甲型肝炎重组痘苗病毒(VMS11HAV25)的构建及其某些生物学性质

将5′端经过剪切的甲型肝炎病毒全部开放读码框架cDNA连接于痘苗病毒晚期启动子P11下游,重组于痘苗病毒天坛株的HiodⅢM片段Spb Ⅰ位点获得了重组病毒VMS11HAV25。对其生物学性质的研究表明,该重组病毒诱生痘苗抗体的能力、对鸡红细胞的血凝性质、空斑大小及对温度的稳定性等均与原天坛株相同。重要的区别是,重组病毒在家兔皮内和小鼠脑内的毒力都比原天坛株低约1个对数。病毒在人胚肺二倍体细胞连续传15代的表达水平与传代早期者相同。连续传20代后提取病毒DNA做Southern blot杂交表明,甲型肝炎病毒基因仍稳定地存在于原插入位置。     

火鸡疱疹病毒在鸡胚成纤维细胞上有效代次的测定及其免疫原性恢复的研究

本试验证明,火鸡疱疹病毒(HVT)FC126株在鸡胚成纤维细胞(CEF)传55代的病毒,仍有预防马立克氏病(MD)的效力,至第70代效力明显下降。若将HVT CEF第35代病毒复归火鸡连续传4代,能明显恢复其免疫原性,用C细胞传代则有效代次可达到75代。因而,此方法能有效地解决免疫原性下降的种毒的更新问题。本试验还为该种毒和疫苗的有效代次的使用范围提供了可靠依据。     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes.     

Cell culture and infection system for hepatitis C virus

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro-transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.     

Production of infectious hepatitis C virus in cell culture

Hepatitis C virus (HCV) is a major public health problem, infecting an estimated 170 million people worldwide. Current therapy for HCV-related chronic hepatitis is based on the use of interferon. However, virus clearance rates are insufficient. Investigations to develop the anti-viral therapy or to understand the life cycle of this virus have been hampered by the lack of viral culture systems. We isolated the JFH-1 strain from a patient with fulminant hepatitis, and the JFH-1 subgenomic replicon could replicate efficiently in culture cell without adaptive mutation. Recently, we developed the HCV infection system in culture cells with this JFH-1 strain. The full-length JFH-1 RNA was transfected into Huh7 cells. Subsequently, viral RNA efficiently replicated in transfected cells and viral particles were secreted. Furthermore, secreted virus displayed infectivity for naive Huh7 cells. This system provides a powerful tool for studying the viral life cycle and constructing anti-viral strategies.     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

Isolation of the novel agent from human stool samples that is associated with sporadic non-A, non-B hepatitis.

The agent(s) responsible for sporadic non-A, non-B hepatitis in humans was serially transmitted in rhesus monkeys by intravenous inoculation of the stool extract from a patient. A novel agent called HFV (hepatitis French [origin] virus) was present as 27- to 37-nm particles in the infectious stool extract. Hepatopathic lesions were noticed in infected monkeys during the acute phase of illness. The purified viral 27- to 37-nm particles consist of a double-stranded DNA of approximately 20 kb and are detected in infected monkey liver. Analysis of cell culture detects the approximately 20-kb-long viral DNA in stool samples from infected monkeys and sporadic enteric non-A, non-B hepatitis patients. Furthermore, the 27- to 37-nm viral particles were able to protect monkeys challenged with infectious stool extract. Our results indicate that 27- to 37-nm virus like particles are responsible for sporadic non-A, non-B hepatitis in rhesus monkeys.     

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine.

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5' noncoding (NC) region. These 5' NC mutations and a separate group of 5' NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5' NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

Construction of probes for hybridization with hepatitis A virus RNA based on phage M13

Three fragments of a DNA copy complementary to tre hepatitis A virus RNA have been isolated from the pHAV-VPI-22 plasmid and cloned in M 13 mp 8 vector. The 140, 250 and 390 b. p. fragments corresponded to the viral genome fragment coding for VP1 protein. Single-stranded DNAs of the hybrid phages with 250 b. p. and 390 b. p. inserts have been used for radioactive probe synthesis. The specificity of the probes for viral RNA was confirmed in hybridization with hepatitis A virus RNA purified from the virus-infected cell culture. The hybridization was shown to detect up to 10(-12)-10(-13) g of the virus. A principal possibility to use the probes in diagnostic purposes was demonstrated by the virus detection in blood samples obtained from patients with hepatitis A infection.     

