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Pathogenicity of a fowl adenovirus serotype 4 isolated from chickens associated with hydropericardium-hepatitis syndrome in China
Pathogenicity of a fowl adenovirus serotype 4 isolated from chickens associated with hydropericardium-hepatitis syndrome in China
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Key Laboratory of Biotechnology and Drug Manufacture for Animal Epidemic Prevention, Ministry of Agriculture, Zhaoqing 526238, China; † Zhaoqing Institute of Biotechnology Co., Ltd, Zhaoqing, China; and ‡ College of Veterinary Medicine, South China Agriculture University, Guangzhou 510642, China flabby with amber-colored and jelly-like fluid in the pericardial sacs. The kidneys were swollen and congested. Histologically eosinophilic intranuclear inclusion body could be seen in the hepatic cell. The result of histopathological examination also revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging. Other tissues like kidney, bursa of Fabricius, thymus, and spleen were observed degeneration and necrosis. Virus-specific antibodies appeared in serum beginning at day 14 and reached statistically significant levels at 21, 28, 35, and 42 dpi (P < 0.001). In conclusion, we identified a highly virulent FAdV-4 virus as causative agent of the HHS outbreak reported here. The FAdV-4 GX-1 strain will be valuable for vaccine evaluation and development to prevent and reduce the spread of HHS in the poultry industry.
ABSTRACT Hydropericardium-hepatitis syndrome (HHS) is characterized by pericardial effusion and hepatitis and causes huge economic losses in the poultry industry in China. In this study, a strain of fowl adenoviruses (FAdV-4) (GX-1) was isolated from liver samples of diseased chickens with HHS. Phylogenetic analysis based on complete genome gene revealed that GX-1 clustered with the C-type fowl adenovirus and was serotyped as FAdV-4. Pathogenicity testing showed that the GX-1 strain caused 100% mortality in 10-day-old specific pathogen-free chickens at a dose of 104 tissue culture infective doses (TCID50 ) within 3 d post-infection. A viral dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were
Key words: fowl adenovirus serotype 4, hydropericardium-hepatitis syndrome, pathogenicity 2019 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pez042
INTRODUCTION
tis causes the mortality to reach levels of 30 to 90% (Nakamura et al., 1999; Kim et al., 2008; Schonewille et al., 2008; Asthana et al., 2013). Fowl adenoviruses (FAdV) are classified into five species (FAdV-A to FAdV-E) with 12 serotypes (FAdV1 to 8a, 8b thru 11) based on restriction enzyme digestion patterns and serum cross-neutralization tests (Hess, 2000). Most cases of HHS are caused by FAdVC species of serotype 4 (FAdV-4) (Zhao et al., 2015; Niu et al., 2016; Zhang et al., 2016). All 12 FAdV serotypes have been associated with outbreaks of syndromes similar to HHS known as inclusion body hepatitis (IBH) (Maartens et al., 2014). In general, only FAdV-4 causes HHS (Asthana et al., 2013). FAdV members are non-enveloped isometric viral particles with their major capsid protein hexon containing type-, group- and subgroup-specific antigenic determinants that are serotype-specific (Steer et al., 2009). FAdV-4 serotypes have recently emerged in China since 2015 and possess novel genotypes (Ye et al., 2016). In this study, we isolated a FAdV-4 strain GX-1 from the livers of commercial broiler chickens infected with HHS in the GuangXi province of China. The goals of
Hydropericardium-hepatitis syndrome (HHS) or hydropericardium syndrome is an infectious disease of young chickens that was originally reported in Pakistan in 1987 (Anjum et al., 1989). It currently has a global presence causing huge economic losses in the poultry industry (Abe et al., 1998; Kim et al., 2008; VeraHernandez et al., 2016; Guan et al., 2018). Interestingly, there is a recent trend towards more epidemic, rather than sporadic outbreaks in many countries (Schachner et al., 2018). HHS is commonly first noticed in young birds aged 3 to 6 wk and occasionally in older animals (Niu et al., 2016). The predominant findings are gross lesions in the hydropericardium accompanied by the accumulation of a clear or straw-colored fluid of aqueous or gelatinous consistency in the pericardial sac. The heart is deformed and associated nephritis and hepati C 2019 Poultry Science Association Inc. Received August 2, 2018. Accepted January 24, 2019. 1 Corresponding author: [email protected] 2 These authors are co-first authors on this work.
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Guangcai Ren,∗,†,1 Han Wang,‡,1 Yuanyuan Yan,‡ Fan Liu,‡ Miaorong Huang,∗,† and Ruiai Chen∗,‡,†,2
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the current study were to (1) investigate its complete genome and relatedness to other FAdV and (2) examine its pathogenicity in specific pathogen free (SPF) chickens.
Ethics Statement Our study was approved by Animal Care and Use Committee of Guangdong Province, China. All animal procedures were performed according to guidelines developed by the China Council on Animal Care and protocols approved by Animal Care and Use Committee of Guangdong, Province, China.
Virus Isolation and Purification The GX-1 strain of FAdV-4 causing HHS was isolated from a commercial broiler farm in Guangxi Province, China in 2017. The virus was isolated from the livers of infected birds and propagated in primary cultures of chicken embryo liver cells (Vera-Hernandez et al., 2016) Medium 199 (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, CA, USA), 100 IU/mL penicillin (Sigma-Aldrich, USA), and 100 μg/mL streptomycin (Sigma-Aldrich, USA) was used for chicken embryo liver cell culture. After isolation, virus dilutions were made for plaque purification in the chicken liver cell line (LMH) (ATCC, USA). Each virus was plaque purified three times. Briefly, virus dilutions from 10−2 to 10−7 were inoculated onto LMH monolayers. One hour after infection the cell layer was overlaid with Medium 199 containing 2% agarose (Sigma-Aldrich, USA) and incubated 3 to 5 d at 37◦ C in a 5% CO2 humidified atmosphere. Isolated plaques were then picked and transferred to a new fresh culture of LMH cells. Molecular identification of virus isolates used PCR with primers specific for the FAdV hexon gene (GenBank GU188428); 5 -CAA CTA CAT CGG GTT CAG GG-3 and 5 -TGG CGT TTC TCA GCA TCA-3 . PCR reactions were performed using the following protocol: 95◦ C for 5 min, followed by 34 cycles of 95◦ C for 30 s, 58◦ C for 30 s, and 72◦ C for 30 s, followed by a final elongation step of 10 min at72◦ C. Other viruses including avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Avian Leukemia virus (ALV), reticuloendotheliosis virus, and Marek’s disease virus (MDV) were examined by PCR or RT-PCR using primers and methods as previously described (Jiang et al., 2013; Liu et al., 2013; Liu et al., 2014; Wang et al., 2014; Lu et al., 2015).
