Research Note. Molecular characterization of chicken infectious anemia viruses detected from breeder and broiler chickens in South Korea

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1 Research Note Molecular characterization of chicken infectious anemia viruses detected from breeder and broiler chickens in South Korea H.-R. Kim,* 1 Y.-K. Kwon, Y.-C. Bae,* J.-K. Oem,* and O.-S. Lee * * Animal Disease Diagnosis Center, and Avian Disease Division, National Veterinary Research and Quarantine Service, 335 Joongangro, Manangu, Anyangsi, Gyeonggido , Korea ABSTRACT In South Korea, 32 sequences of chicken infectious anemia virus (CIAV) from various flocks of breeder and commercial chickens were genetically characterized for the first time. Phylogenetic analysis of the viral protein 1 gene, including a hypervariable region of the CIAV genome, indicated that Korean CIAV strains were separated into groups II, IIIa, and IIIb. Strains were commonly identified in great-grandparent and grandparent breeder farms as well as commercial chicken farms. In the field, CIAV strains from breeder farms had no clinical effects, but commercial farm strains were associated with depression, growth retardation, and anemia regardless of the group from which the strain originated. In addition, we identified 7 CIAV genomes that were similar to vaccine strains from vaccinated and unvaccinated breeder flocks. These data suggest that further studies on pathogenicity and vaccine efficacy against the different CIAV group are needed, along with continuous CIAV surveillance and genetic analysis at breeder farms. Key words: chicken infectious anemia, hypervariable region, viral protein 1, phylogenetic analysis 2010 Poultry Science 89 : doi: /ps INTRODUCTION Chicken infectious anemia virus (CIAV), the only member of the genus Gyrovirus in the family Circoviridae, is a small DNA virus with a circular, covalently linked, negative-sense single-stranded genome. The genome has 3 open reading frames encoding viral protein 1 (VP1), viral protein 2, and viral protein 3 (Noteborn et al., 1991, 1998; Phenix et al., 1994). Among the viral proteins, VP1 is the major capsid protein and has a hypervariable region spanning 13 amino acids (139 to 151; Renshaw et al., 1996). According to a previous report, the amino acid at position 394 in VP1 could be a major genetic determinant of virulence (Yamaguchi et al., 2001). Chicken infectious anemia virus is the causative agent of an immunosuppressive disease of young chickens. The clinical symptoms are generally observed at 10 to 14 d of age and include increased mortality, anemia, thymic atrophy, and subcutaneous hemorrhages. Despite the lack of clinical disease, older birds infected with CIAV have been found to have a decreased immune response as evidenced by poor vaccine response and increased severity of other infections (Adair, 2000). Infection in 2010 Poultry Science Association Inc. Received May 25, Accepted July 28, Corresponding author: dvmkim77@korea.kr commercial flocks is spread either vertically from the hen and possibly the rooster to their offspring (Hoop, 1992, 1993) or horizontally by the oral route. Chicken infectious anemia (CIA) has been found in the poultry industries of many countries (Schat, 2003) and previous studies have identified only one serotype (Yuasa and Imai, 1986; McNulty et al., 1990). However, CIAV was clustered with several genetic groups (Islam et al., 2002; Ducatez et al., 2006), and an antigenically different isolate (CIAV-7) in the United States has been reported, which could be the serotype 2 prototype (Spackman et al., 2002a,b). In Korea, CIAV was detected in 1989 and has continually caused economic losses due to secondary infections of chickens suffering from CIAV-induced immunosuppression. Although a few studies on the isolation of Korean CIAV and outbreaks of CIA in South Korea have been reported, a study of the molecular epidemiology of Korean CIAV based on genetic sequences has not been reported. Therefore, we performed the first genetic analysis of the VP1 gene from Korean CIAV genomes detected in samples of breeder and commercial chicken flocks. Samples MATERIALS AND METHODS To survey the avian diseases in breeder farms located in several provinces of Korea in 2009, six samples (3 2426

