Emergence of a Nephropathogenic Avian Infectious B
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微生物临床杂志 2005年第2期
Poultry Diagnostic and Research Center, Division of Venkateshwara Hatcheries Limited, Loni-Kalbhor, Pune, India
Department of Animal and Food Sciences, University of Delaware, Newark, Delaware
The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, United Kingdom
ABSTRACT
We describe the emergence of a nephropathogenic avian infectious bronchitis virus (IBV) with a novel genotype in India. The Indian IBV isolate exhibited a relatively high degree of sequence divergence with reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).
TEXT
Infectious bronchitis virus (IBV) is prevalent in all countries with an intensive poultry industry, with the incidence of infection approaching 100% in most locations. The virus is belong to group 3 coronavirus (4). IBV has always been something of a moving target for many reasons, such as wide variations in the serotypes and virulence of strains that have developed from time to time, a highly contagious nature, and the evolution of specific tissue tropism and recombinants due to simultaneous infection of multiple virus types and use of live vaccines. Vaccination is only partially successful due to continual emergence of antigenic variants and requires the application of multiple vaccines at many sites due to the simultaneous presence of multiple antigenic types. Although many countries share some common antigenic types, IBV strains within a geographic region are unique and distinct; examples of this include Europe, the United States, and Australia (1, 2, 6-9, 21).
IBV genome consists of ca. 27 kb and codes for three structural proteins: the spike (S) glycoprotein, the membrane (M) glycoprotein, and the nucleocapsid (N) phosphoprotein. The S glycoprotein is composed of two glycopolypeptides: S1 and S2 (3). Neutralizing and serotype-specific antibodies are directed against the S1 glycoprotein, and the greatest divergence in the amino acid sequence is concentrated between residues 53 and 148 of S1 (17).
Until recently, the Indian subcontinent was free of variant forms of IBV. The most prevalent form of IB was only the respiratory form related to the Massachusetts strain (13, 15). However, genotyping and phylogenic analysis of Indian isolates were previously not been done. We investigated outbreaks of visceral gout and nephrosis in commercial broiler chicks in the western parts of India for evidence of IBV. The disease was reported mostly in 1- to 2-week-old broiler chicks in unvaccinated flocks. The clinical signs observed are typical of IB: gasping, upward respiration, and tracheal rales. Grossly, the birds presented with distended ureters filled with uric acid and visceral gout. Kidney lesions were principally those of an interstitial nephritis: granular degeneration, vacuolation, and desquamation of tubular epithelium.
The IBV was isolated by intra-allantoic passage of clinical material (kidneys) from affected chicks. After the third passage in specific-pathogen-free embryonated eggs, we observed lesions in the specific-pathogen-free embryos: mortality of embryos, stunting, curling, and uric acid deposition in the kidneys and ureter. The allantoic fluid of inoculated eggs were found to be negative for Newcastle disease virus and avian influenza virus by spot hemagglutination assay. The presence of coronavirus-like particles in allantoic fluid was confirmed by electron microscopic examination of infectious allantoic fluid.
Direct automated cycle sequencing of a reverse transcription-PCR product of the S-1 subunit of the spike peplomer gene was used to identify IBV genome with degenerate primers CK4 and CK2 (10, 11). The S-1 subunit nucleotide sequences generated by direct automated cycle sequencing were aligned and analyzed with commercial software to determine their relationship to the S-1 nucleotide sequences of IBV strains on deposit in the GenBank and EMBL databases. The sequence reported here has been submitted to the GenBank nucleotide database and has the accession number AY091551.
Indian IBV isolate PDRC/Pune/Ind/1/00 was found to have a unique S-1 sequence compared to selected reference strains from different countries and continents (Fig. 1). The reference IBV strains included in the analysis were from the United States: Massachusetts, Arkansas, Connecticut, Delaware O72, JMK, PA/Wolgemuth/98, MD/106/00, CV-56B, and SE-17. The European strains included were UK/7/93, 6/82, D207, H120, B1648, and D1466. The Mexican IBV strains included were Mex/7483/98 and Mex/1765/99. The Australian strains were V5/90, Vic S, N1/62, and Q3/88. The sequence analysis of the S1 gene demonstrates that Indian isolate PDRC/Pune/Ind/1/00 possesses a unique genotype compared to other reference strains of various countries and is unrelated to North American, European, and Australian strains (Fig. 2). The Indian isolate exhibited <40% similarity in S1 protein sequence to strains D1466, Mex/1765/99, and DE/072/92 but shared 53 to 68% relatedness in S1 protein sequence with rest of the reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).