Transmission Electron Microscopy FAdV cell culture lysates from LMH cultures were used for electron microscopy. Cells at 48-h post-
Genome Sequencing Partial genes were amplified by PCR using 50 pairs of primers specific for the ON1 strain of FAdV (GenBank: GU188428). Overlapping sequences were assembled into complete genome sequences using the SeqMan program in the DNAStar software package (DNAStar, Madison, WI, USA).
Sequence Alignment and Phylogenetic Analysis The complete genome DNA sequence of GX-1 and predicted amino acid sequences of the hexon gene were aligned with other available FAdV sequences using the MegAlign Clustal W method. Phylogenetic trees were constructed using MEGA (Version 7) with neighbor-joining method with 1000 bootstrap replicates (https://www.megasoftware.net). The complete genome sequence of GX-1 was submitted to the GenBank database to obtain a GenBank accession number MH454598.
Pathogenicity Assay in SPF Chickens Two hundred 10-day-old SPF White Leghorn chickens were randomly divided into 4 groups of 50 birds. One group of birds were inoculated with sterile PBS and used as negative controls. The second group was infected with FAdV-4 GX-1 via the intramuscular injection route with 200 μl of inoculum containing 104 tissue culture infective doses (TCID50 ) of virus. The third group was infected with 103 TCID50 of the same virus and the fourth group was infected with 102 TCID50 . All birds were observed twice daily and scored for clinical signs for 42 d. Clinical signs were scored as: 0 for normal, 1 for mild sign of lethargy and ruffled feathers, 2 for reluctant to move, lethargy, trembling, and loss of appetite, 3 for paralysis/prostration, and 4 for death. A total of 4 chickens from each group were euthanized at 0, 7, 14, 21, 28, 35, and 42 d post-infection (dpi). The livers, hearts, kidneys, thymus, bursa, and spleens of the dead birds were collected and processed for histopathologic examination and blood samples were collected for serology. Some birds died between the intervals and tissue samples were also collected for histopathologic
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MATERIALS AND METHODS
infection (hpi) were lysed by three freeze thaw cycles and centrifuged at 1000 × g for 10 min and 8000 × g for 30 min to remove of debris. The supernatant was centrifuged at 13,000 × g for 1 h to pellet virions. Virus pellets were suspended in 50 μl of distilled water and negatively stained with 1% phosphotungstic acid (Sigma-Aldrich, USA) for 30 s and then mounted onto 200-mesh Formvar-coated copper grids (Pan et al., 2017).The samples on grids were examined using a transmission electron microscope (JEM-2010HR, Japan).
PATHOGENICITY OF A FOWL ADENOVIRUS SEROTYPE 4 IN CHINA
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RESULTS Virus Isolation
Genome Size and Phylogenetic Analysis Figure 1. PCR results using purified virus as templates. Agarose gel electrophoresis of PCR amplicons visualized by EtBr staining and UV light. Lane M: DL 2000 bp marker, Lane 1: FAdV-4 GX-1, Lane2: AIV, Lane 3: NDV, Lane 4: IBDV, Lane 5: ALV, Lane 6: ARV, Lane 7: MDV. The solid arrows indicate the target gene.
examination. Tissue samples were fixed in 10% formalin for 48 h at room temperature and processed for haematoxylin and eosin (HE) staining as previously described (Bogomoletz et al., 1980). Serum samples were tested for the presence of FAdV-specific antibodies using a commercial ELISA kit (BioChek, Scarborough, ME, USA).
Statistical Analysis SPSS 16.00 software (SPSS, Chicago, IL, USA) was used to analyze the result of antibody response. Results were represented as the means ± standard error of mean. Statistically significant differences were analyzed by one-way analysis of variance followed by Duncan’s post hoc test. Statistical significance was set as P < 0.05.
The genome of FAdV-4 GX-1 strain was 43,721 base pairs (bp) with a G+C content of 54.87% and contained 38 open reading frames. The complete nucleotide sequences were deposited to the GenBank with accession numbers MH454598. Phylogenetic analysis showed that strain GX-1 belongs to a cluster of FAdV-C with 98.5 and 98.3% identity to FAdV-4 KR5 (GenBank accession No: HE608152) and ON1 (GenBank accession No: GU188428) (Figure 3). The GX-1 complete sequence identity among the 9 Chinese FAdV-4 isolates was 99 to 100% and the isolates showed identity of 98 to 99% to B1 to 7/India, MX-SHP95/Mexico (Figure 3).The entire hexon gene of FAdV GX-1 strain 2814 bp in length. The phylogenetic analysis based on amino acid sequence of hexon gene was similar to that of complete genome (data not show).
Clinical Signs, Antibody Response, Gross Pathology, and Histology The pathogenicity of FAdV-4 GX-1 was examined by infection of young SPF chickens at 3 virus doses.
Figure 2. (A) Cytopathic effects induced by GX-1 in LMH cells 72 h after infection. (B) Non-infected LMH cells. (C) Electron micrograph of GX-1 viral particles.
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A young chicken with a clinical diagnosis of an HHS was used to identify the viral pathogen causing the infection. A plaque-forming virus was identified in an LMH cell culture infected with a liver homogenate and was plaque purified for DNA isolation. PCR analysis indicated the virus was a FAdV member and coinfections with AIV, NDV, IBDV, ALV, ARV, and MDV were absent (Figure 1). During serial passage in cell culture, the LMH cells showed severe cytopathic effects at 48 h post-infection (Figure 2A) when compared with liver homogenates from non-infected chickens were used (Figure 2B). We also identified virus particles in cell culture supernatants (Figure 2C). The isolate was designated GX-1.
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Figure 4. Survival rates (A), clinical scores (B), and antibody response (C) of chickens after inoculation with strain GX-1. Clinical scoring: 0 for normal, 1 for mild sign of lethargy and ruffled feathers, 2 for reluctant to move, lethargy, trembling, and loss of appetite, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars indicate means ± standard error of mean. Asterisks (∗) mark the days in which the antibody titres were significantly different between inoculated and negative control groups (P < 0.001).