2 RESEARCH NOTE 2427 thymuses and 3 livers) per flock were collected. Samples from 45 flocks of great-grandparent (GGP) breeder farms with chickens of different ages were tested by the Animal Disease Diagnosis Center (ADDC) of the National Veterinary Research and Quarantine Service. Sampling from grandparent (GP) breeder farms was performed under the same method. Of the 1,366 samples from flocks of GP breeder farms tested by local veterinary diagnostic laboratories, 178 flock samples were sent to the ADDC to confirm CIAV positivity. In addition, thymus and liver samples from cases of commercial chickens suspected of CIA were inspected in this study. The chickens submitted to the ADDC for diagnosis were less than 5 wk of age and showed clinical symptoms. Virus Detection The CIAV DNA was extracted from thymus and liver samples pooled from each flock using the Viral Gene-Spin Viral DNA/RNA Extraction Kit (Intron Biotechnology, Seongnam, Korea; Kim et al., 2007). Polymerase chain reaction was conducted using the i- star Taq Maxime PCR Premix Kit (Intron Biotechnology; Lee et al., 2009) with primers that flank a 675- bp DNA fragment encompassing part of the putative gene for the capsid protein of CIAV (Todd et al., 1992). Polymerase chain reaction amplification was performed under the following conditions in a T-gradient thermal cycler (Biometra, Gottingen, Germany): an initial denaturation step at 94 C for 5 min followed by 35 cycles of 94 C for 1 min, 50 C for 1 min, and 72 C for 2 min with a final extension at 72 C for 10 min. Sequence and Phylogenetic Analysis After CIAV DNA detection, the viral DNA of positive samples was amplified following a previously reported method (Natesan et al., 2006) to cover the entire coding region of CIAV. The DNA fragments were extracted and purified with a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany; Kiso et al., 2004) and sequencing of the PCR product was performed at Cosmo (Seoul, South Korea) with an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, CA). The sequences of the strains were aligned and edited with Vector NTI (Invitrogen, Carlsbad, CA) and BioEdit ( software. The phylogenetic trees were generated by the neighbor-joining method using the MEGA 4.0 software (Tamura et al., 2007) with 1,000 bootstrap replications. Sequence data were submitted to GenBank with accession numbers HM to HM RESULTS AND DISCUSSION Nineteen flocks (42.2%) of GGP breeder farms and 11 cases of commercial chicken farms were determined to be positive for CIAV by using PCR analysis. In addition, 170 out of the 178 flock samples from GP breeder farms determined to be positive by local veterinary institutes were confirmed positive by ADDC. We carefully selected 32 CIAV consisting of 8 strains from GGP, 15 strains from GP, and 9 strains from commercial chickens to understand the genetic diversity of these Korean strains (Table 1). The phylogenetic branching pattern of VP1 genes of the CIAV strains showed that they belonged to groups II (n = 10), IIIa (n = 6), and IIIb (n = 16) (Figure 1), based on recently proposed nomenclature (Figure 1; Ducatez et al., 2006). No identified Korean CIAV strain belonged to group I, which was isolated only from Australia. The 8 CIAV strains from GGP breeder farms were classified into groups II and IIIb, whereas CIAV strains from GP breeder farms were clustered into groups II, IIIa, and IIIb. In commercial chicken farms, 9 CIAV associated with depression, anemia, and growth retardation clustered to group II (2 strains), IIIa (2 strains), and group IIIb (5 strains) (Table 1). Only commercial chicken flocks showed clinical signs such as depression, anemia, and growth retardation. Seven CIAV strains from breeder farms (GGP and GP) were genetically close to the 26P4 vaccine strain (D10068), showing more than 99.5% homology (Figure 1). Although the number of samples was limited, our phylogenetic analysis revealed that CIAV strains detected in Korea were clustered into 3 groups that show similarity to several strains in Nigeria, Malaysia, and India (Chowdhury et al., 2003; Ducatez et al., 2006; Natesan et al., 2006). In addition, although the Korean CIAV from commercial chickens presenting clinical signs were separated into groups II, IIIa, and IIIb, there was no difference in clinical symptoms between groups. The difference in pathogenicity among viral strains of genetic variation is not clear at this time (Miller and Schat, 2004). Eleven variable amino acid positions of VP1 alignment confirming findings from earlier studies were compared with our 32 strains and the 26P4 