Our data, together with those of previous publications, document several outbreaks of emerging IBV virus infection in the regions where intense poultry farming is practiced (2, 6-9, 20, 21). Because widespread vaccination has been conducted to control IBV, this immune selection pressure, together with the high mutation rate of the genome, may explain the existence of many serotypes and variants. The differences of as little as 5% between S1 sequences of IBV can result in poor cross-protection offered by currently used vaccines (4). Several variants, as well as isolates, related to European or American strains have previously been reported in China and other parts of Asia, with which India either shares political borders or trade exchange (14, 16, 18, 19). Thus, the Indian isolate PDRC/Pune/Ind/1/00 could have emerged from recombination of these variants with the local circulating virus or vaccine strains. Although not confirmed by virus isolation and sequencing of genome, the presence of European 793/B related virus in India has been reported by serological methods (5). The measures to restrict the introduction of exotic IBV strains should therefore be considered since the disease has a significant economic impact; in broilers, these production losses are due to poor weight gains, condemnation at processing, and mortality, whereas in laying birds losses are due to suboptimal egg production and downgrading of eggs. Although previous vaccines in India were mainly imported from the United States, several vaccines from Europe were also used in the past. In view of the emergence of novel variants due to recombination during mixed infection of IBV (12), the policy of importing live attenuated IBV vaccines for domestic vaccination purposes in India also needs to be redefined.
ACKNOWLEDGMENTS
We thank the chairperson of the Venkateshwara Hatcheries group of companies for providing necessary facilities to carry out this study. We thank S. G. Deshmukh for helpful discussion, A. Basu for electron microscopic examination of the virus, and the staff of Poultry Diagnostic and Research Center for technical support.
REFERENCES
Adzhar, A., R. E. Gough, D. Haydon, K. Shaw, P. Britton, and D. Cavanagh. 1997. Molecular analysis of the 793/B serotype infectious bronchitis virus in Great Britain. Avian Pathol. 126:625-640.
Callison, S. A., M. W. Jackwood, and D. A. Hilt. 2001. Molecular characterization of infectious bronchitis virus isolates foreign to the United States and comparison with United States isolates. Avian Dis. 45:492-499.
Cavanagh, D. 1983. Coronavirus IBV: structural characterization of the spike protein. J. Gen. Virol. 64:2577-2583.
Cavanagh, D. 2003. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus. Avian Pathol. 32:567-582.
Elankumaran, S., C. Balachandran, N. D. Chandran, P. Roy, A. Albert, and R. Manickam. 1999. Serological evidence for a 793/B related avian infectious bronchitis virus in India. Vet. Rec. 144:299-300.
Gelb, J., Jr., B. S. Ladman, M. Tamayo, M. Gonzalez, and V. Sivanandan. 2001. Novel infectious bronchitis virus S1 genotypes in Mexico 1998-1999. Avian Dis. 45:1060-1063.
Gelb, J., Jr., J. B. Wolff, and C. A. Moran. 1991. Variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens. Avian Dis. 35:82-87.
Gough, R. E., W. J. Cox, E. Gutierrez, G. MacKenzie, A. M. Wood, and M. D. Dagless. 1996. Isolation of "variant" strains of infectious bronchitis virus from vaccinated chickens in Great Britain. Vet. Rec. 139:552.
Ignjatovic, J., D. F. Ashton, R. Reece, P. Scott, and P. Hooper. 2002. Pathogenicity of Australian strains of avian infectious bronchitis virus. J. Comp. Pathol. 126:115-123.
Jackwood, M. W., N. M. Yousef, and D. A. Hilt. 1997. Further development and use of a molecular serotype identification test for infectious bronchitis virus. Avian Dis. 41:105-110.
Kingham, B. F., C. L. Keeler, Jr., W. A. Nix, B. S. Ladman, and J. Gelb, Jr. 2000. Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Dis. 44:325-335.
Lee, C. W., and M. W. Jackwood. 2000. Evidence of genetic diversity generated by recombination among avian coronavirus IBV. Arch. Virol. 145:2135-2148.
