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. 2010 Oct 19;33(3):321–327. doi: 10.1080/0307945042000220697

A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and non-vaccinated flocks in China

Shengwang Liu 1, Xiangang Kong 1,*
PMCID: PMC7154297  PMID: 15223561

Abstract

Five strains of infectious bronchitis virus (IBV) were isolated from five layer flocks that had nephropathogenic infection in four provinces in China. Among them, three of the five flocks had been vaccinated against infectious bronchitis. Virulence studies indicated that the five Chinese IBV isolates caused 10 to 30% mortality in 15-day-old specific pathogen free chickens and gross lesions were mainly confined to the kidneys in all of the dead chickens. Two oligonucleotide pairs, S1Uni2 and S1Oligo3′ or S1Oligo5′ and S1Oligo3′, were used after propagation of the isolates in embryonated eggs to amplify the S1 protein genes of the spike protein. The cDNA derived by reverse transcriptase-polymerase chain reaction was cloned and sequenced. The nucleotide and amino acid sequence of S1 protein gene had a similar degree of identity (≥92%) among the five Chinese IBV isolates. The nucleotide and amino acid identity of the S1 protein gene between the five Chinese IBV isolates and 16 strains of other IBVs varied from 60 to 81%. This clearly showed that the five Chinese IBV isolates comprised a separate genotype. These results demonstrated, for the first time, that there is a new genotype of nephropathogenic IBV circulating in vaccinated and non-vaccinated flocks in China.

Introduction

Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is an acute and highly contagious disease in chickens. The disease is characterized by respiratory signs including gasping, coughing, sneezing, tracheal rales, and nasal discharge. In young chickens severe respiratory distress may occur, while in layers respiratory distress, decrease in egg production, and loss of internal and shell quality of eggs are reported. Some strains of the virus cause severe kidney damage, urolithiasis and may be associated with high mortality. IB is a major health problem affecting the chicken industry in most countries of the world. Through the use of attenuated live as well as inactivated virus vaccines, economic losses due to this disease have been significantly reduced. However, IBV variants may continue to circulate among vaccinated and non-vaccinated flocks and cause severe economic problems (reviewed by Cavanagh & Naqi, 2003).

IBV is the prototype virus of the genus Coronavirus, family Coronaviridae (Cavanagh, 1997). The genome of IBV contains an enveloped, single-stranded, positive-sense RNA of 27.6 kb. The virion has three major virus-encoded structural proteins, namely the spike (S) glycoprotein, the membrane (M) protein, and the nucleocapsid (N) protein. The spikes of IBV are formed by post-translational cleavage into two polypeptide components, designated S1 and S2. The molecular identification of IBV is based mainly on analysis of the S1 protein gene.

In China, as in other countries, IB has occurred frequently in vaccinated and non-vaccinated flocks and has caused severe economic losses in recent years. Vaccines based on Massachusetts strains such as H120 and H52 and other strains such as 4-91 (Nobilis IB 4-91) have been used for many years on poultry farms (Farsang et al., 2002; Gough et al., 2002). However, nephropathogenic IBV strains related to the Massachusetts type have been isolated in China in recent years (Wang et al., 1997; Wu et al., 1998; Li & Yang, 2001). Furthermore, other IBV strains that had partial or no relationship to Massachusetts type in antigenic and immunogenic characterization have also been isolated in China (Wang et al., 1997; Wu et al., 1998; Yu et al., 2001).

In order to investigate whether there are other genotype(s) of nephropathogenic IBV besides the Massachusetts type in flocks in China, we tested five IBV isolates from layer flocks showing clinical signs of IB by sequencing and analysis of the S1 protein genes. This allowed us to evaluate the prevalence of nephropathogenic IB type(s) in vaccinated and non-vaccinated chickens in recent years in China.

Materials and Methods

Viruses

Tissue samples of kidney were collected from layers showing clinical signs suspected to be related to IB. All flocks investigated in this study contained at least 10,000 layers. Three samples were taken from chickens vaccinated with H120 Massachusetts-type vaccine (Nobilis IB H120) in Xinjiang, Shandong and Heilongjiang provinces, China (Figure 1). The other two samples were from non-vaccinated flocks in Gansu and Heilongjiang provinces, China (Table 1). Obvious nephropathogenic lesions were found in all the diseased layers.

Figure 1. Approximate location of provinces and capitals (★) from where the IBV strains were isolated.

Figure 1.

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Table 1. IBV strains isolated from flocks in different provinces of China.

