A Chinese isolate of avian infectious bronchitis virus (IBV) designated HH06 was isolated from the kidney tissues of a chicken flock experiencing an outbreak of nephritis. In vivo pathogenicity of the IBV isolate HH06 was determined by inoculating specific pathogen-free (SPF) chickens. The clinical signs and related gross lesions of HH06 infected chickens were similar with those of the field-infected chickens. SPF embryonated eggs were inoculated with virus suspension for serial passage and their genomic RNA was extracted. RT-PCR technique was utilized to amplify the M gene sequence encoding membrane protein of IBV. Recombinant plasmid named T-vector-M was constructed via inserting the M gene into the TA cloning vector, pMD 18-T. The sequenced M gene and its deduced amino acid (aa) sequences were compared with the published sequences of reference strains. The M gene is of 687 bp in length encoding the M protein of 228 amino acids with a predicted molecular weight of 25.4 kDa. The sequences of the M gene and M protein share 83.9–97.9% and 83.6–96.5% homologous identities, respectively, compared with 29 IBV reference strains derived from different regions or countries, which revealed that there are still significant variations between strains. Furthermore, a phylogenetic tree based on these M DNA sequences was generated, and the tree topology suggests that some Chinese IBV strains may have a common ancestor; however, HH06 is a new local IBV isolate that is responsible for the field outbreak of nephritis.
Avian infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis (IB) and leads to a great economic loss to the poultry industry worldwide. IB has been reported frequently in China since it first appeared in 1965 [
IBV is a member of the family
The M protein, a polytopic protein, is the most abundant component of coronavirions [
Recently, we isolated an IBV strain (HH06) from northeastern China and cloned its S1 gene, and found this virus has over 98.8% S1 identity with 19 other Chinese IBV isolates. However, the IBV S1 region displays very distinctive difference with the vaccine strain H120 that is used for vaccination in northeastern China. There are several papers describing the epidemic situation regarding IBV in China, based on the S1 gene sequences [
An avian IBV was isolated from the suburb of Harbin, capital of Heilongjiang Province, P.R. China. The IBV designated HH06 was isolated from the kidney tissues of suspected young chicken flock experiencing an outbreak of nephritis, depression, and slight respiratory signs. The virus suspension was prepared from homogenized kidney tissue after freezing and thawing three times. Nine-day-old SPF embryonated eggs were inoculated with the virus suspension (0.3 ml/embryo) containing 100 U penicillin and 100 mg streptomycin/ml via the allantoic cavity and then were incubated at 37°C for 72 h. The harvested allantoic fluid was cleared by centrifugation at 3,000 rpm at 4°C for 15 min and used as inoculum for serial passage (0.1 ml/embryo) up to eight times. Egg lethal dose (ELD)50 of the 8th passage viruses was detected using the embryonated eggs.
For in vivo pathogenicity analysis, 20-day-old SPF chickens were randomly divided into two groups with 15 birds in the experimental group and 5 birds in the control group. Each bird in the experimental group was orally inoculated with 0.5 ml of the allantoic fluid containing the isolate HH06 (105.7 ELD50/ml), and the birds in the control group were orally inoculated with 0.5 ml sterilized physiological saline. All chickens were reared in the isolaters. The infected birds were observed for clinical signs and disease course, and were later killed by intravenous inoculation of barbiturate for observation of gross lesions and sampling of kidney, trachea, and lung.
Viral RNA was extracted using TRIzol reagent (TaKaRa, Japan) according to the manufacturer’s instructions. A pair of primers was designed using Primer Premier 5.0 software, and synthesized by TaKaRa Biotechnology Company (Dalian, China) based on the sequence of Beaudette strain of IBV (GenBank accession No. AJ311362). The sense primer, pM1: 5′-TAACGAGTTTCCTAAGAACGGTTGG-3′, and the anti-sense primer, pM2: 5′-ACATTTATGTGTAAAGACTACTTCC-3′, flanking the M gene were used to amplify a 735 bp fragment.
RT-PCR was performed using an RT-PCR Kit (TaKaRa, Japan) according to the manufacturer’s instructions. Each reverse transcription volume had a total volume of 10 μl and used 1 μl total RNA (1 μg/μl) as template. The cDNA obtained was amplified with primers pM1 and pM2. PCR was in 50 μl volumes containing 5 μl of 10× buffer, 3 μl dNTP mixture (2.5 mM), 10 μl cDNA, 1 μl pM1, 1 μl pM2, lμl ExTaq polymerase (TaKaRa, Japan), and 29 μl sterile water. The cycling parameters for the PCR included 94°C for 1 min, 30 cycles at 94°C for 1 min, 49.9°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products were run in 1% agarose gel electrophoresis containing ethidium bromide (0.5 μg/ml) and visualized by subsequent UV transillumination.