Noncytopathogenic pestivirus strains generated by nonhomologous RNA recombination: alterations in the NS4A/NS4B coding region

Several studies have demonstrated that cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In addition, two recent reports showed the rapid emergence of noncp Bovine viral diarrhea virus (BVDV) after a few cell culture passages of cp BVDV strains by homologous recombination between identical duplicated viral sequences. To allow the identification of recombination sites from noncp BVDV strains that evolve from cp viruses, we constructed the cp BVDV strains CP442 and CP552. Both harbor duplicated viral sequences of different origin flanking the cellular insertion Nedd8*; the latter is a prerequisite for their cytopathogenicity. In contrast to the previous studies, isolation of noncp strains was possible only after extensive cell culture passages of CP442 and CP552. Sequence analysis of 15 isolated noncp BVDVs confirmed that all recombinant strains lack at least most of Nedd8*. Interestingly, only one strain resulted from homologous recombination while the other 14 strains were generated by nonhomologous recombination. Accordingly, our data suggest that the extent of sequence identity between participating sequences influences both frequency and mode (homologous versus nonhomologous) of RNA recombination in pestiviruses. Further analyses of the noncp recombinant strains revealed that a duplication of 14 codons in the BVDV nonstructural protein 4B (NS4B) gene does not interfere with efficient viral replication. Moreover, an insertion of viral sequences between the NS4A and NS4B genes was well tolerated. These findings thus led to the identification of two genomic loci which appear to be suited for the insertion of heterologous sequences into the genomes of pestiviruses and related viruses.     

人轮状病毒的细胞培养分离

用CV-1细胞从急性腹泻病儿7份粪便标本中直接分离出4株人轮状病毒(HRV),并适应传代14代。感染细胞出现特征性细胞病变,经电镜、ELISA、免疫荧光染色及病毒RNA电泳等试验证实HRV毒株在CV-1细胞中的繁殖及抗原特异性。病毒滴度为10~(6.0)TCID_(60)。分离的4株HRV毒株均为RNA电泳型长型,经CV-1细胞传7～14代后,RNA图型与原粪样相比未见变异。     

用人胚肺二倍体细胞株分离一株甲型肝炎病毒

自甲型肝炎患者粪便中分离到一株甲型肝炎病毒(TZ-84)。病毒不产生细胞病变,胞浆中有颗粒性荧光。细胞中的病毒抗原能被抗甲肝病毒抗体阳性血清所中和。 TZ-84株病毒耐酸,耐乙醚。免疫电镜见有大量直径27～30mm的甲肝病毒颗粒,以空心为主。将其接种细胞后的黑暗期随传代而缩短,病毒滴度则随传代而增加。将细胞粉碎后获得的病毒。ELISA滴度为1:16。用其检测45份人血清中抗HAV-IgM抗体,并与Abbott ELISA药盒比较,符合率为91.1％,敏感度为87.1％。     

Dynamics of the accumulation of virus particles with different physico-chemical properties in FRhK-4 cells infected with hepatitis A virus

The propagation time-course of hepatitis A virus (HAV, strain HAS-15) in continuous culture of the foetal rhesus monkey kidney cells (FRhK-4) was investigated. The HAV infectivity and viral RNA content in the infected cells reached the maximal level 5-8 days after infection, while accumulation of hepatitis A antigen (HAAg) continued for 2-3 weeks more. Viral particles with the densities 1.27-1.28 g/cm3 and 1.18-1.22 g/cm3 were isolated from the infected cells as well as the mature virions with the buoyant density 1.33-1.34 g/cm3 in CsCl. The concurrent accumulation of mature virus and "light" particles (1.18-1.22 g/cm3) was registered during infection. Viral particles with the density 1.27-1.28 g/cm3 accumulated predominantly from the 14th to the 21st-24th days after infection. The mature virions (1.34 g/cm3) as well as the particles with the density 1.24-1.25 g/cm3 were isolated from supernatant precipitated by ammonium sulphate. The HAAg activity of both fractions increased progressively in equal proportion in course of infection.     

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus.

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.     

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.