Strain isolate Province (capital)a Yearb Vaccinated Nephropathogenic lesions Chicken embryo passagec
LX4 Xinjiang (Ulumuqi) 1999 Yes Yes 4
LS2 Gansu (Lanzhou) 2002 No Yes 2
LD3 Heilongjiang (Haerbin) 2001 No Yes 3
LHI10 Shandong (Jinan) 2003 Yes Yes 10
LH2 Heilongjiang (Haerbin) 2001 Yes Yes 2
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a

Province (capital) where the viruses were isolated.

b

Year when viruses were isolated.

c

Different passages were performed until the dwarfing and death of embryos were observed between 2 and 7 days after inoculation.

For virus isolation, samples of kidney were pooled and 10% w/v tissue suspensions were made in 0.1% phosphate-buffered saline containing 100 u penicillin and 100 μg streptomycin/ml. After 12 h at 4°C, 200 μl supernatant from the suspensions was inoculated into the allantonic cavity of 9-day-old to 11-day-old embryos of specified pathogen free chickens (Haerbin Veterinary Research Institute, China). Five eggs were used for each sample. The inoculated eggs were incubated at 37°C and candled daily. Two eggs were killed after 72 h incubation and five other eggs were inoculated with the harvested allantoic fluids. Two to 10 blind passages were performed until the dwarfing and death of embryos were observed between 2 and 7 days after inoculation. All the allantoic fluids of inoculated eggs were harvested and tested for the presence of IBV using electron microscopy. The different passages of allantoic fluids containing IBV isolates were used in subsequent experiments (Table 1).

Electron microscopy

Samples of allantoic fluids after egg passages were submitted for electron microscopy. Briefly, after low-speed centrifugation at 1500 × g for 30 min (Allegra™ 21R centrifuge; Beckman), the supernatant of the 1.5 ml allantoic fluids were centrifuged at 12 000 × g for 30 min. The resulting pellet was resuspended in a minimal volume of deionized water and examined by negative contrast electron microscope (JEM-1200, EX).

Virulence studies in chickens

Six groups each of 10 White Leghorn SPF chickens (Haerbin Veterinary Research Institute, China) were kept in isolators with negative pressure. At 15 days of age, groups 1 to 5 were inoculated intranasally with the five isolates (log104.2 to log105.0 median embryo infectious doses, per chick; Table 2). The remaining group 6 was mock-inoculated with sterile allantoic fluid and served as a control. The chicks were examined daily for signs of infection for 30 days after inoculation.

Table 2. Mortality in 15-day-old specific pathogen free chickens inoculated with five isolates of IBV.

Group number Inoculated IBV isolate Chicken embryo passagea Dose, median embryo infectious doses (log10)b Dead chicks (%)c
1 LX4 4 4.2 1
2 LS2 2 4.5 3
3 LD3 3 4.5 2
4 LHI10 10 5.0 2
5 LH2 2 5.0 2
6d Control      
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a

Different passages used as in Table 1.

b

Dose per chick, 200 μl.

c

Ten chicks per group.

d

Inoculated with 200 μl sterile allantoic fluid per chick.

Extraction of RNA

Allantoic fluid (200 μl) containing virus (confirmed by electron microscopy) isolated from flocks of Xinjiang, Gansu, Heilongjiang or Shandong provinces in China, was mixed with 400 μl TRIzol Reagent (Gibco BRL) and RNA was isolated according to the description of the manufacturer. The RNA was air-dried for 2 to 10 min and re-dissolved in 25 μl Rnase-free water.

Generation of cDNA

The same general procedure was used for all the reverse transcriptase-polymerase chain reactions (RT-PCRs). S1Oligo3′ was used for the both of the RT reactions and, subsequently, the PCR with oligonucleotide S1Uni2 or S1Oligo5′ (Kwon et al., 1993; Adzhar et al., 1997). Lyophilized oligonucleotides obtained from the manufacturer were dissolved in RNase-free water at 0.1 μmol/μl to form the stock solutions.

Twenty-five microlitres of viral RNA was mixed with 50 ng S1Oligo3′ and incubated at 70°C for 10 min followed by 2 min on ice. After adding a reaction mixture consisting of 8 μl 5 × First Strand Buffer (250 mM Tris–HCl, 375 mM KCl, 15 mM MgCl2), 4 μl of 2.5 mM dNTPs (Gibco BRL), 200 u RnaseH– murine Moloney leukaemia virus RT (Gibco BRL), 40 u RNAsin (Gibco BRL), the mixture was incubated at 37°C for 2 h. The reaction was terminated by heating at 98°C for 7 min and chilling on ice.