PCR products of each RT-PCR were purified using a Gel Extraction Mini Kit (Omega, USA). Purified PCR products ligated with a TA cloning vector, pMD18-T (TaKaRa, Japan) were transformed into competent cells (strain JM109). Cells carrying recombinant plasmid were screened on Luria-bertani (LB) agar plates containing Ampicillin (50 μg/ml). Colony PCR was utilized to select recombinant bacteria. The PCR conditions were the same as that for the above-mentioned PCR amplification. Three positive clones were randomly selected and cultured. Plasmid DNA prepared by Plasmid Extraction Kit (Omega, USA) was verified with restriction enzyme digestion. The resulting plasmids were designated T-vector-M and sequenced by Sangon bio-company (Shanghai, China).
Sequences of T-vector-M and their deduced amino acids were compared with that of 29 IBV reference strains derived from different regions or countries using the Lasergene software package V5.0 (DNASTAR Inc, USA). The composition of the nucleotide sequence of M gene was analyzed with DNAMAN software version 4.0 (Lynnon BioSoft, Quebec, Canada). The IBV origin places and the GenBank accession numbers of these sequences are summarized in Table Information on IBV strains used in the experiment
Sequence number IBV isolate Virus origin Accession number 1 HH06 Heilongjiang, China EF397564 2 Jilin Jilin, China EF602442 3 CK/CH/LLN/98I Liaoning, China EF602451 4 HK China AY761141 5 LDT3 Guangdong, China AY702975 6 SAIB20 Sichuan, China AY302749 7 SAIBWJ Sichuan, China AY302742 8 SAIBK Sichuan, China DQ288927 9 CK/CH/LSC/99I Sichuan, China EF602450 10 H52-GD Guangdong, China AY044184 11 CK/CH/LGD/04II Guangdong, China EF602444 12 Partridge/GD/S14/2003 Guangdong, China AY646283 13 H120 Guangdong, China AY028295 14 N.A. Qingdao, China AF221667 15 TW1171/92 Taiwan, China DQ646406 16 TW2575/98 Taiwan, China DQ646405 17 Gray USA AF286180 18 SO4-8842 USA AY942744 19 K01-1699 USA AY942743 20 Connecticut USA AY942741 21 Ca199 USA AY514485 22 PA/Wolgemuth/98 USA AF363610 23 JMK USA AF363608 24 Conn USA AF942174 25 CU-T2 USA AIU46035 26 Mass41 USA AF363609 27 Beaudette US USA AJ311362 28 Alabama 1 USA AF470628 29 Beaudette CK UK AJ311317 30 Vic Australia DQ490221
Ten-day-old SPF embryonated eggs were inoculated with virus suspension for serial passage. The gross lesions of the infected embryos were hemorrhage on the legs, mottled necrosis of the liver, swelling of the kidneys, dwarfing, and curling. In the eighth passage, egg lethal dose (ELD)50 of the virus isolate was 105.7 ELD50/ml (Table Determination of ELD50Virus dilution Observed result Cumulative result Mortality rate (%) Death Survival Death Survival 10−3
5 0 16 0 100 10−4
4 1 11 1 92 10−5
4 1 7 2 78 10−6
2 3 3 5 38 10−7
1 4 1 9 10 10−8
0 5 0 14 0
Twenty-day-old chickens inoculated with the 8th passage viruses exhibited clinical symptoms including depress, cough, sneeze, dyspnea, and water-like feces 5 days post-inoculation. Two chickens died 7 days post-inoculation (PI) and another two chickens died 9 days PI. Then more chickens died continuously, with a mortality rate of 60%. Necropsy showed that the kidney of the dead birds was swollen and exhibited severe urate deposition. In addition, there were large volumes of mucilage in the bronchi of the dead chickens, and their throats appeared slightly hyperemic. The control chickens did not show any clinical symptoms.
The integrity of the RNA extracted was confirmed by denaturing agarose gel electrophoresis, and the total RNA concentration was 1 μg/μl approximately. Using the specific primers for the M gene of IBV, RT-PCR products of approximate 750 bp were successfully amplified (data not shown). The amplicons were cloned into pMD18-T vector, and three positive clones identified by colony PCR were subjected to plasmid extraction and DNA sequencing.
