Isolation, Identification and Immunogenicity of a Chicken-derived Strain of Fowl Avidenovirus Type 4

Feng WEI Wentong ZHANG Na TANG Yan WANG Lizhong MIAO Jinliang WANG Zhiqiang SHEN

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].

At present， FADV-4 is prevalent in various countries in the world. Since 2015， fowl avidenovirus has been widespread in Shandong， Jiangsu， Henan， Anhui and other places in China. They can infect chickens of different ages and breeds. The morbidity and mortality of chickens vary by region and breed. The fatality rate of broilers aged 3 to 5 weeks is as high as 40% to 100%， which has caused huge economic losses to Chinas poultry production[6-8]. In 2018， a chicken farm in Binzhou City， Shandong Province showed sick and dead chickens with liver enlargement， necrosis， and pericardial effusion as the main clinical symptoms. We collected the livers of the sick and dead chickens from the chicken farm for the isolation and identification of an FADV strain， and the immunogenicity of the isolated strain was studied， hoping to provide reference for further FADV epidemiological investigation and disease prevention and control.

Materials and Methods

Disease samples， SPF chickens and cells

Liver tissues of dead chickens were collected from chickens suspected of FADV infection in a chicken farm in Binzhou City， Shandong Province; 55 21-day-old SPF chickens were purchased from Jinan Saifu Experimental Animal Breeding Co.， Ltd.; and chicken liver cancer （LMH） cells were preserved by the Key Laboratory of Shandong Binzhou Animal Science & Veterinary Medicine Academy.

Main regents and instruments

DMEM medium and DMEM/F-1 purchased from Gibco; newborn bovine serum， purchased from Tianjin Kangyuan Biotechnology Co.， Ltd.; fetal bovine serum， purchased from Hyclone Bioengineering Company; pancreatin， purchased from Sigma; nucleic acid extraction kit， agarose gel DNA recovery kit （spin column type） and DL-2 000 Marker， all purchased from TaKaRa Biotechnology （Dalian） Co.， Ltd.; carbon dioxide incubator （model MC015AC）， purchased from Shandong Aibo Technology & Trading Co.， Ltd.; inverted microscope （model XDS-IB）， purchased from Chongqing Optoelectronic Instrument Co.， Ltd.; electric heating constant temperature incubator， purchased from Huangshi Hengfeng Medical Equipment Co.， Ltd.

Isolation and passage of viruses

The collected liver tissues of sick and dead chickens were cut into small pieces， and added with sterile PBS （pH 7.2） at a ratio of 1∶10. The mixture was shaken and ground， frozen and thawed repeatedly for 3 times， and centrifuged at 4 ℃， 8 000 r/min for 10 min. The supernatant was filtered with a 0.45 μm filter membrane and inoculated with LMH cells that had grown into a monolayer. The cells were cultured in a 5% CO2 incubator at 37 ℃ for 72 to 96 h， and the cell status was observed every day. The cells were harvested after 85% of the cells showed infection， and the harvested cell culture was frozen and thawed 3 times and then re-inoculated with LMH cells. The cells were cultured to the 8th generation， and the cell cultures of the 6th， 7th and 8th generations were frozen for later use.

PCR identification and sequencing of viruses

Primers were designed based on the sequence of MF496037 in GenBank for PCR detection of FADV. The primer sequences were as follows： upstream 5-CCCAAGGAGTCCATGTTTAA-3; downstream 5-CGTGGTGCCTATGTTAT-3. The nucleic acid extraction kit was used to extract DNA genome from the 8th generation of cell culture harvested under "Isolation and passage of viruses". The PCR amplification system （30 μl） included 10×PCR Buffer 2.5 μl， dNTP （10 mmol /L） 1.0 μl， the upstream and downstream primers 0.5 μl each， rTaq 0.5 μl， DNA 4 μl， sterile water 21 μl. The PCR amplification program was started with 94 ℃ for 3 min， followed by 35 cycles of 94 ℃ for 30 s， 55 ℃ for 30 s， and 72 ℃ for 30 s， and completed with final extension at 72 ℃ for 10 min. The PCR products were detected by 1.0% agarose gel electrophoresis， and the target fragments were recovered with an agarose gel DNA recovery kit （spin column type）， and sent to Sangon Biotech （Shanghai） Co.， Ltd. for sequencing. The sequencing results were subjected to BLAST alignment in NCBI.