Infection of birds with 104 TCID50 resulted in 100% mortality at 2 and 4 dpi. A dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6 (Figure 4A). During the experimental period, depression, ruffled feathers, huddling in corners, reluctant to move, loss
of appetence, and a crouching position were prominent clinical signs. All chickens in the control group remained alive and did not show any clinical signs. Chickens inoculated with 104 TCID50 of GX-1 showed mild or severe depression after 2 dpi and died within 2 to 4 d postinoculation. The clinical scores for 104 TCID50 of GX-1
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Figure 3. Fowl adenovirus phylogenetic tree based on the complete genome sequence. GX-1, red triangle. The number in parenthesis indicates the GenBank accession number for each fowl adenovirus sequence.
PATHOGENICITY OF A FOWL ADENOVIRUS SEROTYPE 4 IN CHINA
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infections were greater than 103 TCID50 at 2 to 4 dpi and at 103 TCID50 were greater than 102 TCID50 at 5 to 9 dpi (Figure 4B). An antibody response of SPF chickens against FAdV was detected at 7 different time points during postinfection. FAdV-specific antibodies appeared at 14 dpi, and continued to rise until 35 dpi. Differences between inoculated and negative control groups were statistically significant (P < 0.001) at 21, 28, 35, and 42 dpi (Figure 4C). The peak antibody titre was reached at 35 dpi and began to decrease at 42 dpi. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were flabby with amber-colored and jelly-like fluid in
the pericardial sacs (Figure 5A and B). The kidneys were swollen and congested (Figure 5D and E). No significant gross lesions were present in the control chicken tissues (Figure 5C and F). Histopathological examination revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging (Figure 6A, solid arrows). The most characteristic histological lesions in the liver were eosinophilic intranuclear inclusion bodies (Figure 6B, solid arrows). In addition, small multifocal areas of coagulative necrosis and hydropic degeneration were observed in hepatocytes (Figure 6B). The kidneys were edematous with renal tubule degeneration and necrosis (Figure 6C). The lymphocytes were degenerated and
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Figure 5. Gross lesions in liver, heart, and kidney from dead chickens. (A and D) Controls. (B and C). Examples of swollen and friable livers with multifocal areas of necrosis and petechial hemorrhages and hydropericardia. (E and F). Mild and severe kidney enlargement in GX-1infected chickens.
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necrotic in the bursa of Fabricius (Figure 6H). The pathological changes in thymus included degeneration and necrosis of the endotheliocytes as well as lymphopenia (Figure 6I). Fewer lymphocytes and degeneration and necrosis of endotheliocytes were present in spleen tissues compared with controls (Figure 6J). No lesions were observed in the corresponding tissues of chickens in the control group (Figure 6D–F, K-M).
DISCUSSION In recent years, FAdV strains related to IBH, HHS, or gizzard erosion have been isolated from most clinical cases in many countries. Clinical presentations of severe hydropericardia have recently emerged and this infection has rapidly spread through broiler and layer farms in many regions of China. FAdV serotype 4 GX1strain isolated from hydropericardium syndrome in the current study was judged to be highly pathogenic for chicks, killing all virus-infected chicks within 2 to 4 d at a dose of 104 TCID50. The dead animals showed severe hydropericardium and IBH, which were almost identical to those of naturally infected chickens. At a dose of 103 TCID50 resulted in a 16% survival and
at 102 TCID50 , an 80% rate. This result indicated that the mortality in chickens were time- and dosagedependent. These results were similar to other studies with other FAdV-4 isolates (Vera-Hernandez et al., 2016). Compared with other highly pathogenic FAdV-4 strains, the infectious dose which caused 100% mortality in SPF chickens was lower or similar to other experimental studies (Mase et al., 2010; Vera-Hernandez et al., 2016). HHS caused by FAdV-4 is characterized by accumulation of transparent or straw-colored fluid in the pericardial sac and hepatitis and our results were similar (see Figure 3). We also found either mild or severe pathologic changes in the spleen, thymus, bursa of Fabricius, and kidney. Previous reports hypothesized that the systemic distribution of the virus in lymphoid tissues might be associated with the mortality effect in chickens at a high viral dose (Naeem et al., 1995). Additionally, a severe depletion of lymphocytes and a generalized suppression of the humoral and cellular immune systems were observed after infection with a highly pathogenic FAdV-4 strain in chickens (Schonewille et al., 2008). In our study, lymphocytes showed degeneration and necrosis in immune organs that may be associated with
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Figure 6. Histopathology of tissues from chickens infected with strain GX-1. Heart: solid arrows indicate muscle fibers in heart are fracture. Liver: solid arrows indicate viral inclusion bodies.
PATHOGENICITY OF A FOWL ADENOVIRUS SEROTYPE 4 IN CHINA
ACKNOWLEDGMENTS This study was supported by Guangdong Wen’s Dahuanong Biotechnology Co., Ltd.
REFERENCES Abe, T., K. Nakamura, H. Tojo, M. Mase, T. Shibahara, S. Yamaguchi, and N. Yuasa. 1998. Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks. Avian Dis. 42:606–612. Anjum, A. D., M. A. Sabri, and Z. Iqbal. 1989. Hydropericarditis syndrome in broiler chickens in Pakistan. Vet. Rec. 124:247–248. Asthana, M., R. Chandra, and R. Kumar. 2013. Hydropericardium syndrome: current state and future developments. Arch. Virol. 158:921–931. Bogomoletz, W. V., J. J. Adnet, S. Widgren, M. Stavrou, and J. E. McLaughlin. 1980. Cystadenoma of the pancreas: a histological, histochemical and ultrastructural study of seven cases. Histopathology 4:309–320. Guan, R., Y. Tian, X. Han, X. Yang, and H. Wang. 2018. Complete genome sequence and pathogenicity of fowl adenovirus serotype 4 involved in hydropericardium syndrome in Southwest China. Microb. Pathog. 117:290–298. Hess, M. 2000. Detection and differentiation of avian adenoviruses: a review. Avian Pathol. 29:195–206. Jiang, L., X. Qi, Y. Gao, Y. Hua, K. Li, X. Deng, Q. Wang, L. Zhang, H. Chai, Y. Chen, C. Yin, H. Gao, L. Qin, Y. Wang, Y. Qu, Q. Chen, Z. Fan, and X. Wang. 2013. Molecular characterization and phylogenetic analysis of the reticuloendotheliosis virus isolated from wild birds in Northeast China. Vet. Microbiol. 166:68–75. Kim, J. N., S. H. Byun, M. J. Kim, J. Kim, H. W. Sung, and I. P. Mo. 2008. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates. Avian Dis. 52:526–530. Liu, D., X. B. Zhang, Z. Q. Yan, F. Chen, J. Ji, J. P. Qin, H. Y. Li, J. P. Lu, Y. Xue, J. J. Liu, Q. M. Xie, J. Y. Ma, C. Y. Xue, and Y. Z. Bee. 2013. Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011. Trop. Anim. Health Prod. 45: 1107–1112.
