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Abstract In order to obtain a fowl avidenovirus type 4 strain with good immunogenicity， chicken liver tissues suspected of adenovirus infection in a chicken farm in Binzhou were treated and then inoculated to chicken liver hepatocellular carcinoma cells （LMH）. The cell cultures were extracted for DNA， which was subjected to PCR identification and sequencing analysis， and animal regression test and immunogenicity test were also carried out. The results showed that one fowl avidenovirus strain was successfully isolated. The isolated strain was inoculated to LMH cells， and the first generation showed obvious cytopathic changes. The PCR identification result of the 8th generation cell culture of the isolated virus strain on LMH cells was positive. The sequencing result and NCBI sequence alignment analysis showed that the isolated virus strain had the highest nucleotide similarity with fowl avidenovirus type 4， reaching 100%， indicating that the isolated strain was of fowl avidenovirus type 4. The strain could cause the death of 21-day-old SPF chickens， with a mortality rate of 100%， and could completely replicate the same symptoms as clinically infected chickens after being challenged. The three batches of oil vaccine prepared with the isolated strain had a protection rate of 100%， and the geometric mean values of serum agar expansion titers were 1∶4.6， 1∶4.9， and 1∶4.6， respectively. It can be seen that the isolated virus is of fowl avidenovirus type 4 in group I， and has good immunogenicity.
Key words Fowl avidenovirus; Isolation and identification; Immunogenicity; Cytopathic effect; SPF chicken
Received： April 16， 2021 Accepted： June 18， 2021 2
Supported by Poultry Innovation Team Project of Agriculture Research System in Shandong Province （SDAIT-11-16）; 2017 Shandong Province Foreign Experts Double Hundred Plan Project （2017 Double Hundred Program for Chinese and British Foreign Experts）; Key Project of Natural Science Foundation of Shandong Province （ZR2020KC006）.
Feng WEI （1979-） ， female， P. R. China， research assistant， devoted to research about biological products.
*Corresponding author. E-mail： bzshenzq@163.com.
Fowl avidenovirus （FADV） is a double-stranded DNA virus without an envelope， 60 to 90 nm in diameter， belonging to the genus of Aviadenovirus in the family of Adenoviridae. FADV can be divided into 3 groups， among which fowl avidenovirus group I （FADV I） can be divided into 5 species A， B， C， D， E and 12 serotypes[1]， which can cause a variety of diseases in poultry， including inclusion body hepatitis-anaemia syndrome. At present， FADV I prevalent in chickens in China is mainly fowl avidenovirus type 4 （FADV-4）. Chicken hepatitis and hydropericardium syndrome （HHS） is a viral infectious disease caused by FADV-4 which mainly affects broilers[2]. Both Ma chickens and laying hens can be infected with fowl avidenovirus. In addition， ostriches and ducks have also been reported to be infected[3-5]. Immunogenicity test
On the 10th d after the challenge， all chickens in the 20190601 group， 20190602 group， and 20190603 group were alive， and all 10 chickens in the control group died. A necropsy of the dead chickens revealed a large amount of yellow effusion in the pericardium， and the liver was enlarged with alternated yellow and red colors. On the 21st d after immunization， the geometric mean values of the serum agar expansion titers of the three batches of vaccine were 1∶4.6， 1∶4.9， and 1∶4.6， as shown in Table 1.