strain (Table 2; Renshaw et al., 1996; Chowdhury et al., 2003; Natesan et al., 2006; Hailemariam et al., 2008). Because the Korean CIAV clustered with groups II and IIIa had a similar amino acid pattern of I75, L97, I125, Q139, Q144, V157, and A413, except for the JB BJS 1 strain, classification of these 2 groups was not possible using amino acid substitution. However, group IIIb, distinct from the other groups, showed an L125, S287, G370, and S413 amino acid pattern. Like this result, several reports showed that groupings of CIAV based on nucleotide sequence comparisons and amino acid sequence comparisons differ (Chowdhury et al., 2003; Ducatez et al., 2006). Seven Korean CIAV similar to the vaccine strain had the same amino acid at variable positions as the 26P4 strain. Based on phylogenetic analysis of the nucleotide sequences of the VP1 gene, the CIAV detected in CN KRfarm chickens was closer to the vaccine strain (98.9%, nucleotide homology of VP1 gene)

3 2428 Kim et al. Table 1. Korean chicken infectious anemia viruses tested in this study Farm/type 1 Strain Clinical sign Vaccination Group GGP/B HKYJ DP-1 None Yes IIIb HKYJ DP-3 Yes vac 2 HKYK KC-2 Yes vac SHJE 2 No vac SHJE 6B No vac HH D No II HKYJ HI-1D Yes II HKYJ HI-4A Yes II GP/B CN BR-37 None Yes IIIa CN MJfarm No IIIa CN HJH No IIIb CN KRfarm No IIIb CN TBfarm No vac CN KKT No vac CN SHCK Yes vac JB AH 4 6 No IIIa JB YKS No IIIa JB KYS14 No IIIb CN YJS No II 103farm-8 No II JB BJS 1 No II JB KYS No II JB LCK2 No II Gf/Br 09Q174 Depression No IIIb 09Q227 Anemia No IIIa 09D243 Depression No IIIa 09D301 Depression/growth retardation No IIIb 09D318 Depression No IIIb 09D354 Depression/death No IIIb 09D359 Depression/death No IIIb 08Q017 Depression/growth retardation No II 09D367 Depression No II 1 GGP = great-grandparent; GP = grandparent; Gf = growing farm, B = breeder; Br = broiler. 2 vac = vaccine strain. compared with the other CIAV characterized, but there were 2 amino acid variations at positions 75 and 287. This amino acid substitution may be due to vaccine strain mutation with genomes clustered the group II and IIIa and unknown CIAV genomes. In addition, 4 Indian CIAV isolates and 2 Malaysian isolates showed similar pathogenicity results in spite of a different genetic grouping (Chowdhury et al., 2003; Natesan et al., 2006). Further investigations are necessary to evaluate the pathogenicity of Korean CIAV strains by group. In Korea, a live-attenuated vaccine (26P4 strain) has been granted by the government and used in several chicken breeder farms since 2009 for control of the continuous economic losses caused by CIAV. Our investigation showed that 5 flocks in GGP farms and 2 flocks in GP farms were vaccinated (Table 1). The HKYJ HI- 4A, HKYJ HI-1D, CN BR-37, and HKYJ DP-1 strains detected from vaccinated breeder farms were clustered to groups II, II, IIIa, and IIIb, respectively. If the vaccine is effective in preventing wild-type CIAV infection without regard to molecular differences, these flocks may have been infected with wild-type CIAV strains before they developed vaccine antibodies. Of the 7 viruses clustered to the vaccine strain (26P4), CN KKT, SHJE 6B, CN TB, and SHJE 2 strains were detected from unvaccinated flocks, whereas CN SHCK, HKYJ DP-3, and HKYK KC-2 were found in the vaccinated flocks (Table 1). The 7 Korean CIAV strains were detected in breeder chicken flocks without clinical signs, and these strains were not found in commercial chickens. Ducatez et al. suggested that the Nigerian CIAV with low mortality clustered to the vaccine but was not a vaccine strain (Ducatez et al., 2006). Therefore, this means that a vaccine strain may be circulating in some breeder farms or that these CIAV may not be the same strain as the vaccine strain despite the close genetic similarities. This first molecular study of Korean CIAV showed that strains from breeder and commercial chickens were separated into 3 distinct groups. Moreover, our results suggest that an additional study of the CIA vaccine may be needed to elucidate the relationship between vaccine efficacy and wild strains in South Korea, as well as further studies related to reversion of vaccine virus to virulence. ACKNOWLEDGMENTS We thank Hyuk-Man Kwon (ADDC) for excellent technical assistance. This work was supported by a grant from the National Animal Disease Control Project of the Ministry of Food, Agriculture, Forestry and Fisheries of Korea.