Liu, L., Y. Qu, T. Wang, G. Wang, F. Wang, and S. Liu. 2014. Skin involvement in lymphomas caused by Marek’s disease virus infection in Silkie chickens. J. Vet. Diagn. Invest. 26:302–307. Lu, Z., L. Zhang, N. Wang, Y. Chen, L. Gao, Y. Wang, H. Gao, Y. Gao, K. Li, X. Qi, and X. Wang. 2015. Naturally occurring reassortant infectious bursal disease virus in northern China. Virus Res. 203:92–95. Maartens, L. H., H. W. Joubert, H. Aitchison, and E. H. Venter. 2014. Inclusion body hepatitis associated with an outbreak of fowl adenovirus type 2 and type 8b in broiler flocks in South Africa. J. S. Afr. Vet. Assoc. 85:e1–5. Mase, M., K. Nakamura, and T. Imada. 2010. Characterization of Fowl adenovirus serotype 4 isolated from chickens with hydropericardium syndrome based on analysis of the short fiber protein gene. J. Vet. Diagn. Invest. 22:218–223. Naeem, K., T. Niazi, S. A. Malik, and A. H. Cheema. 1995. Immunosuppressive potential and pathogenicity of an avian adenovirus isolate involved in hydropericardium syndrome in broilers. Avian Dis. 39:723–728. Nakamura, K., M. Mase, S. Yamaguchi, T. Shibahara, and N. Yuasa. 1999. Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome. Avian Dis. 43:414–423. Niu, Y., Q. Sun, G. Zhang, W. Sun, X. Liu, Y. Xiao, Y. Shang, and S. Liu. 2018. Epidemiological investigation of outbreaks of fowl adenovirus infections in commercial chickens in China. Transbound Emerg. Dis. 65:e121–e126. Niu, Y. J., W. Sun, G. H. Zhang, Y. J. Qu, P. F. Wang, H. L. Sun, Y. H. Xiao, and S. D. Liu. 2016. Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015. J. Gen. Virol. 97:2684–2690. Pan, Q., L. Liu, Y. Gao, C. Liu, X. Qi, Y. Zhang, Y. Wang, K. Li, L. Gao, X. Wang, and H. Cui. 2017. Characterization of a hypervirulent fowl adenovirus 4 with the novel genotype newly prevalent in China and establishment of reproduction infection model of hydropericardium syndrome in chickens. Poult. Sci. 96:1581–1588. Schachner, A., M. Matos, B. Grafl, and M. Hess. 2018. Fowl adenovirus-induced diseases and strategies for their control - a review on the current global situation. Avian Pathol. 47:111–126. Schonewille, E., A. Singh, T. W. Gobel, W. Gerner, A. Saalmuller, and M. Hess. 2008. Fowl adenovirus (FAdV) serotype 4 causes depletion of B and T cells in lymphoid organs in specific pathogenfree chickens following experimental infection. Vet. Immunol. Immunopathol. 121:130–139. Steer, P. A., N. C. Kirkpatrick, D. O Rourke, and A. H. Noormohammadi. 2009. Classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region. J. Clin. Microbiol. 47:311–321. Vera-Hernandez, P. F., A. Morales-Garzon, D. V. Cortes-Espinosa, A. Galiote-Flores, L. J. Garcia-Barrera, E. T. Rodriguez-Galindo, A. Toscano-Contreras, E. Lucio-Decanini, and A. E. Absalon. 2016. Clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl Aviadenovirus serotype 4. Avian Pathol. 45:73–81. Wang, Q., X. Li, X. Ji, J. Wang, N. Shen, Y. Gao, X. Qi, Y. Wang, H. Gao, S. Zhang, and X. Wang. 2014. A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies. PLoS One 9:e115422. Ye, J., G. Liang, J. Zhang, W. Wang, N. Song, P. Wang, W. Zheng, Q. Xie, H. Shao, Z. Wan, C. Wang, H. Chen, W. Gao, and A. Qin. 2016. Outbreaks of serotype 4 fowl adenovirus with novel genotype, China. Emerg. Microbes Infect. 5:e50–e50. Zhang, T., Q. Jin, P. Ding, Y. Wang, Y. Chai, Y. Li, X. Liu, J. Luo, and G. Zhang. 2016. Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China. Virol. J. 13:188. Zhao, J., Q. Zhong, Y. Zhao, Y. X. Hu, and G. Z. Zhang. 2015. Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China. PLoS One 10:e0133073.
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immunosuppression. These results provide a more detailed insight into the pathogenesis of GX-1. An epidemiological survey showed that FAdV infections were caused by different FAdV species in China, such as species C, D, and E (Niu et al., 2018). In the current study, the representative strains of 5 species of FAdV were downloaded from GenBank to perform the phylogenetic analysis. Based on the complete genome sequence, strain GX-1 belongs to a cluster of FAdV-C. As reported, more than one-half of the FAdVs isolated were genetically related to serotype 4 proving that this serotype was dominant in China from 2015 to 2016 (Niu et al., 2018). A wide range of phylogenetic analyses at the whole genome level would be necessary to better understand the evolutionary relationships between avian adenovirus isolates (Zhao et al., 2015). Our phylogenetic analysis based on the hexon gene showed GX-1 strain classified into the same cluster. This result revealed the FAdV-4 isolates obtained from chickens with HHS in China have a common ancestor and belong to the most predominant serotype in China.
