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。     

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.     

Characterization of a simian hepatitis A virus (HAV): antigenic and genetic comparison with human HAV.

PA21, a strain of hepatitis A virus (HAV) recovered from a naturally infected captive owl monkey, is indistinguishable from human HAV in polyclonal radioimmunoassays and cross-neutralization studies. However, cDNA-RNA hybridization has suggested a significant difference at the genomic level between PA21 and a reference human virus, HM175. Further characterization of this unique HAV was undertaken in an effort to determine the extent of genetic divergence from human HAV and its relation to the conserved antigenic structure of the virus. The close similarity between PA21 and HM175 antigens was confirmed with an extended panel of 18 neutralizing murine monoclonal antibodies: a reproducible difference in binding to the two viruses was detected with only one antibody (B5-B3). The nucleotide sequence of the P1 region of the PA21 genome had only 83.2% identity with HM175 virus, a difference approximately twice as great as that found between any two human strains. Most nucleotide changes were in third base positions, and the amino acid sequences of the capsid proteins were largely conserved. Amino acid replacements were clustered in the carboxy terminus of VP1 and the amino-terminal regions of VP2 and VP1. These data indicate that PA21 virus represents a unique genotype of HAV and suggest the existence of an ecologically isolated niche for HAV among feral owl monkeys.     

Molecular characterization of China human rabies vaccine strains

To understand the molecular characteristics of China human rabies vaccine strains, we report the full-length genome of the aG strain and present a comprehensive analysis of this strain and almost all available lyssavirus genomes (58 strains) from GenBank (as of Jan 6, 2011). It is generally considered that the G protein plays a predominant role in determining the pathogenicity of the virus, to this end we predicted the tertiary structure of the G protein of aG strain, CTN181 strain and wild type strain HN10 based on the crystal structure of Vesicular stomatitis virus (VSV) G. The predicted RABV G structure has a similar topology to VSV G and the ectodomain can be divided into 4 distinct domains DI — DIV. By mapping the characterized mutations to this structure between China vaccine strains and their close street strains, we speculate that the G303(P-H) mutations of CTN181 and HN10 causing DII 3D change may be associated with the II attenuated virulence in both strains. Specifically, the two signature mutations (G165P and G231P) in the aG strain are withinßsheets, suggesting that both sites are of structural importance.     

Biophysical and Biochemical Characterization of Lymphocytic Choriomeningitis Virus: IV. Strain Differences

Biological, biochemical, and biophysical properties of three lymphocytic choriomeningitis (LCM) virus strains were compared. The biological property examined was the concentration range of virus which would, when injected into neonates, cause a carrier state. The dosage range for the CA1371 and Traub strains was found to be as broad as the limits examined (5 to 100 ld(50) units/mouse). The WCP strain, however, would only produce carriers within a 3 to 5 ld(50) range. The biochemical properties examined were the growth rates in tissue culture and the effect of varying the input ratio of virus to cells. With identical input ratios, the Traub strain reached a peak titer 32 hr after infection. The CA1371 and WCP strain reached their peaks at the 40th hr. With a 10-fold decrease in the amount of CA1371 virus per cell, peak titer (as high as in the above experiments) was not obtained until 56 hr postinfection. The biophysical properties examined were stability in density gradients and inactivation rates at 4C. In potassium tartrate gradients, full recovery of the CA1371 and WCP strain could be achieved. However, inactivation kinetics showed that only the CA1371 strain was much more stable than the Traub-LCM. The realization that marked differences in LCM strains exist is discussed in relation to certain inconsistencies in the literature.     

Genomic heterogeneity among human and nonhuman strains of hepatitis A virus.

Cloned cDNA probes derived from the P1 and P2 regions of the genome of HM175 virus, a reference strain of human hepatitis A virus (HAV), failed to hybridize under standard stringency criteria with RNA from PA21 and PA33 viruses, two epizootiologically related HAV strains recovered from naturally infected New World owl monkeys. Hybridization of these probes to PA21 RNA was only evident under reduced stringency conditions. However, cDNA representing the 5' nontranslated region of the HM175 genome hybridized equally to HM175 and PA21 RNA under standard stringency conditions, while a probe derived from the 3' 1,400 bases of the genome yielded a reduced hybridization signal with PA21 RNA. In contrast, no differences could be discerned between HM175 virus and three other HAV strains of human origin (GR8, LV374, and MS1) in any region of the genome, unless increased stringency conditions were used. These results suggest that PA21 and PA33 are unique among HAV isolates and may represent a virus native to the owl monkey. Despite extremely poor homology within the P1 region, which encodes capsid polypeptides, monoclonal antibody analysis confirmed that the immunodominant neutralization epitopes of HAV were highly conserved between HM175 and PA21 viruses. Furthermore, experimental challenge of the owl monkey with successive PA33 and HM175 inocula confirmed a high but incomplete degree of cross-protection. Only one of six monkeys previously infected with PA33 developed recurrent hepatitis 28 days after intravenous HM175 challenge, while none of six monkeys previously infected with HM175 had demonstrable hepatitis following PA33 challenge. These data provide molecular evidence for the existence of HAV strains unique to nonhuman primate species and indicate that strict conservation of antigenic function may accompany substantial genetic divergence in HAV.     