Pradhan, H. K., G. C. Mohanty, and B. S. Rajya. 1982. Reproductive tract pathology of chickens following natural exposure to infectious bronchitis virus (IBV). Ind. J. Poultry Sci. 17:107-116.
Seyfi Abad Shapouri, M. R., M. Mayahi, K. Assasi, and S. Charkhkar. 2004. A survey of the prevalence of infectious bronchitis virus type 4/91 in Iran. Acta Vet. Hung. 52:163-166.
Sharma, K., S. N. Sharma, D. Sambyal, and K. K. Baxi. 1984. Isolation and characterization of some avian viruses from ovaries of domestic fowl. Ind. J. Anim. Sci. 54:977-979.
Wang, C. H., M. C. Hsieh, and P. C. Chang. 1996. Isolation, pathogenicity, and H120 protection efficacy of infectious bronchitis viruses isolated in Taiwan. Avian Dis. 40:620-625.
Wang, L., D. Junker, L. Hock, E. Ebiary, and E. W. Collisson. 1994. Evolutionary implications of genetic variations in the S1 gene of infectious bronchitis virus. Virus Res. 34:327-338.
Yu, L., Y. Jiang, S. Low, Z. Wang, S. J. Nam, W. Liu, and J. Kwangac. 2001. Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens. Avian Dis. 45:416-424.
Yu, L., Z. Wang, Y. Jiang, S. Low, and J. Kwang. 2001. Molecular epidemiology of infectious bronchitis virus isolates from China and Southeast Asia. Avian Dis. 45:201-209.
Ziegler, A. F., B. S. Ladman, P. A. Dunn, A. Schneider, S. Davison, P. G. Miller, H. Lu, D. Weinstock, M. Salem, R. J. Eckroade, and J. Gelb, Jr. 2002. Nephropathogenic infectious bronchitis in Pennsylvania chickens 1997-2000. Avian Dis. 46:847-858.
Zwaagstra, K. A., B. A. van der Zeijst, and J. G. Kusters. 1992. Rapid detection and identification of avian infectious bronchitis virus. J. Clin. Microbiol. 30:79-84.(Jagadeesh Bayry, Mallikar)
Department of Animal and Food Sciences, University of Delaware, Newark, Delaware
The Edward Jenner Institute for Vaccine Research, Compton, Berkshire, United Kingdom
ABSTRACT
We describe the emergence of a nephropathogenic avian infectious bronchitis virus (IBV) with a novel genotype in India. The Indian IBV isolate exhibited a relatively high degree of sequence divergence with reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).
TEXT
Infectious bronchitis virus (IBV) is prevalent in all countries with an intensive poultry industry, with the incidence of infection approaching 100% in most locations. The virus is belong to group 3 coronavirus (4). IBV has always been something of a moving target for many reasons, such as wide variations in the serotypes and virulence of strains that have developed from time to time, a highly contagious nature, and the evolution of specific tissue tropism and recombinants due to simultaneous infection of multiple virus types and use of live vaccines. Vaccination is only partially successful due to continual emergence of antigenic variants and requires the application of multiple vaccines at many sites due to the simultaneous presence of multiple antigenic types. Although many countries share some common antigenic types, IBV strains within a geographic region are unique and distinct; examples of this include Europe, the United States, and Australia (1, 2, 6-9, 21).
IBV genome consists of ca. 27 kb and codes for three structural proteins: the spike (S) glycoprotein, the membrane (M) glycoprotein, and the nucleocapsid (N) phosphoprotein. The S glycoprotein is composed of two glycopolypeptides: S1 and S2 (3). Neutralizing and serotype-specific antibodies are directed against the S1 glycoprotein, and the greatest divergence in the amino acid sequence is concentrated between residues 53 and 148 of S1 (17).
Until recently, the Indian subcontinent was free of variant forms of IBV. The most prevalent form of IB was only the respiratory form related to the Massachusetts strain (13, 15). However, genotyping and phylogenic analysis of Indian isolates were previously not been done. We investigated outbreaks of visceral gout and nephrosis in commercial broiler chicks in the western parts of India for evidence of IBV. The disease was reported mostly in 1- to 2-week-old broiler chicks in unvaccinated flocks. The clinical signs observed are typical of IB: gasping, upward respiration, and tracheal rales. Grossly, the birds presented with distended ureters filled with uric acid and visceral gout. Kidney lesions were principally those of an interstitial nephritis: granular degeneration, vacuolation, and desquamation of tubular epithelium.