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Key Laboratory of Biotechnology and Drug Manufacture for Animal Epidemic Prevention, Ministry of Agriculture, Zhaoqing 526238, China; † Zhaoqing Institute of Biotechnology Co., Ltd, Zhaoqing, China; and ‡ College of Veterinary Medicine, South China Agriculture University, Guangzhou 510642, China flabby with amber-colored and jelly-like fluid in the pericardial sacs. The kidneys were swollen and congested. Histologically eosinophilic intranuclear inclusion body could be seen in the hepatic cell. The result of histopathological examination also revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging. Other tissues like kidney, bursa of Fabricius, thymus, and spleen were observed degeneration and necrosis. Virus-specific antibodies appeared in serum beginning at day 14 and reached statistically significant levels at 21, 28, 35, and 42 dpi (P < 0.001). In conclusion, we identified a highly virulent FAdV-4 virus as causative agent of the HHS outbreak reported here. The FAdV-4 GX-1 strain will be valuable for vaccine evaluation and development to prevent and reduce the spread of HHS in the poultry industry.
ABSTRACT Hydropericardium-hepatitis syndrome (HHS) is characterized by pericardial effusion and hepatitis and causes huge economic losses in the poultry industry in China. In this study, a strain of fowl adenoviruses (FAdV-4) (GX-1) was isolated from liver samples of diseased chickens with HHS. Phylogenetic analysis based on complete genome gene revealed that GX-1 clustered with the C-type fowl adenovirus and was serotyped as FAdV-4. Pathogenicity testing showed that the GX-1 strain caused 100% mortality in 10-day-old specific pathogen-free chickens at a dose of 104 tissue culture infective doses (TCID50 ) within 3 d post-infection. A viral dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were
Key words: fowl adenovirus serotype 4, hydropericardium-hepatitis syndrome, pathogenicity 2019 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pez042
INTRODUCTION
tis causes the mortality to reach levels of 30 to 90% (Nakamura et al., 1999; Kim et al., 2008; Schonewille et al., 2008; Asthana et al., 2013). Fowl adenoviruses (FAdV) are classified into five species (FAdV-A to FAdV-E) with 12 serotypes (FAdV1 to 8a, 8b thru 11) based on restriction enzyme digestion patterns and serum cross-neutralization tests (Hess, 2000). Most cases of HHS are caused by FAdVC species of serotype 4 (FAdV-4) (Zhao et al., 2015; Niu et al., 2016; Zhang et al., 2016). All 12 FAdV serotypes have been associated with outbreaks of syndromes similar to HHS known as inclusion body hepatitis (IBH) (Maartens et al., 2014). In general, only FAdV-4 causes HHS (Asthana et al., 2013). FAdV members are non-enveloped isometric viral particles with their major capsid protein hexon containing type-, group- and subgroup-specific antigenic determinants that are serotype-specific (Steer et al., 2009). FAdV-4 serotypes have recently emerged in China since 2015 and possess novel genotypes (Ye et al., 2016). In this study, we isolated a FAdV-4 strain GX-1 from the livers of commercial broiler chickens infected with HHS in the GuangXi province of China. The goals of
Hydropericardium-hepatitis syndrome (HHS) or hydropericardium syndrome is an infectious disease of young chickens that was originally reported in Pakistan in 1987 (Anjum et al., 1989). It currently has a global presence causing huge economic losses in the poultry industry (Abe et al., 1998; Kim et al., 2008; VeraHernandez et al., 2016; Guan et al., 2018). Interestingly, there is a recent trend towards more epidemic, rather than sporadic outbreaks in many countries (Schachner et al., 2018). HHS is commonly first noticed in young birds aged 3 to 6 wk and occasionally in older animals (Niu et al., 2016). The predominant findings are gross lesions in the hydropericardium accompanied by the accumulation of a clear or straw-colored fluid of aqueous or gelatinous consistency in the pericardial sac. The heart is deformed and associated nephritis and hepati C 2019 Poultry Science Association Inc. Received August 2, 2018. Accepted January 24, 2019. 1 Corresponding author: [email protected] 2 These authors are co-first authors on this work.
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Guangcai Ren,∗,†,1 Han Wang,‡,1 Yuanyuan Yan,‡ Fan Liu,‡ Miaorong Huang,∗,† and Ruiai Chen∗,‡,†,2
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the current study were to (1) investigate its complete genome and relatedness to other FAdV and (2) examine its pathogenicity in specific pathogen free (SPF) chickens.
Ethics Statement Our study was approved by Animal Care and Use Committee of Guangdong Province, China. All animal procedures were performed according to guidelines developed by the China Council on Animal Care and protocols approved by Animal Care and Use Committee of Guangdong, Province, China.
Virus Isolation and Purification The GX-1 strain of FAdV-4 causing HHS was isolated from a commercial broiler farm in Guangxi Province, China in 2017. The virus was isolated from the livers of infected birds and propagated in primary cultures of chicken embryo liver cells (Vera-Hernandez et al., 2016) Medium 199 (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, CA, USA), 100 IU/mL penicillin (Sigma-Aldrich, USA), and 100 μg/mL streptomycin (Sigma-Aldrich, USA) was used for chicken embryo liver cell culture. After isolation, virus dilutions were made for plaque purification in the chicken liver cell line (LMH) (ATCC, USA). Each virus was plaque purified three times. Briefly, virus dilutions from 10−2 to 10−7 were inoculated onto LMH monolayers. One hour after infection the cell layer was overlaid with Medium 199 containing 2% agarose (Sigma-Aldrich, USA) and incubated 3 to 5 d at 37◦ C in a 5% CO2 humidified atmosphere. Isolated plaques were then picked and transferred to a new fresh culture of LMH cells. Molecular identification of virus isolates used PCR with primers specific for the FAdV hexon gene (GenBank GU188428); 5 -CAA CTA CAT CGG GTT CAG GG-3 and 5 -TGG CGT TTC TCA GCA TCA-3 . PCR reactions were performed using the following protocol: 95◦ C for 5 min, followed by 34 cycles of 95◦ C for 30 s, 58◦ C for 30 s, and 72◦ C for 30 s, followed by a final elongation step of 10 min at72◦ C. Other viruses including avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Avian Leukemia virus (ALV), reticuloendotheliosis virus, and Marek’s disease virus (MDV) were examined by PCR or RT-PCR using primers and methods as previously described (Jiang et al., 2013; Liu et al., 2013; Liu et al., 2014; Wang et al., 2014; Lu et al., 2015).