PCR, cloning and sequencing of the S1 protein genes

For the PCR reaction, the following mix was made: 15 nmol oligonucleotide S1Oligo3′ and 15 nmol oligonucleotide S1Uni2 or S1Oligo5′; 1 μl cDNA; 5 μl of 10 × PCR buffer (Mg2+ Plus; TaKaRa, Japan); 4 μl of 2.5 mmol dNTPs; 2 u Taq polymerase (TaKaRa, Japan); and 34 μl water. The PCR reaction was performed using the following conditions: denaturation (94°C, 1 min), annealing (50°C, 1 min), and extension (72°C, 2 min), 35 cycles followed by a final extension step (72°C, 10 min).

A product, detectable by ethidium bromide staining, of about 1700 base pairs (bp) was generated with isolates LX4 and LH2 using S1Oligo3′ and S1Oligo5′, but no product was seen with isolates LHI10, LS2 and LD3 (Table 1). Therefore, the PCR was performed for LS2, LD3 and LHI10 using S1Oligo3′ and S1Uni2, to generate a similar size product of about 1700 bp. DNA generated by PCR amplification was cloned using a T-tailed vector, pMD18-T (TaKaRa), and transformed using JM109 competent cells (TaKaRa) according to the manufacturer's instructions. Three clones of each isolate were sequenced.

Sequence analysis of the S1 protein genes

The sequences of the S1 protein gene of the five Chinese IBV isolates were assembled, aligned and compared with 16 strains of other IBV with DNAMAN version 5.2.2 (http://www.lynnon.com/). The 16 strains of IBV and their GenBank accession numbers are: Beaudette (Boursnell et al., 1987; accession number NC_001451), M41 (Niesters et al., 1987; accession number A24863), KB8523 (Sutou et al., 1988; accession number M21515), B1648 (Shaw et al., 1996; accession number X87238), Connecticut (Wang et al., 1994; accession number L18990), Ark99 (Jia et al., 1995; accession number L10384), D274 (Jordi et al., 1989; accession number X15832), H120 (Kusters et al., 1989; accession number M21970 J04329), H52 (Zhou et al., 2001; accession number AF352315; unpublished work), 793/B (Adzhar et al., 1997; accession number Z83975), Georgia GA/2787/98 (Lee et al., 2001; accession number AF274438), A1211 (Wang et al., 1996; accession number AF250006), A1171 (Wang et al., 1996; accession number AF250005), Q1 (Yu et al., 2001; accession number AF286302), T3 (Yu et al., 2001; accession number AF227438), and J2 (Yu et al., 2001; accession number AF286303).

Accession numbers

The S1 protein gene sequences of the five Chinese IBV isolates have been submitted to the GenBank database and have been assigned the following accession numbers: LX4, AY189157; LH2, AY180958; LS2, AY278246; LD3, AY277632 and LHI10, AY273193.

Results

Detection of IBV

IB isolates suspected to be related to IB infection were analysed in this study. The isolates were collected from flocks in four different provinces in China, showing clinical signs of IB infection and with 10 to 30% mortality. The nephritis was observed in both the vaccinated and non-vaccinated flocks and was characterized by enlarged and pale kidneys, frequently with urate deposits in the tubules, severe dehydration and weight loss. Typical signs including dwarfing and death of embryo were observed in the different passages when each of the five Chinese isolates was inoculated into 9-day-old to 11-day-old chicken embryos (Table 1). Diagnoses based on electron microscopy examination performed on allantoic fluids of different passages showed all five isolates had typical coronavirus morphology and were free of other agents such as Newcastle disease virus (results not shown).

Virulence studies

Clinical signs were observed in all of the chicks of groups 1 to 5 about 3 to 10 days after inoculation. The chicks are listless and huddled together, showed ruffled feathers and a dark, shrunken comb. Some of the chicks died during the experiment (Table 2). Gross lesions of dead chicks were mainly confined to the kidneys. The kidney parenchyma of the dead birds was pale, swollen and mottled; tubules and urethras were distended with uric acid crystals. In addition, mild respiratory signs (sneezing, rales) were also observed. The clinical signs of the inoculated birds tended to disappear gradually by 20 days of inoculation.

Analysis of the S1 protein gene

The pair of oligonucleotides S1Oligo3′ and S1Oligo5′ was used to attempt the amplification of the S1 region of the spike gene from all five isolates. The RT-PCR amplified a ∼1700 bp cDNA from LX4 and LH2, but not from LS2, LD3 and LHI10. Therefore, the PCR was performed for LS2, LD3 and LHI10 using S1Oligo3′ and S1Uni2.

