Development Of Detective Methods And Epidemiological Investigation For Novel Goose Parvovirus-related Virus

Short beak and dwarfism syndrome(SBDS)has been constantly breaking out in China since the second half of 2014.The main clinical symptoms were strong growth retardation with beak atrophy,tongue exposure,enteritis,and paralysis.The disease has been reported in cherry valley ducks and mule ducks.Nearly 10% to 50% ducks were affected in the infected flocks.The infected ducks often became marasmus because of the difficulty in ingestion.The weight of ducks with SBDS disease can be 20%-30% less than the weight for normal ducks,which caused great economic losses to meat duck breeding industry.After virus isolation and identification by researchers,people found that it was caused by a novel goose parvovirus-related virus(NGPV).In this research,we developed two different virus detection method,and these two methods were used to perform an investigation for clinical samples,which helped to explore the tissue organism of NGPV.Second,this study isolated and cultured several NGPV strains.We designed primers to amplify the complete genome of NGPV and conduct comprehensive analysis for the isolated NGPV strain,which provided new idea for the genomic feature and pathogenetic mechanism.Thirdly,we collected a large number of clinical samples from different regions,to conduct a comprehensive research on the co-infection of NGPV and DuCV.The research can be divided into four parts:1.Development and application of a duplex semi-nested PCR assay for detection of classical GPV and NGPVAfter analysis of the conservative region in GPV and NGPV,we designed two universal primers and two specific primer aimed to GPV and NGPV,to develop a duplex semi-nested PCR assay which can detect GPV and NGPV in one reaction.The best PCR parameters were as follows: a denaturation step at 95 °C for 7 min and then 33 cycles of a denaturation step at 95 °C for 45 s,an annealing step of 51 °C for 45 s,and extension at 72 °C for 55 s,with a final extension at 72 °C for 10 min.The duplex semi-nested PCR assay we developed was with high specificity,which can only detect NGPV and GPV,but do not react with other common pathogens including DHBV,DuCV,DEV,MDPV,E.coli and RA;the PCR method was also sensitive,which can detect lowest 100 copies DNA.Using the duplex PCR assay,NGPV was tested positive in all the 15 duck flocks with SBDS,whereas classical GPV was not detected in any of the 133 sick and dead ducks collected from East China.A total of 87(91.58%)Cherry Valley ducks aged from 5 to 18 days and 35(92.11%)mule ducks aged from 17 to 25 days were positive for NGPV DNA.In the NGPV-positive cherry valley ducks and mule ducks,the virus detection rates were 85.08% to 9.20% and 74.29% to 5.71% in heart,liver,spleen,lung,kidney,pancreas,bile,thymus,bursa of Fabricius,and brain.The results indicated that NGPV was prevalent in the duck flocks of East China,whereas classical GPV was not detected in Cherry Valley ducks and mule ducks with SBDS.NGPV has extensive tissue tropism in Cherry Valley duck and mule duck,which could invade both the central and peripheral immune organs and break through the blood-brain barrier of ducks.2.Development and application of DNA probe of NGPVA length of 390 bp DNA that located in VP3 of NGPV was cloned to make DNA probe.After the specificity test of DNA probe,the method was demonstrated highly specific and the probe can only combine and react with goose parvovirus rather than other pathogen DNAs.The specificity test showed that the minimum detection limit was determined to be 6pg viral DNA of NGPV.We test 870 tissues from 87 diseased ducks which were collected from Shandong,Jiangsu and Anhui provinces using the DNA probe.The results indicated that the individual positive ratio of NGPV was 89.7%(78/87)and the detection rate of heart(74.4%)and spleen(50.0%)were higher than other organs.The lowest detection rate was found in brain(9.0%).Furthermore,we also detected these samples using PCR assay and the rate of accordance between the two methods was almost same.All these results indicated that the probe had high specificity and sensitivity and was suitable to be applied in clinical diagnosis and epidemical investigation.3.The isolation of NGPV and characterization of complete genomeIn this study,we isolated and cultured seven NGPV stains from different regions in China during 2015 and 2016.To better understand the correlation between NGPV and goose parvovirus(GPV),we conducted complete genome sequencing and a comprehensive analysis of the NGPV genome.The phylogenetic and alignment analysis showed that NGPV is a branch of GPV,sharing 92.2%-97.1% nucleotide identity with GPV.Compared with classical GPV,five consensus nucleotide mutations in all the seven NGPV isolates and two 14-nucleotide-pair deletions in six NGPV isolates(SDLY1512,SDHZ1604,SDDY1605,AH1605,AH1606,JS1603)were found in the inverted terminal repeats,twelve and eight synchronous amino acid changes were found in the replication protein and capsid protein of NGPV respectively,which might be important for viral gene regulation,humoral immune responses,and host transfer.Notably,SDLY1602 was demonstrated a recombinant strain,with the potential major parent GPV vaccine strain(Taiwan)82-0321 v and the minor parent GPV wild strain GDaGPV.This is the first report showing that the recombination between two classical GPV strains generated a NGPV strain circulating in nature.This study will advance our understanding of NGPV molecular biology.4.The co-infection of NGPV and DuCV in ducks with SBDS.Novel goose parvovirus-related virus(NGPV)is believed the main pathogen of SBDS disease,however,SBDS is rarely reproduced by infecting ducks with NGPV alone.As avian circovirus infection causes clinical symptoms which resemble to SBDS,duck circovirus(DuCV)is suspected the minor pathogen of SBDS disease.In this study,an investigation using two duplex semi-nested PCR method was carried out to determine the co-infection of NGPV and DuCV in 170 meat ducks from 20 flocks and 200 duck embryos in different ages.According to our investigation,the co-infection rate of emerging NGPV and DuCV was 100%(20/20)in flocks of East China(Shandong,Jiangsu and Anhui province).The co-infection rate of NGPV and DuCV in meat ducks was 63.53%(108/170),in which the detection rate of DuCV-1 was twice as DuCV-2.This means the DuCV-1 was more important than DuCV-2 in SBDS disease.The co-infection rate of NGPV and DuCV in duck embryos was 3.5%,which means these two viruses could be vertical transmitted.The investigation of NGPV and DuCV in ducks with SBDS disease indicated the cooperative roles of NGPV and DuCV in duck SBDS disease.