Bioassay of Clostera anastomosis Granulosis virus

The second-instar healthy larvae of Clostera. anastomosis were collected in the artificial woodland of poplar in Shuangcheng Town, Heilongjiang Province, China. The dead larvae of C. anastomosis infected by granulosis virus （GV） of Clostera anastomosis were grinded to obtain GV. The GV viral pesticide was diluted to seven concentrations, 1.58×10^3PIB·mL^-1, 1,58×10^4PIB·mL^-1, 1.58×10^5PIB·mL^-1 1.58×10^6PIB·mL^-1, 1,58×10^7PIB·mL^-1; 1.58×10^8PIB·mL^-1 and 1.58×10^9PIB·mL^-1 and the fresh poplar leaves were dipped in the seven concentrations liquids to feed the larvae. After nine days the mortality of larvae was investigated. The minimum corrected mortality （7.32%） of larvae was observed at concentrations of 1.58×10^3PIB·mL^-1 and the maximal mortality （97,36%） was observed at concentration of 1.58×10^9PIB·mL^-1. The regression equation between the logarithm of the virus concentration and the mortality was y= 1.946＋0.558x The LC50 was 2.97×10^5PIB·mL^-1. The LT50 for the virus concentration of 1.58×10^5, 1.58×10^6, 1.58×10^7, 1,58×10^58, 1.58×10^9 PIB·mL^-1 were 8.55d, 6.89d, 5.9d, 4.65d, and 4.08d, respectively, shorting gradually with the concentration increasing, It is concluded that the toxicity of Clostera anastomosis GV is very strong and as a kind of insecticides it has big potential in practical application.

The Influence of Newcastle Disease Virus Major Proteins on Virulence

The Newcastle disease virus(NDV)negative-strand RNA genome contains six genes.These genes encode nucleoprotein(NP),phosphoprotein(P),matrix protein(M),fusion protein(F),hemagglutinin-neuraminidase(HN),and RNA-dependent RNA polymerase(L)proteins.The six proteins affect the virulence of NDV in different ways,but available information on the six proteins is disparate and scattered across many databases and sources.A comprehensive overview of the proteins determining NDV virulence is lacking.This review summarizes the virulence of NDV as a complex trait determined by these six different proteins.

Development of a stable attenuated double-mutant of tobacco mosaic virus for cross-protection

Tobacco(Nicotiana tabacum)and tomato(Solanum lycopersicum)are two major economic crops in China.Tobacco mosaic virus(TMV;genus Tobamovirus)is the most prevalent virus infecting both crops.Currently,some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus.Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases.However,stable attenuated TMV mutants are scarce.In this study,we found that the substitutions in the replicase p126,arginine at position 196(R^(196))with aspartic acid(D),glutamic acid at position 614(E^(614))with glycine(G),serine at position 643(S^(643))with phenylalanine(F),or D at position 730(D^(730))with S,significantly reduced the virulence and replication of TMV.However,only the mutation of S^(643) to F reduced the RNA silencing suppression activity of TMV p126.A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants.Furthermore,our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants.This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.

African Swine Fever Virus MGF-110-9L-deficient Mutant Has Attenuated Virulence in Pigs

African swine fever virus(ASFV)is the etiological agent of African swine fever(ASF),an often lethal disease in domestic and wild pigs.ASF represents a major threat to the swine industry worldwide.Currently,no commercial vaccine is available because of the complexity of ASFV or biosecurity concerns.Live attenuated viruses that are naturally isolated or genetically manipulated have demonstrated reliable protection against homologous ASFV strain challenge.In the present study,a mutant ASFV strain with the deletion of ASFV MGF-110-9L(ASFV-D9L)was generated from a highly virulent ASFV CN/GS/2018 parental strain,a genotypeⅡASFV.Relative to the parental ASFV isolate,deletion of the MGF-110-9L gene significantly decreased the ability of ASFV-D9L to replicate in vitro in primary swine macrophage cell cultures.The majority of animals inoculated intramuscularly with a low dose of ASFV-D9L(10 HAD50)remained clinically normal during the 21-day observational period.Three of five ASFV-D9L-infected animals displayed low viremia titers and low virus shedding and developed a strong virus-specific antibody response,indicating partial attenuation of the ASFV-D9L strain in pigs.The findings imply the potential usefulness of the ASFV-D9L strain for further development of ASF control measures.