Discussion
In recent years， avian adenovirus infection is one of the important infectious diseases affecting the poultry industry. Multiple types of FADV and immunosuppressive viruses can cause mixed infection of poultry[9]. Generally speaking， adenovirus is a kind of epithelial cell virus， which is easier to multiply and survive in the liver， pancreas and intestines， so the liver is the best place to get materials. FADV I generally does not infect animals other than poultry， and does not survive and reproduce well in cells not derived from poultry. Therefore， it is best to use poultry-derived cells to culture this virus， such as chicken kidney cells and liver cells. FADV-infected cells generally have obvious cytopathic changes. For example， the cells become round， grape-shaped， show a refractive index becoming strong， and finally fall off[10]. Zhao et al.[11] found that LMH cells are liver epithelial cells， and adenovirus tends to infect epithelial cells， which may be related to epithelial cells expressing higher levels of viral receptors. In this study， the clinical disease materials were treated and inoculated to LMH cells， and the first generation produced obvious cytopathic changes. The virus titer of the 6th generation of culture could reach 1×108.5 TCID50/ml. Mansoor et al.[12] obtained an attenuated vaccine strain by passing adenovirus on chicken embryos for 12 consecutive times. The vaccination and challenge comparison test confirmed that compared with the inactivated vaccine， the attenuated vaccine could provide better protection for the immunized chickens， and the survival rate of the experimental chickens was higher. In this study， an oil emulsion inactivated vaccine prepared from the isolated strains could provide 100% protection to chickens， showing that the FADV-4 oil emulsion inactivated vaccine could also provide better protection for immunized chickens. Niu et al.[13] conducted an investigation on FADV infection in chicken flocks in China from 2014 to 2016 and found that FADV infection was widespread in chicken flocks in most areas of China， and it caused morbidity and death in chicken flocks. And the lesions of sick and dead chickens in necropsy were mainly pericardial effusion， liver enlargement and hemorrhage. Clinically， most cases of pericardial effusion have mixed infection with chicken infectious anemia virus， chicken infectious bursal disease virus and other pathogens[14]， which is also an important factor leading to high morbidity and mortality after avian adenovirus infection. The results of the animal regression test in this study showed that the dead chickens infected with the FADV-4 isolate showed a large amount of yellow effusion in the pericardium， and enlarged liver with alternated yellow and red colors in autopsy， which are consistent with the symptoms caused by the virus-causing strain. The results of the immunization test showed that the serum agar expansion titers in various immunization groups were 1∶4.6， 1∶4.9， and 1∶4.6， respectively， which are higher than the antibody agar expansion titer （1∶2） reported by Lu[15]. It showed that the strain had good immunogenicity. Conclusions
In this study， one strain of chicken-derived fowl avidenovirus was obtained through cell isolation and culture. After identification， the isolated virus was determined to be FADV-4. Three batches of oil vaccine prepared from the isolated strain were used to immunize 21-day-old chickens， and the protection rate was 100%. This study provides reference for the isolation， vaccine research and disease prevention and control of chicken-derived fowl avidenovirus.
References
[1] NICZYPORUK JS. Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland[J]. Arch Virol， 2016， 161（1）： 33-42.
[2] SAIF YM. Poultry disease[M]. 12th edition. SU JL， GAO F， SUO X， translated. Beijing： China Agriculture Press， 2012. （in Chinese）
[3] WANG H， CHENG XG， GUO JY， et al. The recent prevalence and control of fowl adenovirus I in chickens[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine， 2016（2）： 62-63. （in Chinese）
[4] LI CJ， WANG DD， WANG JL， et al. Gene sequence analysis of hexon protein and separation identification and serotype identification of fowl adenovirus group I in ostrich[J].Chinese Journal of Veterinary Medicine， 2016， 52（12）： 14-16， 20， 50. （In Chinese）
[5] LIU JS， XIAO YQ， YU X， et al. Prevention and treatment of the disease in Shelduck resulted from a fowl adenovirus type 4 infection[J]. Chinese Veterinary Science， 2017， 47（9）： 1112-1117. （in Chinese）
[6] TORRED DL， NU EZ LFN， SANTANDER PARRA SH， et al. Molecular characterization of fowl adenovirus group I in commercial broiler chickens in Brazil[J]. Virus Disease， 2018， 29（1）： 83-88.
[7] ZHAO J， RUAN S， GUO Y， et al. Serological and phylogenetic analysis indicating prevalence of fowl adenovirus in China[J]. Vet Rec， 2018， 182（13）： 381.
[8] ZHAO L， LI LIN， SHI AH， et al. Isolation and identification of 5 avian adenoviruses of group I and type 4[J]. China Poultry， 2018， 40（22）： 48-51. （in Chinese）
[9] LIU JH， GAN MH. Chinese poultry diseases[M]. 2nd edition. Beijing： China Agriculture Press， 2016. （In Chinese）
[10] NIU YJ. Isolated， identification and pathogenic mechanism of fowl adenovirus[D]. Tai’an： Shandong Agricultural University， 2018. （in Chinese）
[11] ZHAO L， ZHOU J， GAO C， et al. Study on the proliferation of type I fowl adenovirus AV208 strain in chicken liver cancer cells[J]. Microbiology China， 201 39（8）： 1120-1126.（in Chinese）
[12] MANSOOR MK， HUSSAIN I， ARSHAD M， et al. Preparation and evaluation of chickens embryo-adapted fowl adenovirus serotype 4 vaccine in broiler chickens[J]. Trop Anim Health Prod， 201 43（2）： 331-338.