The IBV was isolated by intra-allantoic passage of clinical material (kidneys) from affected chicks. After the third passage in specific-pathogen-free embryonated eggs, we observed lesions in the specific-pathogen-free embryos: mortality of embryos, stunting, curling, and uric acid deposition in the kidneys and ureter. The allantoic fluid of inoculated eggs were found to be negative for Newcastle disease virus and avian influenza virus by spot hemagglutination assay. The presence of coronavirus-like particles in allantoic fluid was confirmed by electron microscopic examination of infectious allantoic fluid.
Direct automated cycle sequencing of a reverse transcription-PCR product of the S-1 subunit of the spike peplomer gene was used to identify IBV genome with degenerate primers CK4 and CK2 (10, 11). The S-1 subunit nucleotide sequences generated by direct automated cycle sequencing were aligned and analyzed with commercial software to determine their relationship to the S-1 nucleotide sequences of IBV strains on deposit in the GenBank and EMBL databases. The sequence reported here has been submitted to the GenBank nucleotide database and has the accession number AY091551.
Indian IBV isolate PDRC/Pune/Ind/1/00 was found to have a unique S-1 sequence compared to selected reference strains from different countries and continents (Fig. 1). The reference IBV strains included in the analysis were from the United States: Massachusetts, Arkansas, Connecticut, Delaware O72, JMK, PA/Wolgemuth/98, MD/106/00, CV-56B, and SE-17. The European strains included were UK/7/93, 6/82, D207, H120, B1648, and D1466. The Mexican IBV strains included were Mex/7483/98 and Mex/1765/99. The Australian strains were V5/90, Vic S, N1/62, and Q3/88. The sequence analysis of the S1 gene demonstrates that Indian isolate PDRC/Pune/Ind/1/00 possesses a unique genotype compared to other reference strains of various countries and is unrelated to North American, European, and Australian strains (Fig. 2). The Indian isolate exhibited <40% similarity in S1 protein sequence to strains D1466, Mex/1765/99, and DE/072/92 but shared 53 to 68% relatedness in S1 protein sequence with rest of the reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).
Our data, together with those of previous publications, document several outbreaks of emerging IBV virus infection in the regions where intense poultry farming is practiced (2, 6-9, 20, 21). Because widespread vaccination has been conducted to control IBV, this immune selection pressure, together with the high mutation rate of the genome, may explain the existence of many serotypes and variants. The differences of as little as 5% between S1 sequences of IBV can result in poor cross-protection offered by currently used vaccines (4). Several variants, as well as isolates, related to European or American strains have previously been reported in China and other parts of Asia, with which India either shares political borders or trade exchange (14, 16, 18, 19). Thus, the Indian isolate PDRC/Pune/Ind/1/00 could have emerged from recombination of these variants with the local circulating virus or vaccine strains. Although not confirmed by virus isolation and sequencing of genome, the presence of European 793/B related virus in India has been reported by serological methods (5). The measures to restrict the introduction of exotic IBV strains should therefore be considered since the disease has a significant economic impact; in broilers, these production losses are due to poor weight gains, condemnation at processing, and mortality, whereas in laying birds losses are due to suboptimal egg production and downgrading of eggs. Although previous vaccines in India were mainly imported from the United States, several vaccines from Europe were also used in the past. In view of the emergence of novel variants due to recombination during mixed infection of IBV (12), the policy of importing live attenuated IBV vaccines for domestic vaccination purposes in India also needs to be redefined.
ACKNOWLEDGMENTS
We thank the chairperson of the Venkateshwara Hatcheries group of companies for providing necessary facilities to carry out this study. We thank S. G. Deshmukh for helpful discussion, A. Basu for electron microscopic examination of the virus, and the staff of Poultry Diagnostic and Research Center for technical support.
REFERENCES
Adzhar, A., R. E. Gough, D. Haydon, K. Shaw, P. Britton, and D. Cavanagh. 1997. Molecular analysis of the 793/B serotype infectious bronchitis virus in Great Britain. Avian Pathol. 126:625-640.