N-terminal domain of the RNA polymerase of very virulent infectious bursal disease virus contributes to viral replication and virulence

Dear Editor,Infectious bursal disease virus （IBDV） causes an economically significant disease of chickens worldwide （Berg,2000）. The molecular basis for the virulence of very virulent IBDV （vvIBDV） is not fully understood. Previous studies have shown that genome segment A, especically VP2 protein, plays the most important role in the tropism and pathogenicity of serotype 1 IBDV （Brandt et al., 2001）. VP2 is,however, unlikely to be the only factor for the virulence of vvIBDV （Boot et al., 2000）.

Cryo-EM structures of infectious bursal disease viruses with different virulences provide insights into their assembly and invasion

Infectious bursal disease virus(IBDV)causes a highly contagious immunosuppressive disease in chickens,resulting in significant economic losses.The very virulent IBDV strain(vvIBDV)causes high mortality and cannot adapt to cell culture.In contrast,attenuated strains of IBDV are nonpathogenic to chickens and can replicate in cell culture.Although the crystal structure of T=1 subviral particles(SVP)has been reported,the structures of intact IBDV virions with different virulences remain elusive.Here,we determined the cryo-electron microscopy(cryo-EM)structures of the vvIBDV Gx strain and its attenuated IBDV strain Gt at resolutions of 3.3 Å and 3.2 Å,respectively.Compared with the structure of T=1 SVP,IBDV contains several conserved structural elements unique to the T=13 virion.Notably,the Nterminus of VP2,which is disordered in the SVP,interacts with the S_(F) strand of VP2 from its neighboring trimer,completing theβ-sheet of the S domain.This interaction helps to form a contact network by tethering the adjacent VP2 trimers and contributes to the assembly and stability of the IBDV virion.Structural comparison of the Gx and Gt strains indicates that H253 and T284 in the VP2 P domain of Gt,in contrast to Gx,form a hydrogen bond with a positively charged surface.This suggests that the combined mutations Q253 H/A284 T and the associated structural electrostatic features of the attenuated Gt strain may contribute to adaptation to cell culture.Furthermore,a negatively charged groove in VP2,containing an integrin binding IDA motif that is critical for virus attachment,was speculated to play a functional role in the entry of IBDV.

Identification of genome-wide nucleotide sites associated with mammalian virulence in influenza A viruses

The virulence of influenza viruses is a complex multigenic trait.Previous studies about the virulence determinants of influenza viruses mainly focused on amino acid sites,ignoring the influence of nucleotide mutations.In this study,we collected>200 viral strains from 21 subtypes of influenza A viruses with virulence in mammals and obtained over 100 mammalian virulence-related nucleotide sites across the genome by computational analysis.Fifty of these nucleotide sites only experienced synonymous mutations.Experiments showed that synonymous mutations in three high-scoring nucleotide sites,i.e.,PB1–2031,PB1–633,and PB1–720,enhanced the pathogenicity of the influenza A(H1N1)viruses in mice.Besides,machine-learning models with accepted accuracy for predicting mammalian virulence of influenza A viruses were built.Overall,this study highlighted the importance of nucleotide mutations,especially synonymous mutations in viral virulence,and provided rapid methods for evaluating the virulence of influenza A viruses.It could be helpful for early warning of newly emerging influenza A viruses.

Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China

[Objective] The paper was to determine the genomic sequence of a very virulent strain of infectious bursal disease virus（IBDV）,and study its molecular characteristics.[Method] A very virulent strain（vvIBDV）（HLJ-0504） of infectious bursal disease virus（IBDV） with special characters was isolated in China and its genome was sequenced.[Result] Sequence analysis showed that segment A of HLJ-0504 was derived from vvIBDV,while segment B was from a distinct ancestor.The morbidity and mortality of HLJ-0504 was 100% and 86.7%to SPF chickens,respectively.[Conclusion] vvIBDV with distinct segment B were still circulating and the evolution of IBDV was diversified in China.Besides,it is hard to imagine that the virulence of IBDV is determined solely by segment A or B.