[13] NIU DY， SHEN Y， WANG R， et al. Molecular epidemiological survey of fowl adenovirus in China in 2015[J].China Poultry， 2016， 38（9）： 65-68. （in Chinese）
[14] HU RL. Modern animal virology[M]. Beijing： China Agriculture Press， 2014. （in Chinese）
[15] LU W. Optimization of multivalent inactivated fowl adenovirus subgroup I in chicken and study on immune efficacy[D].Yangzhou： Yangzhou University， 2018. （in Chinese）
Key words Fowl avidenovirus; Isolation and identification; Immunogenicity; Cytopathic effect; SPF chicken
Received： April 16， 2021 Accepted： June 18， 2021 2
Supported by Poultry Innovation Team Project of Agriculture Research System in Shandong Province （SDAIT-11-16）; 2017 Shandong Province Foreign Experts Double Hundred Plan Project （2017 Double Hundred Program for Chinese and British Foreign Experts）; Key Project of Natural Science Foundation of Shandong Province （ZR2020KC006）.
Feng WEI （1979-） ， female， P. R. China， research assistant， devoted to research about biological products.
*Corresponding author. E-mail： bzshenzq@163.com.
Fowl avidenovirus （FADV） is a double-stranded DNA virus without an envelope， 60 to 90 nm in diameter， belonging to the genus of Aviadenovirus in the family of Adenoviridae. FADV can be divided into 3 groups， among which fowl avidenovirus group I （FADV I） can be divided into 5 species A， B， C， D， E and 12 serotypes[1]， which can cause a variety of diseases in poultry， including inclusion body hepatitis-anaemia syndrome. At present， FADV I prevalent in chickens in China is mainly fowl avidenovirus type 4 （FADV-4）. Chicken hepatitis and hydropericardium syndrome （HHS） is a viral infectious disease caused by FADV-4 which mainly affects broilers[2]. Both Ma chickens and laying hens can be infected with fowl avidenovirus. In addition， ostriches and ducks have also been reported to be infected[3-5]. Immunogenicity test
On the 10th d after the challenge， all chickens in the 20190601 group， 20190602 group， and 20190603 group were alive， and all 10 chickens in the control group died. A necropsy of the dead chickens revealed a large amount of yellow effusion in the pericardium， and the liver was enlarged with alternated yellow and red colors. On the 21st d after immunization， the geometric mean values of the serum agar expansion titers of the three batches of vaccine were 1∶4.6， 1∶4.9， and 1∶4.6， as shown in Table 1.
Discussion
In recent years， avian adenovirus infection is one of the important infectious diseases affecting the poultry industry. Multiple types of FADV and immunosuppressive viruses can cause mixed infection of poultry[9]. Generally speaking， adenovirus is a kind of epithelial cell virus， which is easier to multiply and survive in the liver， pancreas and intestines， so the liver is the best place to get materials. FADV I generally does not infect animals other than poultry， and does not survive and reproduce well in cells not derived from poultry. Therefore， it is best to use poultry-derived cells to culture this virus， such as chicken kidney cells and liver cells. FADV-infected cells generally have obvious cytopathic changes. For example， the cells become round， grape-shaped， show a refractive index becoming strong， and finally fall off[10]. Zhao et al.[11] found that LMH cells are liver epithelial cells， and adenovirus tends to infect epithelial cells， which may be related to epithelial cells expressing higher levels of viral receptors. In this study， the clinical disease materials were treated and inoculated to LMH cells， and the first generation produced obvious cytopathic changes. The virus titer of the 6th generation of culture could reach 1×108.5 TCID50/ml. Mansoor et al.[12] obtained an attenuated vaccine strain by passing adenovirus on chicken embryos for 12 consecutive times. The vaccination and challenge comparison test confirmed that compared with the inactivated vaccine， the attenuated vaccine could provide better protection for the immunized chickens， and the survival rate of the experimental chickens was higher. In this study， an oil emulsion inactivated vaccine prepared from the isolated strains could provide 100% protection to chickens， showing that the FADV-4 oil emulsion inactivated vaccine could also provide better protection for immunized chickens. Niu et al.