Construction Of Infectious Clone Of Novel Goose Parvovirus KC3S3 Strain And Biological Characteristics Of Rescue Strain

Traditionally,waterfowl parvoviruses include Goose Parvovirus（GPV）and Muscovy Duck Parvovirus（MDPV）,Goose parvovirus can cause fibrinous embolism in the intestinal tract of goslings,while duckling microvirus causes symptoms of diarrhea,wheezing and weakness of the feet.From March 2015,an outbreak of Novel Goose parvovirus（NGPV）infection,mainly characterized by short beaks,tongue outgrowth and stunted development of diseased ducks,was reported in Cherry Valley and mule duck flocks in different provinces of Shandong,Anhui and Jiangsu,China,The disease is also known as"short beak and dwarfism syndrome"（SBDS）,The growth of diseased ducks was retarded and the group culling rate reached more than 80%,causing huge economic losses to China’s duck industry.In this study,a strain of NGPV was isolated from the tissues of Cherry Valley ducks with obvious SBDS symptoms from Inner Mongolia and named strain KC3S3.The whole-genome sequence analysis and pathogenicity study of this strain were carried out,and the whole-genome sequence of this virulent spider was used as the basis for constructing an infectious clone of NGPV,and the infectious plasmid was transfected by duck embryo to successfully obtain NGPV virus particles with molecular genetic markers.Subsequently,during the transmission of the rescue strain,as the replication capacity of the strain gradually reached the level of the parental strain,the ability of the virus to infect duck embryos and cells was clearly different from that of the parental strain.In view of this,this experiment selected the parental strain and two different generations of rescue strains for duckling infection tests,and compared the pathogenic ability of the strains by analyzing the differences between different groups,This not only reflects the variability of NGPV infection at the animal level,but also provides a factual basis for the theoretical model of NGPV transmission weakness,and provides technical support for future research on the pathogenesis and immune control of NGPV.The main research contents are as follows:1.In this study,the diseased material of Cherry Valley meat ducks with obvious symptoms of SBDS in Inner Mongolia was collected,treated and inoculated with duck embryos,and the embryos died around 120 h.The dead duck embryos had slight hemorrhage in the anterior breast feather area and serious growth inhibition,PCR detection and NGPV VP2 gene sequencing indicated that the isolated virus was NGPV,which was named strain KC3S3,and then the whole sequence analysis of strain KC3S3 was performed by splicing the genome after segmental sequencing.The results of homology analysis showed that strain KC3S3 had 99.8%nucleotide homology with the 2020 Shandong NGPV isolate JS1212.Nucleotide homology analysis of strain KC3S3 with both the GPV reference strain and the MDPV reference strain,The results showed that the genome-wide nucleotide homology with the GPV reference strain ranged from94.7%to 95.9%,while the nucleotide homology with the MDPV reference strain was only 81.2%to 85.0%.Genetic evolutionary analysis showed that strain KC3S3 was most closely related to the novel goose parvovirus,and this isolate was located in a separate evolutionary branch and shared the same genetic evolutionary cluster with NGPV strains JS1212,JS112 and HN1P.The results of animal regression tests showed that Cherry Valley meat ducks infected with strain KC3S3 exhibited typical SBDS lesions such as stunting,tongue abrasion,tibial fracture,and punctate hemorrhage in the thymus gland.In conclusion,strain KC3S3 is an NGPV isolate with unique evolutionary properties.2.Reverse genetic manipulation of strain KC3S3 using segmented DNA amplification and homologous recombination.The complete genome of strain KC3S3 was amplified by high fidelity DNA polymerase,and the SphⅠenzyme cut site located at the NS2 fragment was overlappingly mutated and used as a molecular tag.Inverted terminal repeats（ITRs）are long complex palindromic sequences containing a large number of repeated GC base sequences located at both ends of the genome,which are common to the genus Minutia.Since intact ITR is highly susceptible to deletion rearrangement during transformation,this experiment modified the mutation-prone region by separating ITR,thus reducing the mutation probability of ITR replication in E.coli,and also reducing the number of ITR replication in the strain,ensuring the correctness of the cloned plasmid.The bottom plasmid was selected from the p Blue Script II SK（+）vector（p BSK）,and the target fragment was sequentially ligated between the KpnⅠand Bam HⅠdigestion sites of the vector by homologous recombination,and the recombinant plasmid was named p KS3.The p KS3 plasmid was mixed with liposomes and transfected into the chorionic allantoic membrane of 10-day-old duck embryos,and the allantoic fluid was collected after 120 h of incubation at 37°C and transmitted blindly for 3 generations.The third generation of duck embryos died around 160 h.The duck embryo allantoic fluid was collected for identification,and PCR results showed that the virus particles were still detected in the third generation of blind passage,and sequencing results indicated that the rescue strain had successfully introduced nucleotide site markers.3.The infectious rescue plasmid was named r KS3-N（N is the generation）,and the biological characterization of the rescued strain was performed using cell and animal infection assays.The parental strains and the rescued strains of different generations were quantified by Real-time Quantitative PCR to standardize the virus particle copy number,and KC3S3,r KS3-3,r KS3-5 and r KS3-10 strains were selected for DEF cell infection assay.At the same time,KC3S3 and r KS3-10 strains were selected for the duck embryo infection test,and the infection results of different strains were very different.KC3S3,r KS3-5 and r KS3-10 strains were selected to construct animal models.120 one-day-old Cherry Valley ducklings were randomly divided into four groups of 30 ducks each,with group A being the KC3S3-infected group,group B being the r KS3-5-infected group,group C being the r KS3-10-infected group and group D being the control group.Each of the KC3S3-infected group and r KS3-10-infected group were inoculated with 0.1 m L(1×10^6.1 copies/0.1m L)of virus solution mixed with 2.4 m L of saline,the r KS3-5-infected group was inoculated with 2.5 m L(1×10^5.4 copies/0.1m L)of virus solution,and each of the control group was inoculated with 2.5 m L of saline,Clinical signs were observed daily.After tapping at 7 dpi,14 dpi,21 dpi and 28 dpi,6 animals were randomly selected from each group,weighed,cotton swabs were collected and serum was separated,dissected and collected from each tissue and organ,while the tongue,rostrum,tibia and femur parts were separated for measurement and identification.Clinical signs and autopsy results showed typical symptoms of SBDS in all infected groups,including stunting,interlingual abrasion,congestion of the liver,spleen and duodenum,and punctate hemorrhage in the thymus.When compared with the infected groups,the KC3S3-infected group had the most obvious disease,with slow weight gain,severe cloacal detoxification and viraemia,high viral load in all tissues and organs,significant differences between the tongue to beak ratio and the control group,and lower than normal bone density content;the r KS3-5-infected group had similar morbidity indicators to the KC3S3-infected group;the r KS3-10-infected group had significantly lower morbidity than the first two strains of virus.The above tests demonstrated that the r KS3-10 strain showed a decrease in virulence during virus transmission.