猪流行性腹泻灭活疫苗毒株的培育

为培育出毒力弱、病毒含量高、免疫原性好的猪流行性腹泻病毒(PEDV)疫苗株,将PEDV-YL株在Vero细胞上连续传代并进行噬斑克隆纯化,测定病毒含量后进行毒力试验及免疫原性试验。结果显示:将健康仔猪接种F22代clone6、clone7毒种后,试验猪均表现出腹泻症状且均能检测到PEDV,剖检后各脏器未见异常;攻毒保护率为60%,各脏器均未出现病变。健康仔猪接种F32代clone10、clone12毒种后,60%试验猪出现腹泻症状且检出PEDV,剖检后各脏器无明显变化;一次免疫组攻毒保护率为60%~80%,二次免疫组为100%,各脏器均未发生病变。F32代clone10病毒含量略低于clone12且两者免疫原性相当。结果说明:PEDV-YL株F32代clone12更适合作为疫苗研制候选毒株,且二次免疫保护效果更好。本研究为猪流行性腹泻灭活疫苗研发提供了技术支撑。

Analysis of the function of D279N mutation of VP2 of infectious bursal disease virus

Infectious bursal disease virus（IBDV）is responsible for the highly contagious infectious bursal disease of chickens.Further understanding the gene-function is necessary to design the tailored vaccine.The amino acid residue 279,located on strand P_F of VP2,is one of the three residues that have been reported to be involved in cell-tropism but with some inconsistency.In this study,to further clarify the amino acids involved in the cell tropism of IBDV,a series of mutations about residue 279were introduced into the VP2 of vv IBDV Gx strain.With the reverse genetic system,we found single mutation of D279N,double mutations of D279N/A284T or Q253H/D279N were not enough to adapt IBDV to chicken embryo fibroblast（CEF）cell.To evaluate whether residue 279 could influence the replication and virulence of IBDV,the virus r Gx HT-279 with three mutations（Q253H/D279N/A284T）was rescued and evaluated.Results showed that the mutation of residue 279 in VP2had no efficient effects on both the replication efficiency in vitro and the virulence to SPF chickens of IBDV.In summary,the results demonstrated that residue 279 of VP2 did not contribute efficiently to cell tropism,replication efficiency,and virulence of IBDV at least in some strains.These findings provided further information for understanding the gene function of IBDV.

鸭坦布苏病毒E蛋白功能的研究进展

鸭坦布苏病毒(Duck Tembusu virus,DTMUV)病是由DTMUV引起以病毒性脑炎(雏鸭、育成鸭)及产蛋下降(产蛋鸭)为主要特征的传染性疾病,该病的暴发和传播给水禽养殖业造成了巨大的经济损失。包膜蛋白E(Envelope,E)作为DTMUV关键的免疫原性蛋白,在介导病毒与宿主细胞融合、侵入等方面发挥作用。文章总结了近年来DTMUV E蛋白结构特点、关键毒力位点、潜在抗原表位、蛋白互作及生物制品等最新研究进展,以期为鸭坦布苏病毒病的特异性诊断、疫苗研究和抗病毒药物的设计提供参考。

猴痘病毒B.1谱系遗传分支、毒力基因及蛋白功能

2022年以来,猴痘疫情在全球暴发和流行。相较以往的猴痘病毒,2022年流行的猴痘毒株传播能力和宿主适应性等明显增强,猴痘B.1谱系毒株已成为全球猴痘疫情流行的主要毒株。为此,本文对猴痘病毒B.1谱系遗传分支、毒力基因及蛋白功能进行综述,并就部分基因产物的蛋白功能进行了注释,以期为猴痘疫情的科学防控提供参考。

Effect of amino acid mutation at position 127 in 3A of a rabbitattenuated foot-and-mouth disease virus serotype Asia1 on viral replication and infection

An amino acid mutation(R127→I) in the 3A non-structural protein of an FMDV serotype Asia1 rabbit-attenuated ZB strain was previously found after attenuation of the virus. To explore the effects of this mutation on viral replication and infection, the amino acid residue isoleucine(I) was changed to arginine(R) in the infectious cDNA clone of the rabbit-attenuated ZB strain by sitedirected mutagenesis, and the R127-mutated virus was rescued. BHK monolayer cells and suckling mice were inoculated with the R127-mutated virus to test its growth property and pathogenicity, respectively. The effects of the R127 mutation on viral replication and virulence were analyzed. The data showed that there was a slight difference in plaque morphology between the R127-mutated and wild-type viruses. The growth rate of the mutated virus was lower in BHK-21 cells and its virulence in suckling mice was also attenuated. This study indicates that the R127 mutation in 3A may play an important role in FMDV replication in vitro and in pathogenicity in suckling mice.

