| OsHV-1 is a double stranded DNA virus belonging to Malacoherpesviridae,a family of mollusc herpesviridae.It can infect a variety of bivalves,and has a very high mortality rate of shellfish larvae and juveniles,up to more than 90%.Os HV-1 has been widely prevalent worldwide since its discovery and has been introduced to China since the 21 st century,becoming one of the main infectious pathogens that restrict the healthy development of China’s shellfish farming industry.In recent years,the frequent outbreak of oyster herpesvirus disease has led to a series of problems such as slow growth of shellfish,low egg laying and hatching rates,and high mortality rates,causing huge economic losses to China’s shellfish breeding industry.This study investigated the prevalence of oyster herpesvirus in Shandong Province from2021 to 2022 by using the nested PCR method that has been established.The raft and mudflat cultured shellfish samples from seven coastal cities in Shandong Province were collected for detection,which improved the epidemiological law of oyster herpesvirus in Shandong Province in the past two years.In 2021,287 samples were collected in Weihai,Yantai,Binzhou,Dongying,Weifang,Rizhao and Qingdao,with a positive rate of 21.25%.Among them,the positive rate of samples from Dongying was the highest,reaching 72.40%,the positive rate of clams was the highest,reaching 50.00%,the detection rate of raft cultured shellfish was 14.29%,and the detection rate of mudflat cultured shellfish was 23.21%;In2022,384 samples were collected in Weihai,Weihai Wendeng,Yantai Binzhou,Dongying,Weifang,Rizhao and Qingdao,with a positive rate of 16.14%.Among them,the positive rate of samples from Binzhou was the highest,reaching 75%.The positive rate of bay scallop was the highest,reaching 25%.The detection rate of raft cultured shellfish was 23.33%,and the detection rate of mudflat cultured shellfish was 14.81%.Based on the laboratory testing results from 2017 to 2022,the detection rates from 2017 to 2022 were 11.5%,15.7%,17.83%,17.2%,21.25%,and 16.14%,respectively.It can be seen that the overall detection rate is showing a slow fluctuation and upward trend.The positive rates vary in different regions,and there are also differences in Os HV-1 infection among different varieties of shellfish.In order to study the changes of oyster herpesvirus at the molecular level,this study used a combination of second-generation sequencing and first-generation sequencing to sequence the genome of a oyster herpesvirus(Clam strain)that caused large-scale death of clam larvae in Yangkou Town,Shandong in 2017,and named it Os HV-1-YK.Firstly,based on the publicly available Os HV-1 genome sequence(Gen Bank: AY509253)and other data,a long fragment PCR amplification primer was designed and cloned to obtain the gene sequence fragments of the oyster herpesvirus strain.The ends of these fragments were overlapped with each other,and finally,the genome sequence of the oyster herpesvirus strain was obtained by splicing.The structure and function of the genome of the oyster herpesvirus strain were explored using annotations from different databases.In this study,long segment PCR amplification and molecular cloning were used to measure the total genome length of oyster herpesvirus clam strain,199,267 bp,8,172 bp shorter than Os HV-1 genome sequence(207,439 bp),11,726 bp shorter than AVNV genome sequence(210,993 bp),and 87 bp shorter than Os HV-1-SB genome sequence(199,354 bp).The GC content is 38.41%,which is similar to the GC content of AVNV(38.5%)and Os HV-1-SB(38.5%),but slightly lower than the 38.7% of Os HV-1.The similarity between the clam strain and Os HV-1,AVNV,and Os HV-1-SB at the DNA level was 99.61%,99.48%,and 99.66%,respectively.By analyzing the gene sequence of the clam strain,two sets of long reverse repetitive sequences were found.The first set was 3,533 bp in length,with positions ranging from 1 to 3,533 bp and 176,261 to 179,794 bp;The second group has a length of7,458 bp,with positions ranging from 178,776 to 187,233 bp and 191,810 to 199,267 bp.Its structure is TRL UL IRL US TRS,which conforms to the D type of the six structural types of herpes virus.A phylogenetic tree was constructed using 32 open reading frame sequences,including Os HV-1,AVNV,and Os HV-1-SB.The results showed that the clam strain had the closest genetic relationship with the clam strain Os HV-1-SB,and was closely related to Os HV-1 μ Var and other French variants have the farthest relationship.The annotation analysis results of different databases show that the Pfam domain classification has six identical domains: BIR(ubiquitin protein ligase),zf-C3HC4-3(related to protein and zinc ion binding);There are two identical domains: DNA primase S(deoxyribonucleic acid primer enzyme)and Zf-RING-5(zinc finger protein).The GO annotation classifies 47 genes into three aspects: viral DNA genome replication(1),DNA replication repair and recombination(1),DNA replication(3),and DNA packaging(1)in biological processes;Molecular functions include DNA exonuclease(1),nucleotide binding(1),metal ion binding(8),DNA polymerase(1),DNA primer enzyme(2),DNA binding(1);Among the cell components,there are membrane components(19)and interactions with the host cell membrane(8).The KEGG database annotates four B-level pathways involved in nucleotide metabolism and two global and overview maps under the A-level pathway of metabolism.The Uniprot database predicted a total of 135 functional genes,but 9 of them were not confirmed by BLASTp(including ORF＿PT1,PT2,PT4,PT5,PT5,PT6,IN1,IN4,and 2 genes that did not match other database annotations).The Refseq annotation identified a total of 123 hypothetical genes,corresponding to a total of 123 ORFs,all of which were confirmed in the ORF finder.Through the analysis of Tigrfam database,five genes corresponding to three groups of gene sequences were found,namely: class 1b ribonucleoside diphosphate reductase β Subunit,1b class ribonucleoside diphosphate reductase α Subunit,ribonucleoside diphosphate reductase α Subunit,adenosylmethionine dependent ribonucleoside diphosphate reductase and d UTP diphosphase,in which adenosylmethionine dependent ribonucleoside diphosphate reductase can convert ribonucleoside triphosphate(NTP)to deoxynucleoside triphosphate(d NTP).In summary,this study compared the differences in DNA levels between the clam strain and other mutant strains.On the one hand,it improved the genome of Os HV-1 with different variations,promoted the study of Os HV-1 genome sequence diversity,variability,and evolutionary characteristics,and provided data support for studying its origin;On the other hand,by establishing the molecular epidemiology of Os HV-1,we can sequence and analyze the genome of an Os HV-1 strain.At the same time,this study conducted an epidemiological investigation on shellfish and oyster herpesvirus disease in Shandong Province from 2021 to2022,grasping the epidemic situation of oyster herpesvirus disease,providing data basis for studying the epidemiological changes and prevention and control warnings of shellfish and oyster herpesvirus disease,and promoting the healthy development of shellfish breeding industry. |
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