4 RESEARCH NOTE 2429 Figure 1. Phylogenetic diagram of viral protein 1 genes among chicken infectious anemia virus (CIAV) strains detected in Korea. The numbers above and below the branches indicate neighbor-joining distances with 1,000 bootstrap replicates. The CIAV strains isolated at great-grandparent farms in Korea are highlighted in blue and boldface, CIAV strains isolated at grandparent farms in Korea are in blue, and CIAV strains from commercial chickens displaying clinical symptoms are marked in red. The vaccine strain (26P4) is indicated in green.

5 2430 Kim et al. Table 2. Eleven variable amino acid positions of Korean chicken infectious anemia viruses viral protein 1 Amino acid position Strain Group P4_D10068 vac 1 H V M I K E M T A S A 08Q017 II Q I L Q Q V A 09D367 II I L Q Q V P JB KYS II Q I L Q Q V P JB LCK2 II Q I L Q Q V A HKYJ HI-4A II Q I L Q Q V T HKYJ HI-1D II Q I L Q Q V HH D II I L Q Q V A 103 farm-8 II N I L Q Q V S CN YJS II Q I L Q Q V JB BJS 1 II I L V P T 09D243 IIIa N I L Q Q V A CN MJfarm IIIa N I L Q Q V A JB AH 4 6 IIIa N I L Q Q V A JB YKS IIIa N I L Q Q V A CN BR-37 IIIa I L Q Q V A 09Q227 IIIa Q I L Q Q V A P 09D301 IIIb L V S G S 09D354 IIIb L V S G S CN HJH IIIb L V S G S HKYJ DP-1 IIIb L V S G S 09D359 IIIb L V S G S 09Q174 IIIb L V S G S 09D318 IIIb L L V S G S JB KYS14 IIIb Q V S G S CN KRfarm IIIb I N CN SHCK vac HKYJ DP-3 vac HKYK KC-2 vac CN KKT vac SHJE 6B vac CN TBfarm vac SHJE 2 vac 1 vac = vaccine strain. REFERENCES Adair, B Immunopathogenesis of chicken anemia virus infection. Dev. Comp. Immunol. 24: Chowdhury, S., A. Omar, I. Aini, M. Hair-Bejo, A. Jamaluddin, B. Md-Zain, and Y. Kono Pathogenicity, sequence and phylogenetic analysis of Malaysian Chicken anaemia virus obtained after low and high passages in MSB-1 cells. Arch. Virol. 148: Ducatez, M., A. Owoade, J. Abiola, and C. Muller Molecular epidemiology of chicken anemia virus in Nigeria. Arch. Virol. 151: Hailemariam, Z., A. R. Omar, M. Hair-Bejo, and T. C. Giap Detection and characterization of chicken anemia virus from commercial broiler breeder chickens. Virol. J. 5:128. Hoop, R Persistence and vertical transmission of chicken anaemia agent in experimentally infected laying hens. Avian Pathol. 21: Hoop, R Transmission of chicken anaemia virus with semen. Vet. Rec. 133: Islam, M., R. Johne, R. Raue, D. Todd, and H. Muller Sequence analysis of the full-length cloned DNA of a chicken anaemia virus (CAV) strain from Bangladesh: Evidence for genetic grouping of CAV strains based on the deduced VP1 amino acid sequences. J. Vet. Med. B Infect. Dis. Vet. Public Health 49: Kim, M., Y. Kwon, S. Joh, J. Kwon, J. Kim, and S. Kim Development of one-step reverse transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1. Avian Dis. 51: Kiso, M., K. Mitamura, Y. Sakai-Tagawa, K. Shiraishi, C. Kawakami, K. Kimura, F. Hayden, N. Sugaya, and Y. Kawaoka Resistant influenza A viruses in children treated with oseltamivir: Descriptive study. Lancet 364: Lee, G., C. Chong, C. Lee, and S. Lee Cell-culture-based immunochromatography for rapid detection of group A human rotaviruses in aquatic environments. Environ. Technol. 