Molecular evolution of hepatitis A virus in a human diploid cell line

AIM： To investigate the hoLspoLs, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells.METHODS： Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the fulllength genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells.RESULTS： Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-l, other than its progenitor H2w.CONCLUSION： Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.

Molecular Differentiation of Different Pathogenic Phenotypes of Infectious Bursal Disease Viruses by RT-PCR Combined with Restriction Fragment Length Polymorphism(RFLP) Assay

Accurate differentiation of the pathogenic phenotypes of infectious bursal disease viruses(IBDVs) will instruct effective vaccination programs and improve the study of the molecular epidemiology of IBDVs. In this study, an 833 bp hypervariable nucleotide region was identified in VP2 genes of known IBDVs with different virulences through multiple sequence alignment.Moreover, using NEBcutter software analysis, two restriction enzyme sites, SpeⅠ(generating 531 and 302 bp fragments) and StuⅠ(generating 242 and 591 bp fragments) were found presented in very virulent but not attenuated IBDVs. Moreover, the restriction enzyme site SacⅠ(generating 218 and 615 bp fragments) presented in attenuated IBDVs but not very virulent IBDVs. Therefore,a reverse-transcription(RT)-PCR combined with a restriction fragment length polymorphism(RFLP) assay was developed to differentiate attenuated and very virulent IBDVs. The RT-PCR assay was used to confirm 282 IBDV positive samples from 310 suspicious dead chicken samples. The 60 IBDV positive samples were used to evaluate the assay, followed by confirmation via gene sequencing and histopathological examinations of the bursas of Fabricius from chickens infected by these IBDVs. The results showed that 24 viral strains with SpeⅠand StuⅠsites were very virulent, causing severe pathological damage in the bursas of Fabricius, while36 viral strains with the SacⅠsite were attenuated IBDVs, exhibiting only slight pathological damage. The combined RT-PCR and RFLP assay provided a useful approach for differentiating the pathogenic phenotypes of IBDVs.

A study on pathogenicity of hepatitis G virus

AIM To study the pathogenicity of hepatitis G virus (HGV) and observe the genesis and pathological process of hepatitis G.METHODS HGV-RNA in serum was detected by RT-PCR assay. The immunohistochemical assays of liver tissue were performed with HGV monocoloned antibody (McAb)expressed from the region of HGV NS5 nucleic acid sequence. The clinical and pathological data of 52 patients with hepatitis G were discussed. In animal experiment,the Chinese Rhesus monkeys were infected with the serum of a patient with HGV infection. And the dynamic changes in serology and liver histology of animals were observed.RESULTS One hundred and fifty-four patients with HGVRNA positive were selected from 1552 patients with various kinds of hepatitis. Of 154 patients with HGV infection, 52 were infected with HGV only, which accounted for 33.8% (52/154) and 102 with positive HGVRNA were super-infected with other hepatitis viruses,which accounted for 66.2% (102/154). The clinical and pathological observation showed that the acute and chronic hepatitis could be induced by HGV. The slight abnormality of transaminases ALT and AST in serum of monkeys lasted nearly 12 months and histological results showed a series of pathological changes.CONCLUSION HGV is a hepatotropic virus and has pathogenicty.