Transmission Electron Microscopy FAdV cell culture lysates from LMH cultures were used for electron microscopy. Cells at 48-h post-
Genome Sequencing Partial genes were amplified by PCR using 50 pairs of primers specific for the ON1 strain of FAdV (GenBank: GU188428). Overlapping sequences were assembled into complete genome sequences using the SeqMan program in the DNAStar software package (DNAStar, Madison, WI, USA).
Sequence Alignment and Phylogenetic Analysis The complete genome DNA sequence of GX-1 and predicted amino acid sequences of the hexon gene were aligned with other available FAdV sequences using the MegAlign Clustal W method. Phylogenetic trees were constructed using MEGA (Version 7) with neighbor-joining method with 1000 bootstrap replicates (https://www.megasoftware.net). The complete genome sequence of GX-1 was submitted to the GenBank database to obtain a GenBank accession number MH454598.
Pathogenicity Assay in SPF Chickens Two hundred 10-day-old SPF White Leghorn chickens were randomly divided into 4 groups of 50 birds. One group of birds were inoculated with sterile PBS and used as negative controls. The second group was infected with FAdV-4 GX-1 via the intramuscular injection route with 200 μl of inoculum containing 104 tissue culture infective doses (TCID50 ) of virus. The third group was infected with 103 TCID50 of the same virus and the fourth group was infected with 102 TCID50 . All birds were observed twice daily and scored for clinical signs for 42 d. Clinical signs were scored as: 0 for normal, 1 for mild sign of lethargy and ruffled feathers, 2 for reluctant to move, lethargy, trembling, and loss of appetite, 3 for paralysis/prostration, and 4 for death. A total of 4 chickens from each group were euthanized at 0, 7, 14, 21, 28, 35, and 42 d post-infection (dpi). The livers, hearts, kidneys, thymus, bursa, and spleens of the dead birds were collected and processed for histopathologic examination and blood samples were collected for serology. Some birds died between the intervals and tissue samples were also collected for histopathologic
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MATERIALS AND METHODS
infection (hpi) were lysed by three freeze thaw cycles and centrifuged at 1000 × g for 10 min and 8000 × g for 30 min to remove of debris. The supernatant was centrifuged at 13,000 × g for 1 h to pellet virions. Virus pellets were suspended in 50 μl of distilled water and negatively stained with 1% phosphotungstic acid (Sigma-Aldrich, USA) for 30 s and then mounted onto 200-mesh Formvar-coated copper grids (Pan et al., 2017).The samples on grids were examined using a transmission electron microscope (JEM-2010HR, Japan).
PATHOGENICITY OF A FOWL ADENOVIRUS SEROTYPE 4 IN CHINA
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RESULTS Virus Isolation
Genome Size and Phylogenetic Analysis Figure 1. PCR results using purified virus as templates. Agarose gel electrophoresis of PCR amplicons visualized by EtBr staining and UV light. Lane M: DL 2000 bp marker, Lane 1: FAdV-4 GX-1, Lane2: AIV, Lane 3: NDV, Lane 4: IBDV, Lane 5: ALV, Lane 6: ARV, Lane 7: MDV. The solid arrows indicate the target gene.
examination. Tissue samples were fixed in 10% formalin for 48 h at room temperature and processed for haematoxylin and eosin (HE) staining as previously described (Bogomoletz et al., 1980). Serum samples were tested for the presence of FAdV-specific antibodies using a commercial ELISA kit (BioChek, Scarborough, ME, USA).
Statistical Analysis SPSS 16.00 software (SPSS, Chicago, IL, USA) was used to analyze the result of antibody response. Results were represented as the means ± standard error of mean. Statistically significant differences were analyzed by one-way analysis of variance followed by Duncan’s post hoc test. Statistical significance was set as P < 0.05.
The genome of FAdV-4 GX-1 strain was 43,721 base pairs (bp) with a G+C content of 54.87% and contained 38 open reading frames. The complete nucleotide sequences were deposited to the GenBank with accession numbers MH454598. Phylogenetic analysis showed that strain GX-1 belongs to a cluster of FAdV-C with 98.5 and 98.3% identity to FAdV-4 KR5 (GenBank accession No: HE608152) and ON1 (GenBank accession No: GU188428) (Figure 3). The GX-1 complete sequence identity among the 9 Chinese FAdV-4 isolates was 99 to 100% and the isolates showed identity of 98 to 99% to B1 to 7/India, MX-SHP95/Mexico (Figure 3).The entire hexon gene of FAdV GX-1 strain 2814 bp in length. The phylogenetic analysis based on amino acid sequence of hexon gene was similar to that of complete genome (data not show).
Clinical Signs, Antibody Response, Gross Pathology, and Histology The pathogenicity of FAdV-4 GX-1 was examined by infection of young SPF chickens at 3 virus doses.
Figure 2. (A) Cytopathic effects induced by GX-1 in LMH cells 72 h after infection. (B) Non-infected LMH cells. (C) Electron micrograph of GX-1 viral particles.
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A young chicken with a clinical diagnosis of an HHS was used to identify the viral pathogen causing the infection. A plaque-forming virus was identified in an LMH cell culture infected with a liver homogenate and was plaque purified for DNA isolation. PCR analysis indicated the virus was a FAdV member and coinfections with AIV, NDV, IBDV, ALV, ARV, and MDV were absent (Figure 1). During serial passage in cell culture, the LMH cells showed severe cytopathic effects at 48 h post-infection (Figure 2A) when compared with liver homogenates from non-infected chickens were used (Figure 2B). We also identified virus particles in cell culture supernatants (Figure 2C). The isolate was designated GX-1.
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Figure 4. Survival rates (A), clinical scores (B), and antibody response (C) of chickens after inoculation with strain GX-1. Clinical scoring: 0 for normal, 1 for mild sign of lethargy and ruffled feathers, 2 for reluctant to move, lethargy, trembling, and loss of appetite, 3 for paralysis/prostration, and 4 for death. The mean scores per group per day are shown. The error bars indicate means ± standard error of mean. Asterisks (∗) mark the days in which the antibody titres were significantly different between inoculated and negative control groups (P < 0.001).
Infection of birds with 104 TCID50 resulted in 100% mortality at 2 and 4 dpi. A dose of 103 TCID50 resulted in a 16% survival rate before day 9 and at 102 TCID50 an 80% rate before day 6 (Figure 4A). During the experimental period, depression, ruffled feathers, huddling in corners, reluctant to move, loss
of appetence, and a crouching position were prominent clinical signs. All chickens in the control group remained alive and did not show any clinical signs. Chickens inoculated with 104 TCID50 of GX-1 showed mild or severe depression after 2 dpi and died within 2 to 4 d postinoculation. The clinical scores for 104 TCID50 of GX-1
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Figure 3. Fowl adenovirus phylogenetic tree based on the complete genome sequence. GX-1, red triangle. The number in parenthesis indicates the GenBank accession number for each fowl adenovirus sequence.