Comparison of the S1 protein gene sequences of the five Chinese IBV isolates with those of 16 strains of other IBVs revealed that nucleotide and amino acid identities among the five Chinese IBV isolates were between 92 and 99%, but the nucleotide identity of the five Chinese IBV strains and the 16 strains of other IBV were not more than 79% and amino acid identity was not more than 78% (Table 3).

Table 3. Nucleotide and amino acid similarities of the S1 protein genesa among the five Chinese IBV isolates and other IBV strains.

Strain LD3 LS2 LX4 LH2 LHI10 Q1 T3 J2 A1211 A1171 M41 H120 H52 UK/7/93 D274 B1648 KB8523 Beau Conn Ark99 GA/98
  Amino acid identity (%)
LD3   92 94 98 98 75 76 76 77 77 74 75 74 76 76 76 75 74 73 75 53
LS2 95   93 92 92 76 76 76 78 77 75 76 75 76 75 76 75 75 74 74 53
LX4 96 96   94 94 75 76 76 78 78 75 75 75 77 75 76 75 75 73 75 52
LH2 99 95 96   98 76 76 76 77 77 74 75 75 76 76 76 75 75 73 75 53
LHI10 99 95 96 99   76 76 76 78 77 75 75 74 76 76 76 75 75 72 75 53
Q1 76 76 76 76 76   98 99 77 77 74 74 73 75 80 76 74 73 73 76 53
T3 76 76 76 76 76 99   99 78 78 74 74 74 74 80 76 74 73 73 77 52
J2 76 76 76 76 76 99 99   77 78 82 82 82 75 80 76 75 73 73 77 52
A1211 78 78 79 78 78 77 77 77   92 82 82 82 77 77 75 82 82 79 78 53
A1171 78 78 79 78 78 77 77 77 95   82 82 82 76 77 75 81 81 79 78 53
M41 76 77 77 77 77 77 77 77 81 81   95 96 74 77 74 95 97 88 75 53
H120 76 77 77 77 77 76 76 76 81 81 97   99 74 78 74 99 96 94 76 53
H52 76 77 77 77 77 76 76 76 81 81 97 99   74 78 74 98 95 94 76 52
UK/7/93 78 78 79 78 78 78 78 78 77 77 78 78 78   78 78 75 74 73 76 48
D274 77 77 77 77 77 80 80 80 78 78 80 80 81 78   78 78 78 76 78 53
B1648 77 77 77 77 77 78 78 78 75 76 77 77 77 79 78   75 75 73 78 52
KB8523 76 77 77 77 77 76 76 76 81 81 97 99 99 78 81 77   95 87 76 53
Beau 76 77 77 76 76 76 76 76 81 81 98 97 97 78 81 78 97   86 76 53
Conn 74 74 74 74 73 74 74 74 78 78 91 91 90 75 76 74 90 90   73 51
Ark99 75 75 76 75 75 76 76 76 77 78 78 78 77 77 78 78 78 78 75   51
GA/98 60 60 60 60 60 61 61 61 61 62 62 62 62 60 61 61 62 62 61 61  
  Nucleotide identity (%)
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Top right, amino acid identity (%); bottom left, nucleotide identity (%).

a

The first 1605 nucleotides, starting at the AUG translation start codon, of the S1 protein genes were compared.

Discussion

For isolation of non-egg-adapted IBV field strains, several sequential passages can be given to increase the amounts of virus before performing subsequent experiments. The extent of changes to the infected embryo that are induced by IBV vary greatly. Especially for the field strains, the visible changes in the embryos in the first passage can be minimal. Usually, embryo mortality and dwarfing increase as the number of serial passages increases. Some field strains still caused no dwarfing on the third passage, while other methods could detect IBV using inoculated allantoic cells (reviewed by De Wit, 2000). Of the five IBV isolates in this study, they showed different adaptation to embryos. All the five IBV strains did not cause visible changes in the embryos in the first passage, and four of them caused dwarfing and death of the embryos on the second to fourth passages (Table 1). For one of the IBV isolates, LHI10, obvious changes in the inoculated embryos were observed in the 10th passage. This isolate may not grow well in chicken embryo and the virus titre may be minimal in the first several passages.