Isolation,Identification and Pathogenic Characteristics of Duck Hepatitis Virus Isolates

[ Objective] The aim of this study was to determine and analyze the pathogen and the reasons of duck viral hepatitis which is prevalent recently and difficult to control. [Method] Viruses were isolated from livers and spleens of ducks with typical clinical symptoms in Linyi, Weifang, Binzhou and other regions of Shandong Province. The pathogenic characteristics were observed by inoculation in chicken or duck embryo, RT- PCR, serological test, and duck regression. [ Result] Four duck hepatitis virus （DHV） strains were isolated, and the 5th passage allantoic fluid contained 10^3.41 -10^5.20 ELD50/mI. The serum cross protection rate was 20% -80% between the DHV stains and DHV type I. The mortalities of 4- day-old healthy ducks challenged by these four stains were 50% -100%. All challenged ducks had typical lesions of duck viral hepatitis, and the death peak appeared after 24-48 h. [Conclusion] The virulence of different DHV isolates has regional difference.

Biological Characterization and Whole Genome Sequencing of a Genotype VII Newcastle Disease Virus Strain

[Objective] The aim of this paper was to study the genotype, pathogenicity and nucleotide difference between Newcastle disease virus(NDV) isolates and traditional NDV vaccine strain(La Sota). [Method] A suspected NDV strain was isolated from a chicken farm. The isolate was preliminarily determined by HA and HI tests. A pair of primers was designed based on the partial sequence of NDV F gene published in GenBank(accession No. JF950510.1). F gene was amplified by RT-PCR, cloned and sequenced. The sequencing result was compared with the F gene sequences published in GenBank, and the phylogenetic tree was constructed to analyze the genotypes. The pathogenicity of the virus was determined by mean death time(MDT) of chicken embryos, intracerebral pathogenicity index(ICPI) of one-day-old chicks and intravenous pathogenicity index(IVPI) of six-week-old chickens, respectively. Based on the NDV genome sequence published in GenBank(accession No. JF950510.1), nine pairs of primers were designed to amplify the genome sequence of the isolate, and its structure was analyzed. [Result] The length of F gene was about 500 bp, and a NDV strain of genotype VII was isolated. The MDT, ICPI and IVPI were 52.8 h, 1.675 and 2.46, respectively, indicating the isolate was a virulent strain. The whole genome sequence analysis results showed that the full genome length of the isolate was 15 192 bp, which had 6 more nu-cleotides than that of La Sota strain, and the homology between the two strains was 82.8%. [Conclusion] A virulent NDV strain of genotype Ⅶ was isolated, with low homology to La Sota strain in nucleotide sequence.

Immunological Changes of Chicks Immunized with March＇s Disease Vaccines and Challenged with Virulent MD Virus

The experiment was conducted to inoculate one-day old chicks with March's disease (MD) trivalent and herpesvirus of turkey (HVT) vaccines separately, and then to challenge them with virulent MD virus (vMDV) at the age of 15 days. and 5, 25, 45 and 75 days after the challenge with vMDV, comparing with the control-challenged chicks without immunization, to detect the immunoprotetive efficacy and dynamic changes of the inductive activity of interleukin-2(IL-2), expression of IL-2 receptor and proliferative function of T cells in thymus and spleen; the number of ANAE+T, AP+T cells and IgG, IgM, IgA antibody-producing cells in Bursa Fabricius, spleen,thymus, cecal tonsil and Harder gland; as the amount of T cells and IgG, IgM, IgA in peripheral blood as well as the content of IgG, IgM and IgA in the tear, trachea washings, bile and intestinal fluids of the experimental chicks. The experimental results firstly demonstrate that the immunorcgulation of IL-2, and IL-2 receptor, the cellualr and humoral immune responses were significantly enhanced in the central and peripheral immune organs; the local mucosal immune function were markedly amplified in the respiratory and digestive tracts of the immunized-challenged,chicks, which were closely correlated with the immunoprotection against MD; the immune response and immunoprotective effect of the trivalent vaccine-immunized chicks were much better than those of HVT vaccine-immunized chicks:

猪瘟病毒致弱机制研究进展

猪瘟是由猪瘟病毒(CSFV)引起的一种严重的猪的传染病,其有效控制主要依赖于猪瘟兔化弱毒疫苗株的应用,但该毒株致弱的机制尚未完全阐明。对近年猪瘟病毒结构蛋白、非结构蛋白和非编码区对毒力影响的国内外研究成果进行了综述,以期为猪瘟病毒致弱相关机制的研究以及新型疫苗的研发提供参考。