NC_001451; M41, AY851295; CK/CH/LSD/ 05I, EU637854; A2, EU526388; LX4, AY338732; SAIBK, DQ288927; The accession numbers of IBV gene sequences which are used in this study are as follows: For replicase gene sequences: The details of genome organization of Ark DPI are shown in Fig. 1 . IBV polyprotein is cleaved into 15 cleavage products, among which first two N-terminal products are cleaved by PL pro and rest of the C-terminal products are cleaved by 3CL pro [15] . The putative domains and their cleavage sites ( Fig. 1 ) are predicted by comparison of amino acid sequences of each non-structural protein (nsp) of Ark DPI with those of IBV-Beaudette which is available in Coronavirus Database (CoVDB) [16] . The nucleotide and the amino acid identity of Ark DPI with other IBVs and coronaviruses are listed in Tables 1, 2, 3. The whole structural gene of Jilin is 100% identical to Ark DPI, which suggests that Jilin strain is actually Ark DPI, which is currently used as a vaccine in China [17] . The whole genome comparison of IBV strains reveals a close relationship of Ark DPI with Cal 99 (96% identity), as shown in Fig. 2 . Earlier studies have shown that Cal 99 probably evolved from Ark DPI [18] .

The complete genome sequence analysis of Ark DPI strain shows striking similarity to the Conn, Gray, JMK, and Ark 99 IBV strains, which were circulating during that time period [1, [19] [20] [21] . The 5'UTR, PL pro , M pro and RdRp sequence analysis demonstrates that Ark DPI is 100% identical to Gray strain, except for PL pro which has 87% identity, as shown in Table 1 . It was suggested that PL pro gene has high genetic variation because of selection pressure [22] . From this analysis, it appears that genetic mutation may have occurred at PL pro gene level. The modern strain GA98 maintains 100% identity with Ark DPI in replicase proteins and because of unavailability of sequence information for rest of the genome; we speculate that GA98 may be a derivative of the Ark DPI strain.

Analysis of the structural region of Ark DPI clearly demonstrates that it is a chimera of three strains. The S1 gene of Ark DPI is probably derived from Ark 99 (97% identical) and because of genetic mutations in the S1 region, Ark DPI may have evolved independently. There is an A-T rich sequence TGTGTTGATTATAAT (Fig. 3) at the 3'terminus of S1 gene (~300 nts upstream from the end of S1 gene) which is conserved among most of the IBV strains. The S1 gene of Ark 99 maintains its identity with Ark DPI up to this conserved region, but from this point onwards to the end of S2, the nucleotide sequence is 100% identical to JMK strain. The recombination between JMK and Ark 99 had taken place presumably between above mentioned conserved region and intergenic (IG) region of S gene, which is located 49 nts upstream of start codon of S gene. It is speculated that IG sequences serve as "hot spots" for recombination because of its consensus nature [23] . Gray and JMK strains share 99% homology both in the S1 and S2 genes of Ark DPI, but JMK shows greater identity than Gray strain, as shown in Table 2 . It is interesting that very few residues in the S1 gene make the Gray strain nephrotropic, whereas JMK is pneumotropic [24] .

Out of 174 nts of gene 3a of Ark DPI, last 74 nts are 100% identical to Conn, whereas first 100 nts are only 86% identical. Even though it is not clear whether the 5'-end of 3a was derived from Conn or JMK, but it is evident that the recombination event may have occurred between JMK and Conn at gene 3a. The 3b gene of Ark DPI and Conn differed only by two nucleotides and both share 99% identity, suggesting that 3b belongs to Conn strain. From gene 3c to N gene, Ark DPI shares 100% identity with Conn. It is obvious that the entire structural genome, except spike, belongs to Conn strain. Cross protection studies carried out by Gelb and coworkers [11] demonstrated that the birds immunized with Ark DPI showed Classical Genome Organization of IBV-Ark DPI 95%, 90% and 63% protection against Conn, Ark 99 and JMK strains, respectively. Indeed, these results suggest that major part of Ark DPI genome was derived from Conn. The level of protection for JMK is 80%, when Ark 99 was used as immunogen [25] . On the other hand, Conn and JMK immunization induces inadequate immunity against Ark-type IBV challenge, suggesting that Ark cross-immunity to JMK and Conn is a one-way relationship [10, 11, 26] .

Recombination hot spots have been demonstrated for IBV isolates by many researchers. These hot spots have been detected in the IG region [23] , S1 gene [27] , 3' terminus of S2, N and between N gene and 3'UTR [8, 28] . Some earlier sequencing studies had provided circumstantial evidence of recombination events in field isolates of IBV [7, 29, 30] . More or less recombination sites were detected over the entire genome of coronavirus [31] . Based on these results, we speculate that the Ark DPI strain originated from the Conn strain, but diverged and evolved independently by point mutations and recombination between field strains. These findings suggest that there is high level of genetic diversity among currently circulating IBV serotypes. Most of them come from genetic changes which already exist in the IBV field strains and from IBV live vaccines. So frequent monitoring is highly essential to track the emergence of new variants and is mandatory to develop efficient vaccination strategies to control and prevent IB. a Sequences with > 95% identity are in bold letters b NA-not analyzed c Parental strains of Ark DPI are shown in bold letters and immediate derivative of Ark DPI is indicated by asterisk (*). Phylogenetic tree analysis of complete Ark DPI genome sequence with other IBV strains 

The propagation and cytopathogenic effect of an egg-adapted strain of infectious bronchitis virus in tissue culture

Infectious Bronchitis

Coronaviruses: structure and genome expression

Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus

Binns MM: Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus

Characterization of an efficient coronavirus ribosomal frameshifting signal: Requirement for an RNA pseudoknot

Infectious bronchitis virus: evidence for recombination within the Massachusetts serotype

Experimental evidence of recombination in coronavirus infectious bronchitis virus

S1 gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the United States and Israel

Serologic and crossprotection studies with several infectious bronchitis virus isolates from Delmarva-reared broiler chickens

Prevalence of Arkansas-type infectious bronchitis virus in Delmarva Peninsula chickens

Molecular determinants of virulence, cell tropism, and pathogenic phenotype of infectious bursal disease virus

GeneDoc: analysis and visualization of genetic variation

MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0

Virus-encoded proteinases and proteolytic processing in the Nidovirales

CoVDB: a comprehensive database for comparative analysis of coronavirus genes and genomes

Infectious bronchitis virus: S1 gene characteristics of vaccines used in China and efficacy of vaccination against heterologous strains from China

Genotypic and phenotypic characterization of the California 99 (Cal99) variant of infectious bronchitis virus

Immunological characteristics of a variant of infectious bronchitis virus isolated from chickens

Etiology of an infectious nephritisnephrosis syndrome of chickens

A new serotype of infectious bronchitis virus responsible for respiratory disease in Arkansas broiler flocks

Comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains

Evidence of genetic diversity generated by recombination among avian coronavirus IBV

Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus

Cross-immunity in chickens using seven isolates of avian infectious bronchitis virus

The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures

Using DNA shuffling to create novel infectious bronchitis virus S1 genes: Implications for S1 gene recombination

A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains

Evolution of avian coronavirus IBV: Sequence of the matrix glycoprotein gene and intergenic region of several serotypes

Evidence of natural recombination within the S1 gene of infectious bronchitis virus. Virology 1993

RNA recombination in animal and plant viruses

The authors declare that they have no competing interests.

VNV and JG conceived the study. AA planned the experimental design, AA and CU carried out cloning and sequencing. AA drafted the manuscript. All authors critically reviewed and approved the final manuscript.