Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein

doi:10.3969/j.issn.1004-1524.2022.10.09

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (10): 2149-2159.DOI: 10.3969/j.issn.1004-1524.2022.10.09

• Animal Science • Previous Articles Next Articles

Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein

ZHU Yinchu¹(), WANG Hongyu¹^,², YUN Tao¹, HUA Jionggang¹, YE Weicheng¹, NI Zheng¹, CHEN Liu¹, ZHANG Cun¹^,^*()

1. Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2021-07-26 Online:2022-10-25 Published:2022-10-26
Contact: ZHANG Cun

Abstract

Abstract:

Goose astrovirus (GAstV) is an important pathogen in gosling, which causes viscera gout and death and leads to huge economic loss in the gosling-breeding industry in China. In the present study, a total of 30 gosling samples were collected from Zhejiang Province, China, and were identified by RT-PCR and pathogen isolation and sequencing. The prokaryotic expression plasmid pET-28a-ORF2 was constructed and transformed into BL21 strain for expression of the recombinant protein with induction. The results showed that all samples had typical viscera gout symptoms and were positive for goose astrovirus. Some samples were infected by two different genotypes of goose astrovirus. Strains ZJC14 and ZJLD20 were isolated from goose embryos. ZJLD20 could stably proliferate in both goose embryos and LMH cells, but ZJC14 could not adapt to LMH cell. ZJC14 and ZJLD20 were relatively distantly related. ZJC14 belonged to the genotype of GAstV-Ⅰ, yet ZJLD20 belonged to the genotype of GAstV-Ⅱ. The recombinant protein was purified and used to immunize rabbit for preparation of polyclonal antibodies. The prepared polyclonal antibodies could effectively bind to the protein. In addition, indirect immunofluorescence and Western-blot test results showed that the polyclonal antibody from the ZJLD20 capsid protein could effectively recognize the virus replicated in LMH. These findings would benefit the further study of the pathogenicity of GAstV. Meanwhile, the prepared capsid protein and polyclonal antibody would lay foundation for the development of diagnostic reagents for goose astrovirus infection.

Key words: goose astrovirus, isolation and identification, capside protein, prokaryotic expression, polyclonal antibody

CLC Number:

S858.33

ZHU Yinchu, WANG Hongyu, YUN Tao, HUA Jionggang, YE Weicheng, NI Zheng, CHEN Liu, ZHANG Cun. Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein[J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2149-2159.

Figures/Tables 8

Table 1 Basic information of primers used in study

目的基因 Target gene	片断大小 Fragment length/bp	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
TMUV-E	401	GCCACGGAATTAGCGGTTGT	TAATCCTCCATCTCAGCGGTGTAG
GPV-VP1	779	AGACTTATCAACAACCATTG	TCACTTATTCCTGCTGTAG
GRV-sigma C	380	TGAGACGCCTGACTACGATT	ATGCTTGGAGTGAGACGACT
GAstVⅠ-RbRp	441	GATACTTATGCTTCCACAAC	ATCTTGTTCAAAAGATGCTC
GAstVⅡ-RbRp	277	ACGACAGATGCGTTACTT	GGTGACATTATCCCTGAG
ZJLD20-Capsid	2 112	AGCAAATGGGTCGCGGATCCATGGCAGACAGGGCGGTGGC	CGGAGCTCGAATTCGGATCCCTCATGTCCGCCCTTCTCAA
ZJC14-Spike	834	AGCAAATGGGTCGCGGATCCGTAGTTCATTACCTGCCACT	CGGAGCTCGAATTCGGATCCTTGTCCACCCTTTTCAAAGC

Fig.1 Results of necropsy and double PCR for diseased goslings A, Urate deposition on the surfaces of the heart and liver; B, Severe nephritis with hemorrhage and swelling; C, Urate crystals accumulated in the gallbladder; D. Results of double RT-PCR for some clinical samples. M, DNA marker; N, Negative control.

Fig.2 Isolation of GAstV from goose embryo and LMH cells

Fig.3 RT-PCR results of GAstV replicated in goose embryo and LMH cells M, DNA marker; 1, PCR result of ZJLD20 in goose embryo; 2, PCR result of ZJC14 in goose embryo; 3. PCR result of ZJLD20 in LMH cell; 4, PCR result of ZJC14 in LMH cell. N, Negative control.

Fig.4 Phylogenic analysis of goose astrovirus based on nucleotide sequences

Fig.5 Phylogenetic analysis based on amino acid sequences of ORF2

Fig.6 Construction of ORF2 recombinant plasmid and protein expression A, PCR detection for recombinant vectors; B, SDS-PAGE detection for protein expression; C, Western-blot detection for polyclonal antibody. M, DNA marker; N, Negative control.

Fig.7 Western blot and IFA identification result of ORF2 polyclonal antibody with GAstV ZJLD20 Mock, Blank control.

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Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein

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