Determination of virus titer

The LMH cells were subcultured at a ratio of 1∶3. After the cells were counted， they were transferred to a 96-well cell culture plate at a cell concentration of 1.5×105/ml， 0.1 ml/well， and cultured in a 5% CO2 incubator at 37 ℃ for 24 h. When inoculating the virus， the cell growth medium in the cell culture plate was discarded， and the cell cultures of the 6th， 7th and 8th generations were diluted with DMEM in a 10-fold gradient （1×101-1×108）. Each dilution was inoculated to 8 wells at 0.1 ml/well. Meanwhile， a healthy cell control was set. The cell culture plate was put into a 5% CO2 incubator at 37 ℃ for 7 d， and the cytopathic condition was observed every day. According to the Reed-Muench method， 50% tissue culture infective dose （TCID50） of each generation of virus was calculated.

Animal regression test

Fifteen 21-day-old SPF chickens were divided into two groups， the experimental group and the blank control group. In the experimental group， 10 animals were injected intramuscularly with 0.5 ml of the 8th generation cell culture of chicken-derived fowl avidenovirus isolated and cultured on each chest. Five chickens in the blank control group were injected with the same amount of saline intramuscularly. The morbidity and death of the two groups of chickens were observed and recorded every day. The dead chickens were dissected， and the liver tissues of the dead chickens were taken and detected for the viruses by the PCR method the same as "PCR identification and sequencing of viruses".

Immunogenicity test

The 6th， 7th and 8th generations of virus cell cultures （100 ml each） were added with formaldehyde at 1/1 000， and inactivated at 37 ℃， 120 r/min for 24 h. After the inactivation test， three batches of oil vaccine were prepared according to the ratio of oil to water at 2∶ and they were numbered 2019060 2019060 and 20190603， respectively. The 21-day-old SPF chickens were divided into 4 groups （20190601 group， 20190602 group， 20190603 group， control group）， each with 10 chickens. Among them， the 20190601 group， the 20190602 group and the 20190603 group were the immunization groups， which were injected intramuscularly with the 2019060 20190602 and 20190603 batches of vaccine， respectively， 0.2 ml per chicken. In the control group， 10 chickens were injected with the same amount of saline. On the 21st d after immunization， the blood of each group of chickens was collected， and the serum was separated. The agar titer of each serum was detected by agar gel diffusion precipitin test （AGP）， and the geometric mean was calculated. Meanwhile， the chickens of the immunization groups and the control group were challenged. Specifically， each chicken was injected with 0.5 ml of virus culture （1×108.0 TCID50/ml） into the chest muscles， and observed continuously for 10 d， and the morbidity and death of the chickens were recorded every day.

Results and Analysis

Virus isolation and titer determination

The treated liver tissue material was inoculated to LMH cells. About 60% of the cells showed cytopathy at 24 h after inoculation， and about 80% of cells showed cytopathy at 36 h after inoculation. The cells became round and gathered into irregular grape clusters， and the intercellular spaces increased （Fig. 1）. After testing， the virus titers of the 6th， 7th and 8th generations of cell cultures were 1×108.5， 1×108.625 and 1×108.5 TCID50/ml， respectively.

PCR identification

The 8th generation of the cell culture of the isolated virus strain was identified as positive by PCR， as shown in Fig. 2. The sequencing results of the PCR products were subjected to BLAST alignment in NCBI. The results showed that the nucleotide similarity to FADV-4 was the highest， reaching 100%， indicating that the isolated virus was FADV-4.

Animal regression test

On the 2nd d after the challenge， the chickens in the experimental group began to show depression and reduced feed intake symptoms. The chickens began to die on day 3 and all died on day 5. Chickens in the blank control group were still healthy on day 10. Autopsy of the dead chickens showed a large amount of yellow effusion in the pericardium， and the liver was yellow and red alternately. The PCR method was used to detect viruses in the liver tissues of sick and dead chickens， and the test results were positive， indicating that the dead chickens were infected with FADV-4.

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.

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