PATHOGENICITY OF A FOWL ADENOVIRUS SEROTYPE 4 IN CHINA
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infections were greater than 103 TCID50 at 2 to 4 dpi and at 103 TCID50 were greater than 102 TCID50 at 5 to 9 dpi (Figure 4B). An antibody response of SPF chickens against FAdV was detected at 7 different time points during postinfection. FAdV-specific antibodies appeared at 14 dpi, and continued to rise until 35 dpi. Differences between inoculated and negative control groups were statistically significant (P < 0.001) at 21, 28, 35, and 42 dpi (Figure 4C). The peak antibody titre was reached at 35 dpi and began to decrease at 42 dpi. At necropsy, livers from infected chickens were swollen and yellow brown with necrotic foci. The hearts were flabby with amber-colored and jelly-like fluid in
the pericardial sacs (Figure 5A and B). The kidneys were swollen and congested (Figure 5D and E). No significant gross lesions were present in the control chicken tissues (Figure 5C and F). Histopathological examination revealed that heart muscle fibers were fractured with extensive congestion and hemorrhaging (Figure 6A, solid arrows). The most characteristic histological lesions in the liver were eosinophilic intranuclear inclusion bodies (Figure 6B, solid arrows). In addition, small multifocal areas of coagulative necrosis and hydropic degeneration were observed in hepatocytes (Figure 6B). The kidneys were edematous with renal tubule degeneration and necrosis (Figure 6C). The lymphocytes were degenerated and
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Figure 5. Gross lesions in liver, heart, and kidney from dead chickens. (A and D) Controls. (B and C). Examples of swollen and friable livers with multifocal areas of necrosis and petechial hemorrhages and hydropericardia. (E and F). Mild and severe kidney enlargement in GX-1infected chickens.
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necrotic in the bursa of Fabricius (Figure 6H). The pathological changes in thymus included degeneration and necrosis of the endotheliocytes as well as lymphopenia (Figure 6I). Fewer lymphocytes and degeneration and necrosis of endotheliocytes were present in spleen tissues compared with controls (Figure 6J). No lesions were observed in the corresponding tissues of chickens in the control group (Figure 6D–F, K-M).
DISCUSSION In recent years, FAdV strains related to IBH, HHS, or gizzard erosion have been isolated from most clinical cases in many countries. Clinical presentations of severe hydropericardia have recently emerged and this infection has rapidly spread through broiler and layer farms in many regions of China. FAdV serotype 4 GX1strain isolated from hydropericardium syndrome in the current study was judged to be highly pathogenic for chicks, killing all virus-infected chicks within 2 to 4 d at a dose of 104 TCID50. The dead animals showed severe hydropericardium and IBH, which were almost identical to those of naturally infected chickens. At a dose of 103 TCID50 resulted in a 16% survival and
at 102 TCID50 , an 80% rate. This result indicated that the mortality in chickens were time- and dosagedependent. These results were similar to other studies with other FAdV-4 isolates (Vera-Hernandez et al., 2016). Compared with other highly pathogenic FAdV-4 strains, the infectious dose which caused 100% mortality in SPF chickens was lower or similar to other experimental studies (Mase et al., 2010; Vera-Hernandez et al., 2016). HHS caused by FAdV-4 is characterized by accumulation of transparent or straw-colored fluid in the pericardial sac and hepatitis and our results were similar (see Figure 3). We also found either mild or severe pathologic changes in the spleen, thymus, bursa of Fabricius, and kidney. Previous reports hypothesized that the systemic distribution of the virus in lymphoid tissues might be associated with the mortality effect in chickens at a high viral dose (Naeem et al., 1995). Additionally, a severe depletion of lymphocytes and a generalized suppression of the humoral and cellular immune systems were observed after infection with a highly pathogenic FAdV-4 strain in chickens (Schonewille et al., 2008). In our study, lymphocytes showed degeneration and necrosis in immune organs that may be associated with
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Figure 6. Histopathology of tissues from chickens infected with strain GX-1. Heart: solid arrows indicate muscle fibers in heart are fracture. Liver: solid arrows indicate viral inclusion bodies.
PATHOGENICITY OF A FOWL ADENOVIRUS SEROTYPE 4 IN CHINA
ACKNOWLEDGMENTS This study was supported by Guangdong Wen’s Dahuanong Biotechnology Co., Ltd.
REFERENCES Abe, T., K. Nakamura, H. Tojo, M. Mase, T. Shibahara, S. Yamaguchi, and N. Yuasa. 1998. Histology, immunohistochemistry, and ultrastructure of hydropericardium syndrome in adult broiler breeders and broiler chicks. Avian Dis. 42:606–612. Anjum, A. D., M. A. Sabri, and Z. Iqbal. 1989. Hydropericarditis syndrome in broiler chickens in Pakistan. Vet. Rec. 124:247–248. Asthana, M., R. Chandra, and R. Kumar. 2013. Hydropericardium syndrome: current state and future developments. Arch. Virol. 158:921–931. Bogomoletz, W. V., J. J. Adnet, S. Widgren, M. Stavrou, and J. E. McLaughlin. 1980. Cystadenoma of the pancreas: a histological, histochemical and ultrastructural study of seven cases. Histopathology 4:309–320. Guan, R., Y. Tian, X. Han, X. Yang, and H. Wang. 2018. Complete genome sequence and pathogenicity of fowl adenovirus serotype 4 involved in hydropericardium syndrome in Southwest China. Microb. Pathog. 117:290–298. Hess, M. 2000. Detection and differentiation of avian adenoviruses: a review. Avian Pathol. 29:195–206. Jiang, L., X. Qi, Y. Gao, Y. Hua, K. Li, X. Deng, Q. Wang, L. Zhang, H. Chai, Y. Chen, C. Yin, H. Gao, L. Qin, Y. Wang, Y. Qu, Q. Chen, Z. Fan, and X. Wang. 2013. Molecular characterization and phylogenetic analysis of the reticuloendotheliosis virus isolated from wild birds in Northeast China. Vet. Microbiol. 166:68–75. Kim, J. N., S. H. Byun, M. J. Kim, J. Kim, H. W. Sung, and I. P. Mo. 2008. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates. Avian Dis. 52:526–530. Liu, D., X. B. Zhang, Z. Q. Yan, F. Chen, J. Ji, J. P. Qin, H. Y. Li, J. P. Lu, Y. Xue, J. J. Liu, Q. M. Xie, J. Y. Ma, C. Y. Xue, and Y. Z. Bee. 2013. Molecular characterization and phylogenetic analysis of infectious bursal disease viruses isolated from chicken in South China in 2011. Trop. Anim. Health Prod. 45: 1107–1112.