Callison, S. A., M. W. Jackwood, and D. A. Hilt. 2001. Molecular characterization of infectious bronchitis virus isolates foreign to the United States and comparison with United States isolates. Avian Dis. 45:492-499.
Cavanagh, D. 1983. Coronavirus IBV: structural characterization of the spike protein. J. Gen. Virol. 64:2577-2583.
Cavanagh, D. 2003. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus. Avian Pathol. 32:567-582.
Elankumaran, S., C. Balachandran, N. D. Chandran, P. Roy, A. Albert, and R. Manickam. 1999. Serological evidence for a 793/B related avian infectious bronchitis virus in India. Vet. Rec. 144:299-300.
Gelb, J., Jr., B. S. Ladman, M. Tamayo, M. Gonzalez, and V. Sivanandan. 2001. Novel infectious bronchitis virus S1 genotypes in Mexico 1998-1999. Avian Dis. 45:1060-1063.
Gelb, J., Jr., J. B. Wolff, and C. A. Moran. 1991. Variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens. Avian Dis. 35:82-87.
Gough, R. E., W. J. Cox, E. Gutierrez, G. MacKenzie, A. M. Wood, and M. D. Dagless. 1996. Isolation of "variant" strains of infectious bronchitis virus from vaccinated chickens in Great Britain. Vet. Rec. 139:552.
Ignjatovic, J., D. F. Ashton, R. Reece, P. Scott, and P. Hooper. 2002. Pathogenicity of Australian strains of avian infectious bronchitis virus. J. Comp. Pathol. 126:115-123.
Jackwood, M. W., N. M. Yousef, and D. A. Hilt. 1997. Further development and use of a molecular serotype identification test for infectious bronchitis virus. Avian Dis. 41:105-110.
Kingham, B. F., C. L. Keeler, Jr., W. A. Nix, B. S. Ladman, and J. Gelb, Jr. 2000. Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene. Avian Dis. 44:325-335.
Lee, C. W., and M. W. Jackwood. 2000. Evidence of genetic diversity generated by recombination among avian coronavirus IBV. Arch. Virol. 145:2135-2148.
Pradhan, H. K., G. C. Mohanty, and B. S. Rajya. 1982. Reproductive tract pathology of chickens following natural exposure to infectious bronchitis virus (IBV). Ind. J. Poultry Sci. 17:107-116.
Seyfi Abad Shapouri, M. R., M. Mayahi, K. Assasi, and S. Charkhkar. 2004. A survey of the prevalence of infectious bronchitis virus type 4/91 in Iran. Acta Vet. Hung. 52:163-166.
Sharma, K., S. N. Sharma, D. Sambyal, and K. K. Baxi. 1984. Isolation and characterization of some avian viruses from ovaries of domestic fowl. Ind. J. Anim. Sci. 54:977-979.
Wang, C. H., M. C. Hsieh, and P. C. Chang. 1996. Isolation, pathogenicity, and H120 protection efficacy of infectious bronchitis viruses isolated in Taiwan. Avian Dis. 40:620-625.
Wang, L., D. Junker, L. Hock, E. Ebiary, and E. W. Collisson. 1994. Evolutionary implications of genetic variations in the S1 gene of infectious bronchitis virus. Virus Res. 34:327-338.
Yu, L., Y. Jiang, S. Low, Z. Wang, S. J. Nam, W. Liu, and J. Kwangac. 2001. Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens. Avian Dis. 45:416-424.
Yu, L., Z. Wang, Y. Jiang, S. Low, and J. Kwang. 2001. Molecular epidemiology of infectious bronchitis virus isolates from China and Southeast Asia. Avian Dis. 45:201-209.
Ziegler, A. F., B. S. Ladman, P. A. Dunn, A. Schneider, S. Davison, P. G. Miller, H. Lu, D. Weinstock, M. Salem, R. J. Eckroade, and J. Gelb, Jr. 2002. Nephropathogenic infectious bronchitis in Pennsylvania chickens 1997-2000. Avian Dis. 46:847-858.
Zwaagstra, K. A., B. A. van der Zeijst, and J. G. Kusters. 1992. Rapid detection and identification of avian infectious bronchitis virus. J. Clin. Microbiol. 30:79-84.(Jagadeesh Bayry, Mallikar)