甲肝病毒（龙甲—25株）在Frhk4细胞上传代特征

应用Frhk4细胞增殖HAV,经较系统研究,建立了稳定的细胞传代方法,病毒增殖条件,获得了较大量的甲肝病毒抗原.HAV龙甲-25株在2BS细胞上传至第六代移到Frh4细胞上连续传至九代,用荧光法(IF)、EMISA夹心法,和免疫电镜方法(IEM)进行检测,结果表明龙甲-25不同代次间的病毒增殖情况不同.我们将龙甲-25HAV在Frhk4细胞上从第七代传至第十六代,分别用IF,ELISA,IEM方法检测,结果是细胞在感染病毒后不同天数的IF结果阳性,病毒传代的代次不同在ELISA和IEM检测中出现不同的结果.在第九代至第十一代的HAV′培养过程中我们改变了病毒培养及收获方法,再重复试验时,出现三种实验方法一致的结果.     

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Hepatitis A virus (HAV) contains a single-stranded, plus-sense RNA genome with a single long open reading frame encoding a polyprotein of approximately 250 kDa. Viral structural proteins are generated by posttranslational proteolytic processing of this polyprotein. We constructed recombinant vaccinia viruses which expressed the HAV polyprotein (rV-ORF) and the P1 structural region (rV-P1). rV-ORF-infected cell lysates demonstrated that the polyprotein was cleaved into immunoreactive 29- and 33-kDa proteins which comigrated with HAV capsid proteins VP0 and VP1. The rV-P1 construct produced a 90-kDa protein which showed no evidence of posttranslational processing. Solid-phase radioimmunoassays with human polyclonal anti-HAV sera and with murine or human neutralizing monoclonal anti-HAV antibodies recognized the rV-ORF-infected cell lysates. Sucrose density gradients of rV-ORF-infected cell lysates contained peaks of HAV antigen with sedimentation coefficients of approximately 70S and 15S, similar to those of HAV empty capsids and pentamers. Immune electron microscopy also demonstrated the presence of viruslike particles in rV-ORF-infected cell lysates. Thus, the HAV polyprotein expressed by a recombinant vaccinia virus demonstrated posttranslational processing into mature capsid proteins which assembled into antigenic viruslike particles.     

Direct sequencing of hepatitis A virus strains isolated during an epidemic in France.

Direct sequencing of PCR products was used to study the VP1 region of the hepatitis A virus (HAV) genome (position 2199 to 2356) of nine strains isolated from human stools collected during a hepatitis A epidemic (western France, 1992), three strains from environmental samples (1990, 1991, and 1992), and two HAV cell culture isolates (the French strain CF53/Lyon and strain CLF). These viruses differed from CF53/Lyon (genotype I) by between 1 and 10.3%, and results indicated the existence of two groups of strains belonging to two different subgenotypes (IA and IB). With this sequencing technique it was possible to monitor the epidemiology of HAV and study its relations.     

Application of the Laurell immunoelectrophoresis technique to the study of serological relationships between granulosis viruses

The Laurell technique of two-dimensional immunoelectrophoresis was used to distinguish between isolates of granulosis virus (GV) from Plodia interpunctella (GV strains A and B), Ephestia cautella, Spodoptera littoralis (GV strain 65), Pieris brassicae, and Cydia pomonella. Granules, alkali-soluble proteins, and virus particles of P. interpunctella GV strain A and granules of P. interpunctella GV strain B were used as sources of antigens. They were reacted with the immunoglobulins of antisera prepared against whole granules of each strain of virus. Peaks of precipitation were most clearly defined when antigens were pretreated with 0.1 m Na₂CO₃, 2% Triton X, and succinic anhydride, Granules and alkali-soluble proteins of P. interpunctella GV strain A treated in this way exhibited at least one peak of precipitation when reacted with each of the antisera studied. Four peaks were observed in both the homologous reaction and in the heterologous reaction with antiserum prepared against granules of P. interpunctella GV strain B. Four different peaks were present in the homologous reaction between immunoglobulins and virus particles of P. interpunctella GV strain A. Two peaks were present in the heterologous reaction with antiserum prepared against granules of P. interpunctella GV strain B and one in that with the antiserum prepared against granules of S. littoralis GV strain 65.