Preliminary Identification Of Novel Proteins In Porcine Astrovirus

Astrovirus(AstV) is a single-sense RNA virus that mainly infects mammals and poultry,and is also a zoonotic virus.Porcine astrovirus has a high infection rate of pigs and distributes over the world.Astrovirus mutates rapidly,and a lot of evidence that recombination between different strains can occur.Phylogenetic analysis and proteolysis revealed that astrovirus is a rapidly evolving virus that recombines and exchanges part of its capsid domain by replication,producing an extension of cellular tropism.It is evident that the virus has the ability to spread across species and adapt to new hosts,while the astrovirus is also associated with many new clinical symptoms such as encephalitis and gout.In this study,we used bioinformatics methods to predict the structure and function of two unknown proteins in astroviruses,and explored the effects of two proteins on the biological characteristics of the virus based on the reverse genetics of the porcine astrovirus.1.Prediction of structure and function as ORFX and ORFY proteins of porcine astrovirus and preparation of polyclonal antibodiesIn this study,the genome of astrovirus was analyzed by bioinformatics method.Two potential open reading frames,ORFX and ORFY,were found.ORFX overlaps with the 5' end of ORF2 and is separated from the start codon of ORF2 by 7 amino acids.ORFY is located in the conserved region P1 domain encoded by ORF2,and is separated from the start codon of ORF2 by about 299 amino acids.The protein library and related servers were used to simulate and calculate the structure of the protein,and the physicochemical properties,hydrophilicity,transmembrane region and secondary domain of the protein were known,and the tertiary structure model of the protein was finally established.According to the predicted structure,the 19-45aa amino acid sequence in ORFX is similar to the tertiary structure of the partial amino acid sequence of c3j9dA protein in the protein library.This protein is present in the bluetongue virus VP2,and the role of the ORFX protein is presumed mainly according to the c3j9dA protein.It is a pH sensor when the virus enters the host.The amino acid sequence of 1-62aa in ORFY is similar to the carboxy terminus of c5nbhB protein in the protein library.It can be concluded that the ORFY protein may be involved in the binding of host cells,which may provide a direction for the receptor search of astrovirus.To identify whether ORFX and ORFY encode viral proteins.The gene fragments of ORFX and ORFY were amplified and cloned into the prokaryotic expression vectors,resulting plasmids(pET-B2M-ORFX and pET-32a-ORFY).The pET-B2M-ORFX and pET-32a-ORFY were transformed into E.coli.Rosseta and E.coli.BL21 competent cells,respectively,to express the pET-B2M-ORFX and pET-32a-ORFY coding protein.Both proteins were found to express as inclusion body proteins in E.coli.by SDS-PAGE.The expressed proteins were purified and mixed with an adjuvant to form a suitable immunogen..Finally,New Zealand white rabbits were weekly immunized subcutaneously with ORFX or pET-32a-ORFY protein.Blood was collected from the heart,and serum was isolated to obtain antibodies against ORFX and ORFY after five times of immunization.The prepared antibodies were tested by IFA and found that the two polyclonal antibodies specifically bind to the ORFX and ORFY proteins expressed in the cells infected with the porcine astrovirus.2.Construction and rescue of pT7-ORFX and pT7-ORFY mutantsBased on an infectious porcine astrovirus pT7GX1 infectious clone,two mutant clones,pT7-ORFX and pT7-ORFY carrying ATG mutation of ORFX or ORFY were constricted by site-directed mutagenesis.The wild-type pT7GX1 and pT7-ORFX,pT7-ORFY mutant clones were rescued in PK-15 cells.The results showed that the wild-type pT7GX1 and the mutant strain pT7-ORFX showed CPE in PK-15 cells after five times passages,while the mutant pT7-ORFY showed no CPE until 8th passages.Are these two proteins present in the cells of the pT7-ORFX,pT7-ORFY mutant virus by IFA.It can be speculated that ORFX and ORFY are present in the porcine astrovirus.The replication of mutants in PK-15 cells were detected by qRT-PCR.The results showed that RNA copy number of the wild-type strain rT7GX1 viral RNA was higher than mutant viruses between 12 and 24 h.RNA copy number of mutant viruses increase significantly between 24-48 h.The wild-type strain rT7GX1 has a higher virus titer than the mutant viruses rT7-ORFX and rT7-ORFY.The results of this study showed that the ORFX protein and the ORFY protein are nonessential for virus viability but important for virus replication.