Winterfield & Hitchner (1962) first reported a nephrosis condition associated with IB in the United States. Cumming (1962) reported an IB outbreak causing severe kidney lesion in chickens in Australia in the same year. From this time, various nephropathogenic strains of IBV have been identified throughout the world (reviewed by Meulemans & van der Berg, 1998). In China, nephropathogenic IBV strains of the Massachusetts genotype have isolated in rural chicken farms of Beijing (Li & Yang, 2001). In the present study, we isolated three IBV strains from H120-vaccinated flocks and two from non-vaccinated flocks of layer chickens that experienced nephropathogenic infection in four provinces of north China. Virulence studies showed that severe kidney lesions were observed in the dead chicks that had been inoculated with each of the five Chinese IBV isolates. It was reported that nephropathogenic IB occurred in Pennsylvania from 1997 to 2000 in commercial broiler-type and layer-type chicken flocks, with mortality as high as 20% (Ziegler et al., 2002). Similar mortality was also found in this study by inoculating 15-day-old specific pathogen free chickens with each of the five Chinese IBV isolates (Table 2). Although the results of this experiment were not a true reflection of the situation in the flocks that the five IBV strains came from, they confirmed that the five IBV isolates are nephropathogenic.

The S1 protein genes of the five IBV isolates, LD3, LS2, LX4, LH2, and LHI10, isolated from 1999 to 2003 in China, shared 95 to 99% of nucleotide identity and 92 to 98% amino acid identity. This strikingly high identity implied a close genetic relationship and possibly indicated a common origin. Not more than 76% nucleotide and amino acid identity was shared between the five IBV isolates and three strains, Q1, T3, and J2, which were also isolated in China before 1999 (Yu et al., 2001). Isolates Q1, T3, and J2 were isolated from the proventricular tissues of infected chickens with a syndrome associated with lesions of alimentary and respiratory tracts. Unlike the IBV isolates in this study, nephropathogenic infections were not observed from chickens infected with those three IBV strains (Yu et al., 2001). In addition, nephropathogenic IBV strains that showed more than 99.1 and 97.8% nucleotide and amino acid identity with the S1 protein of standard IBV strain M41 were isolated in Beijing (Li & Yang, 2001). The S1 protein genes were also compared between the IBV strains isolated in this study and three Massachusetts-type IBV strains, M41, H52 and H120. The results showed that they had only 74 to 77% nucleotide and amino acid identity at the level of S1 protein gene and indicated that they belonged to different genotypes. On the basis of the genotype and pathogenicity, we can speculate that there were at least three different genotypes of IBV strains circulating in China.

The spreading of a virus from one area or country to another could be due, at least in part, to its improper introduction by the trading of birds or by the use of attenuated vaccines. With the exception of the Massachusetts strain, a very interesting aspect of IBV epidemiology, as far as it is possible to know, is the presence and the spreading of the various IBV serotypes in different continents. About 20 emergent serotypes in North America did not spread to other continents. Similarly, the European, Australian, and Asiatic serotypes apparently did not spread elsewhere. In this study, the S1 protein genes of the five Chinese isolates were compared with two IBV strains, A1211 and A1171, which were isolated in Taiwan; one Japanese isolate, KB8523; three European isolates, UK/7/93, D274, and B1648; four American isolates, GA/2787/98, Connecticut, Beaudette, and Ark99. These IBV strains shared only 48 to 79% nucleotide and amino acid identity with the S1 protein of the five Chinese isolates. Obviously, they belonged to different genotypes. However, we cannot conclude that other IBV types are not present in China because the research on the matter is rather poor and it would be worthwhile to conduct more studies.

Molecular studies have shown that a new serotype can emerge as a result of only a few changes in the amino acid composition in the S1 part of the virus spike protein, with the majority of the virus genome remaining unchanged (Cavanagh et al., 1992). This could be due to immunologic pressure caused by the widespread use of vaccines, to recombination as a consequence of mixed infections, or to the decrease of dominant serotypes as a result of vaccination, allowing other field strains to emerge. Further to this study, more epidemiological investigations are needed to better understand the origin, diffusion and persistence of the new genotype.

The sharing of antigens between the IBV isolates and vaccinal viruses might suggest that currently available vaccines should be able to provide protection against challenge from viruses belonging to different serotypes from the vaccine. The low nucleotide and amino acid similarities between the H120 Massachusetts vaccine and the new genotype viruses (Table 3) may account for the occurrence of the disease caused by the new genotype viruses in H120 vaccinated layer flocks.

Footnotes

Translations of the abstract in French, German and Spanish are available on the Avian Pathology website.

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