Preparation Of A Goose Embryo Fibroblast Adapted Goose Parvovirus Strain And Molecular Basis Of Cell Adaptation

Goose parvoviral disease,also named gosling plague,is a major pandemic disease threatening goslings within 30 days of age and circulates in geese-raising countries worldwide with high morbidity and mortality rates.The etiological agent of gosling plague is goose parvovirus(GPV),which is now classified in the Dependoparvovirus genus in the Parvovidae subfamily.Till now,lack of an efficient in vitro replication system for GPV limits our deeply elucidating the molecular pathogenesis of GPV.The virulent GPV can efficiently replicate in and kill the embryonated goose eggs,but grows poorly in goose embryo fibroblast(GEF),which is a disadvantage for us to further dissect the interaction between the host cellular proteins and the virally coded proteins.This thesis study aims at creating a GPV strain that can replicate efficiently in goose embryo fibroblast(GEF)and unveiling the molecular basis underlying the cellular adaptability for GPV.1 Creation of a goose embryo fibroblast-adapted strain of GPVSYG61v is an attenuated GPV strain,which has a good propagation capability in embryonated goose embryos.In this study,SYG61v was used as the starting parent to prepare the GEF-adapted strain through blindly passaging in GEF,and a total of 40 passages were performed till now.The result showed that the forty-generation virus has exhibited a good replication ability in GEF.Obvious cell pathogenic effect(CPE),including increased particulates,cell swelling,rough boundary until disintegration can be observed in GEF layers at 72 hours post-infection.To differentiate from the parental virus,the 40-generation viral passage was named with SYG61v-C40.Indirect immunofluorescence assay(IFA)indicated the polyclonal mouse antibody raised against the GPV Rep protein can recognize the viral protein antigen expressed in the GEF at 72 h post-infection of SYG61v-C40,but not in GEF as the mock control.Absolute real-time fluorescence quantity PCR(qPCR)was used to compare the genome copies numbers throughout the passage period.The nucleic acids extracted from the cell lysates at 96 h post-infection was used as the qPCR template.The result indicated that the genomic copies detected in the 20-generation passage reached the highest level with 2.4×109/ml,then slightly decreased in subsequent generations,averaging 108/ml.The medium tissue cell infection dose(TCID50)of SYG61v-C40 was also determined,reaching 2×106.69/ml.The pathogenicity of the GEF-adapted strain SYG61v-C40 was also evaluated in susceptible 9-day-old embryonated goose eggs and 2-day-old goslings,respectively.It was found that SYG61v-C40 killed nine embryonated goose eggs,however,the mean death time(MDT)reached 211 hours,demonstrating the decreased virulence against goose embryos in comparison to the parental strain SYG61v,which generally kills goose embryos in 120 hours.All of 16 goslings survived in the challenge test with SYG61v-C40 during the 21-day observation period.The average body weights of goslings at 7 days and 21 days post-infection were 0.258 kg and 0.657 kg,respectively,which was slightly lower than that of the control group(0.288 kg and 0.690 kg,respectively)(P>0.05),implying that SYG61v-C40 still possess a weak pathogenicity toward goslings.2 Genome sequencing and comparison analysis of the GEF-adapted strain SYG61v-C40Which element alterations in the genome of SYG61v-C40 are involved in the transition of cell adaption deserved to be investigated.In this study,the overlapping genome fragments were amplified,cloned and sequences,then the complete genome sequence was obtained.Comparison between SYG61v-C40 and the parental strain SYG61v was implemented across the genome.The result indicated that a total of 13 nucleotides mutations were produced in SYG61v-C40,among which three mutations lied in the Rep gene,ten in the VP1 gene.These nucleotide mutations totally brought out six amino acid changes in the coding proteins,among which one is located in the carboxyl terminus of Rep1,five in VP1.Further analysis demonstrated that amino acid mutations occurred in site 107 and 142 were located within the N terminal unique region of VP1(VP1u),which has the phospholipase activity and is involved in infectivity of parvovirus.The amino acid mutations(N381K and S449R)were located in the VP3 protein,both of which not only changed the amino acid polarities,but also is supposedly exposed to the surface of capsid,which probably play a key role in cell adaptability transition of SYG61v-C40.Inverted terminal repeats(ITR)sequence comparison revealed that there are 34 nucleotide deletions produced in the palindromic stem of ITR of SYG61v-C40.These consecutive deletions were distributed in the outside of ITR,resulted in formation of an additional loop structure in ITR as analyzed by the DNA/RNA second structure prediction software.ITR not only act as the initiation site for genome replication,but also in involved in the transcription,viral particle assembly and other processes,therefore,whether the change of the ITR secondary structure play an important role in the cell adaptability transition for SYG61v-C40 strain deserved to be investigated.3 Functional analysis of the amino acid point mutations produced in SYG61v-C40The 34-bp deletions in ITR deletion and six amino acid mutations in the coding proteins were two distinctive feature found in the GEF-adapted strain SYG61v-C40,however,whether two aspects of differences contribute equally to the GEF adaption remained unknown.An infectious clone plasmid pSYG61v containing the whole genome of SYG61v was previously constructed in our lab.In this study,the internal coding frame that covered all of the nucleotide differences between SYG61v-C40 and SYG61v was amplified and replaced the corresponding portion in the plasmid pSYG61v,resulting in generation of a chimeric plasmid pSYG61v-C40.Transfection of plasmid pSYG61v-C40 in GEF yielded the infectious virus,which can be passaged in GEF and produce typical CPE at 72 h as manifested by infection of the cell-adapted strain SYG61v-C40.Further tests showed that the rescued virus pSYG61v-C40 exhibited a high TCID50 value of 2×108/ml,and a high viral genome copies of 108?109/ml in the cell lysates as detected by qPCR.Transfection of the parental plasmid pSYG61v in GEF also yielded an infectious virus,however,the rescued virus cannot produce typical CPE when passaged at GEF till 96 h.All of these results demonstrated that it is the six amino acid mutation,not the ITR deletions,that played a key role in the GEF adaptation for SYG61v-C40.