Sequencing results showed that the nucleotide sequences of the three M genes of IBV strain HH06 had no mutation. The consensus sequence was reported to GenBank Database and was assigned an accession number, EF397564. Sequence analysis indicated that the nucleotide sequence of the entire M gene of IBV strain HH06 was 687 pair of bases in length and had a base composition of 196 adenine (29%), 123 cytosine (18%), 151 guanine (22%), and 217 thymine (32%), and a GC content of 40%. Moreover, there were several TATA motifs (possible introns) in the M gene sequence. The M gene sequence is accessible through the website link: Homologous identity percentage at the molecular and the amino acid levels between the IBV isolations
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 N 100 89.4 88.1 89.3 97.2 92.2 89.2 97.9 96.0 89.5 94.8 97.2 89.2 89.1 89.4 90.3 89.6 89.2 89.4 89.4 89.0 89.6 89.9 89.4 83.9 89.7 89.7 89.7 90.0 87.0 A 100 92.9 90.3 92.4 95.6 94.2 92.9 96.5 95.6 92.9 95.6 95.6 92.9 91.2 92.0 92.5 93.8 93.4 91.6 92.9 92.5 92.9 93.8 92.9 83.6 93.4 91.2 92.9 92.0 90.6
To understand the phylogeny of IBV strains, a phylogenetic tree based on the nucleotide sequences of the M genes was generated by the MEGALIGN program in DNAStar with the Jotun Hein method [ Phylogenetic relationships between the IBV isolates are constructed on the nucleotide sequences of the M gene using the MEGALIGN program in DNAStar with the Jotun Hein method (Higgins and Sharp 1988 [
The SPF chickens inoculated with isolate HH06 exhibited very similar clinical signs and disease course with those from field infected birds, giving the evidence that HH06 was a local epidemic IBV isolate. In addition, the SPF embryonated eggs infected with HH06 virus showed the gross lesions such as hemorrhage on the legs, mottled necrosis of the liver, swelling of the kidneys, and dwarf which were similar with those of the infected with nephropathogenic IBVs [
Nephropathogenic IBV usually damages kidneys and reproductive tract of chicken, causing a high mortality rate in young flocks. Currently the major control measures of IB are vaccination of live attenuated IBV vaccine (Mass serotypes) such as H52, H120, and Ma5 strains in China; however, the chicken flocks vaccinated with the live vaccines usually fail to present full protection to field virulent IBV challenge, and the vaccinated chicken flocks were often subjected to the attack of nephropathogenic IBV. [
Genotyping on the basis of the S1 gene sequence is the most common way to classify IBV isolates. There have been several papers describing the epidemic situation regarding IBV in China [
The M protein of coronaviruses is a structural membrane protein and plays an important role in the viral assembly process. Here, we have cloned the full-length M gene of the Chinese IBV isolate HH06 and determined its nucleotide sequence. Sequence comparison with other reference strains, which was revealed that the M gene was conserved among various IBV strains inferred from their high sequence similarity, and can be used as a target to develop a nucleic acid-based test for the diagnosis of IBV infection. Many aspects of IBV have been extensively studied [
There is evidence that IBV recombination occurs in the envelope and membrane genes [
To further elucidate the molecular evolution of IBV, a phylogenetic tree was constructed according to the M gene sequences. In agreement with the sequence comparison, the tree topology showed the IBV isolates have been divided into several branches, and most Chinese isolates including HH06 isolate were closely clustered in the phylogenetic tree. Most American IBV isolates were also located in neighbored branches. Interestingly, many Chinese IBV isolates that were from different places shared the same branch. For example, SAIBK isolated from Sichuan Province, partridge/GD/S14/2003 and LDT3 isolated from Guangdong Province, and HH06 isolated in Northern China shared the same branch and displayed closer relationships. This result, together with the sequence comparison, allows us to hypothesize that some Chinese IBV isolates may have a common ancestor, although they were isolated from the different regions in mainland China. Particularly, it should be noted that the IBV LDT3 is a peafowl isolate [
Our results also highlight the importance of developing in time new IBV vaccines against the IBV variants. However, more data and necessary experiments are required to verify our hypothesis and to delineate why the Chinese isolates (HK and Jilin) locate in other different branches in the phylogenetic tree, respectively. In addition, to really understand the relationships among the IBV isolates, more extensive sequencing of the whole genome, the regional vaccination background, and the epidemiology should be investigated in the future.
We thank the anonymous reviewers and the editors of this journal for their critical review on the paper. The authors’ research works were supported by funds from Heilongjiang Provincial Science and Technology Department (LC06C01) and Harbin Science and Technology Bureau (2006RFLXN004), National Natural Science Foundation of China (30700590; 30700591), Heilongjiang Provincial Education Department SARS Project (2003fz012), Ministry of Education of China (NO706019) and Program for Innovative Research Team of Northeast Agricultural University (CXZ008-1).