[13] conducted an investigation on FADV infection in chicken flocks in China from 2014 to 2016 and found that FADV infection was widespread in chicken flocks in most areas of China， and it caused morbidity and death in chicken flocks. And the lesions of sick and dead chickens in necropsy were mainly pericardial effusion， liver enlargement and hemorrhage. Clinically， most cases of pericardial effusion have mixed infection with chicken infectious anemia virus， chicken infectious bursal disease virus and other pathogens[14]， which is also an important factor leading to high morbidity and mortality after avian adenovirus infection. The results of the animal regression test in this study showed that the dead chickens infected with the FADV-4 isolate showed a large amount of yellow effusion in the pericardium， and enlarged liver with alternated yellow and red colors in autopsy， which are consistent with the symptoms caused by the virus-causing strain. The results of the immunization test showed that the serum agar expansion titers in various immunization groups were 1∶4.6， 1∶4.9， and 1∶4.6， respectively， which are higher than the antibody agar expansion titer （1∶2） reported by Lu[15]. It showed that the strain had good immunogenicity. Conclusions
In this study， one strain of chicken-derived fowl avidenovirus was obtained through cell isolation and culture. After identification， the isolated virus was determined to be FADV-4. Three batches of oil vaccine prepared from the isolated strain were used to immunize 21-day-old chickens， and the protection rate was 100%. This study provides reference for the isolation， vaccine research and disease prevention and control of chicken-derived fowl avidenovirus.
References
[1] NICZYPORUK JS. Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland[J]. Arch Virol， 2016， 161（1）： 33-42.
[2] SAIF YM. Poultry disease[M]. 12th edition. SU JL， GAO F， SUO X， translated. Beijing： China Agriculture Press， 2012. （in Chinese）
[3] WANG H， CHENG XG， GUO JY， et al. The recent prevalence and control of fowl adenovirus I in chickens[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine， 2016（2）： 62-63. （in Chinese）
[4] LI CJ， WANG DD， WANG JL， et al. Gene sequence analysis of hexon protein and separation identification and serotype identification of fowl adenovirus group I in ostrich[J].Chinese Journal of Veterinary Medicine， 2016， 52（12）： 14-16， 20， 50. （In Chinese）
[5] LIU JS， XIAO YQ， YU X， et al. Prevention and treatment of the disease in Shelduck resulted from a fowl adenovirus type 4 infection[J]. Chinese Veterinary Science， 2017， 47（9）： 1112-1117. （in Chinese）
[6] TORRED DL， NU EZ LFN， SANTANDER PARRA SH， et al. Molecular characterization of fowl adenovirus group I in commercial broiler chickens in Brazil[J]. Virus Disease， 2018， 29（1）： 83-88.
[7] ZHAO J， RUAN S， GUO Y， et al. Serological and phylogenetic analysis indicating prevalence of fowl adenovirus in China[J]. Vet Rec， 2018， 182（13）： 381.
[8] ZHAO L， LI LIN， SHI AH， et al. Isolation and identification of 5 avian adenoviruses of group I and type 4[J]. China Poultry， 2018， 40（22）： 48-51. （in Chinese）
[9] LIU JH， GAN MH. Chinese poultry diseases[M]. 2nd edition. Beijing： China Agriculture Press， 2016. （In Chinese）
[10] NIU YJ. Isolated， identification and pathogenic mechanism of fowl adenovirus[D]. Tai’an： Shandong Agricultural University， 2018. （in Chinese）
[11] ZHAO L， ZHOU J， GAO C， et al. Study on the proliferation of type I fowl adenovirus AV208 strain in chicken liver cancer cells[J]. Microbiology China， 201 39（8）： 1120-1126.（in Chinese）
[12] MANSOOR MK， HUSSAIN I， ARSHAD M， et al. Preparation and evaluation of chickens embryo-adapted fowl adenovirus serotype 4 vaccine in broiler chickens[J]. Trop Anim Health Prod， 201 43（2）： 331-338.
[13] NIU DY， SHEN Y， WANG R， et al. Molecular epidemiological survey of fowl adenovirus in China in 2015[J].China Poultry， 2016， 38（9）： 65-68. （in Chinese）
[14] HU RL. Modern animal virology[M]. Beijing： China Agriculture Press， 2014. （in Chinese）
[15] LU W. Optimization of multivalent inactivated fowl adenovirus subgroup I in chicken and study on immune efficacy[D].Yangzhou： Yangzhou University， 2018. （in Chinese）