30: McNulty, M., T. Connor, F. McNeilly, M. McLoughlin, and K. Kirkpatrick Preliminary characterisation of isolates of chicken anaemia agent from the United Kingdom. Avian Pathol. 19: Miller, M., and K. A. Schat Chicken infectious anemia virus: An example of the ultimate host-parasite relationship. Avian Dis. 48: Natesan, S., J. Kataria, K. Dhama, S. Rahul, and N. Bhardwaj Biological and molecular characterization of chicken anaemia virus isolates of Indian origin. Virus Res. 118: Noteborn, M., G. De Boer, D. Van Roozelaar, C. Karreman, O. Kranenburg, J. Vos, S. Jeurissen, R. Hoeben, A. Zantema, and G. Koch Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle. J. Virol. 65: Noteborn, M., C. Verschueren, G. Koch, and A. Van der Eb Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope. J. Gen. Virol. 79: Phenix, K., B. Meehan, D. Todd, and M. McNulty Transcriptional analysis and genome expression of chicken anaemia virus. J. Gen. Virol. 75:

6 RESEARCH NOTE 2431 Renshaw, R., C. Soine, T. Weinkle, P. O Connell, K. Ohashi, S. Watson, B. Lucio, S. Harrington, and K. Schat A hypervariable region in VP1 of chicken infectious anemia virus mediates rate of spread and cell tropism in tissue culture. J. Virol. 70: Schat, K. A Chicken infectious anemia. Pages in Diseases of Poultry. Y. M. Saif, H. J. Barnes, J. R. Glisom, A. M. Fadly, L. R. McDougald, and D. E. Swayne, ed. Iowa State University Press, Ames. Spackman, E., S. Cloud, C. Pope, and J. Rosenberger. 2002a. Comparison of a putative second serotype of chicken infectious anemia virus with a prototypical isolate. I. Pathogenesis. Avian Dis. 46: Spackman, E., S. Cloud, and J. Rosenberger. 2002b. Comparison of a putative second serotype of chicken infectious anemia virus with a prototypical isolate. II. Antigenic and physicochemical characteristics. Avian Dis. 46: Tamura, K., J. Dudley, M. Nei, and S. Kumar MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Mol. Biol. Evol. 24: Todd, D., K. Mawhinney, and M. McNulty Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction. J. Clin. Microbiol. 30: Yamaguchi, S., T. Imada, N. Kaji, M. Mase, K. Tsukamoto, N. Tanimura, and N. Yuasa Identification of a genetic determinant of pathogenicity in chicken anaemia virus. J. Gen. Virol. 82: Yuasa, N., and K. Imai Pathogenicity and antigenicity of eleven isolates of chicken anaemia agent (CAA). Avian Pathol. 15:

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