Liu, L., Y. Qu, T. Wang, G. Wang, F. Wang, and S. Liu. 2014. Skin involvement in lymphomas caused by Marek’s disease virus infection in Silkie chickens. J. Vet. Diagn. Invest. 26:302–307. Lu, Z., L. Zhang, N. Wang, Y. Chen, L. Gao, Y. Wang, H. Gao, Y. Gao, K. Li, X. Qi, and X. Wang. 2015. Naturally occurring reassortant infectious bursal disease virus in northern China. Virus Res. 203:92–95. Maartens, L. H., H. W. Joubert, H. Aitchison, and E. H. Venter. 2014. Inclusion body hepatitis associated with an outbreak of fowl adenovirus type 2 and type 8b in broiler flocks in South Africa. J. S. Afr. Vet. Assoc. 85:e1–5. Mase, M., K. Nakamura, and T. Imada. 2010. Characterization of Fowl adenovirus serotype 4 isolated from chickens with hydropericardium syndrome based on analysis of the short fiber protein gene. J. Vet. Diagn. Invest. 22:218–223. Naeem, K., T. Niazi, S. A. Malik, and A. H. Cheema. 1995. Immunosuppressive potential and pathogenicity of an avian adenovirus isolate involved in hydropericardium syndrome in broilers. Avian Dis. 39:723–728. Nakamura, K., M. Mase, S. Yamaguchi, T. Shibahara, and N. Yuasa. 1999. Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome. Avian Dis. 43:414–423. Niu, Y., Q. Sun, G. Zhang, W. Sun, X. Liu, Y. Xiao, Y. Shang, and S. Liu. 2018. Epidemiological investigation of outbreaks of fowl adenovirus infections in commercial chickens in China. Transbound Emerg. Dis. 65:e121–e126. Niu, Y. J., W. Sun, G. H. Zhang, Y. J. Qu, P. F. Wang, H. L. Sun, Y. H. Xiao, and S. D. Liu. 2016. Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015. J. Gen. Virol. 97:2684–2690. Pan, Q., L. Liu, Y. Gao, C. Liu, X. Qi, Y. Zhang, Y. Wang, K. Li, L. Gao, X. Wang, and H. Cui. 2017. Characterization of a hypervirulent fowl adenovirus 4 with the novel genotype newly prevalent in China and establishment of reproduction infection model of hydropericardium syndrome in chickens. Poult. Sci. 96:1581–1588. Schachner, A., M. Matos, B. Grafl, and M. Hess. 2018. Fowl adenovirus-induced diseases and strategies for their control - a review on the current global situation. Avian Pathol. 47:111–126. Schonewille, E., A. Singh, T. W. Gobel, W. Gerner, A. Saalmuller, and M. Hess. 2008. Fowl adenovirus (FAdV) serotype 4 causes depletion of B and T cells in lymphoid organs in specific pathogenfree chickens following experimental infection. Vet. Immunol. Immunopathol. 121:130–139. Steer, P. A., N. C. Kirkpatrick, D. O Rourke, and A. H. Noormohammadi. 2009. Classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region. J. Clin. Microbiol. 47:311–321. Vera-Hernandez, P. F., A. Morales-Garzon, D. V. Cortes-Espinosa, A. Galiote-Flores, L. J. Garcia-Barrera, E. T. Rodriguez-Galindo, A. Toscano-Contreras, E. Lucio-Decanini, and A. E. Absalon. 2016. Clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl Aviadenovirus serotype 4. Avian Pathol. 45:73–81. Wang, Q., X. Li, X. Ji, J. Wang, N. Shen, Y. Gao, X. Qi, Y. Wang, H. Gao, S. Zhang, and X. Wang. 2014. A recombinant avian leukosis virus subgroup j for directly monitoring viral infection and the selection of neutralizing antibodies. PLoS One 9:e115422. Ye, J., G. Liang, J. Zhang, W. Wang, N. Song, P. Wang, W. Zheng, Q. Xie, H. Shao, Z. Wan, C. Wang, H. Chen, W. Gao, and A. Qin. 2016. Outbreaks of serotype 4 fowl adenovirus with novel genotype, China. Emerg. Microbes Infect. 5:e50–e50. Zhang, T., Q. Jin, P. Ding, Y. Wang, Y. Chai, Y. Li, X. Liu, J. Luo, and G. Zhang. 2016. Molecular epidemiology of hydropericardium syndrome outbreak-associated serotype 4 fowl adenovirus isolates in central China. Virol. J. 13:188. Zhao, J., Q. Zhong, Y. Zhao, Y. X. Hu, and G. Z. Zhang. 2015. Pathogenicity and complete genome characterization of fowl adenoviruses isolated from chickens associated with inclusion body hepatitis and hydropericardium syndrome in China. PLoS One 10:e0133073.
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immunosuppression. These results provide a more detailed insight into the pathogenesis of GX-1. An epidemiological survey showed that FAdV infections were caused by different FAdV species in China, such as species C, D, and E (Niu et al., 2018). In the current study, the representative strains of 5 species of FAdV were downloaded from GenBank to perform the phylogenetic analysis. Based on the complete genome sequence, strain GX-1 belongs to a cluster of FAdV-C. As reported, more than one-half of the FAdVs isolated were genetically related to serotype 4 proving that this serotype was dominant in China from 2015 to 2016 (Niu et al., 2018). A wide range of phylogenetic analyses at the whole genome level would be necessary to better understand the evolutionary relationships between avian adenovirus isolates (Zhao et al., 2015). Our phylogenetic analysis based on the hexon gene showed GX-1 strain classified into the same cluster. This result revealed the FAdV-4 isolates obtained from chickens with HHS in China have a common ancestor and belong to the most predominant serotype in China.
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