ABSTRACT
The recombinant baculocvirus containing genome P1-2A-P3 of hepatitis A virus (HAV) was constructed and used for infecting the Sf9 insect cells. It was demonstrated that the deletion of 2BC from HAV polyprotein and the insertion of a new 3C protease cleavage site between P1-2A and P3 did not interfere with the processing of polyprotein or with forming the 70S-procapsids. The identity of the protein contents as well as of morphological and antigen characteristics, obtained in Sf9-cells, to HAV empty capsids, which take shape in the infected mammal cells, proves that it is possible to use them in making the vaccine and diagnostic preparations.
Subject(s)
Baculoviridae/metabolism , Capsid/metabolism , Hepatitis A/metabolism , Protein Precursors/metabolism , Animals , Baculoviridae/genetics , Baculoviridae/ultrastructure , Capsid/chemistry , Capsid/ultrastructure , Cell Line , Gene Deletion , Genome, Viral , Hepatitis A/genetics , Hepatitis C Antigens/analysis , Hepatitis C Antigens/metabolism , Immunoblotting , Insecta , Microscopy, Immunoelectron , Protein Engineering , Protein Precursors/analysis , Protein Precursors/chemistry , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombination, GeneticABSTRACT
To study the role of the poly(A) tail length during the replication of poly(A)-containing plus-strand RNA virus, we have developed a simple reverse transcription polymerase chain reaction (RT-PCR)-based method that substantially improves the previously reported PAT [poly(A) test] assay. In contrast to the PAT assay, the new method is based on the enzymatic 3' elongation of mRNA with guanosine residues, thus immediately preserving the 3' end of the RNA and creating a unique poly(A)-oligo(G) junction. The oligo(G)-protected full-length poly(A) tail is reverse transcribed using the universal anti-sense primer oligo(dC(9)T(6)) and amplified by PCR with a gene-specific sense primer. After sequencing the resulting RT-PCR product the length of the poly(A) tail was unequivocally deduced from the number of adenosine residues between the oligo(G) stretch and the sequence upstream of the poly(A) tail. The efficiency and specificity of the newly developed assay was demonstrated by analysing the poly(A) tail length of the hepatitis A virus (HAV) RNA. We show here that the poly(A) tail of HAV RNA rescued after transfection of in vitro transcripts was elongated in the course of HAV replication.
Subject(s)
Hepatovirus/genetics , Poly A/analysis , Poly A/genetics , Poly G/biosynthesis , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Viral/genetics , Adenosine/genetics , Adenosine/metabolism , Animals , Base Sequence , Cell Line , DNA Primers/genetics , Genome, Viral , Guanine/metabolism , Hepatovirus/growth & development , Humans , Oligodeoxyribonucleotides/genetics , Poly A/chemistry , Poly G/genetics , RNA, Messenger/chemistry , RNA, Viral/analysis , RNA, Viral/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Sequence Analysis, DNA , Transfection , Virus Replication/geneticsABSTRACT
The cytolytic form of hepatitis E virus reproduction in FRhk-4 cells is described. The cytopathic effect was observed by days 6-7 of the 25th passage in this cell culture. Reverse transcription with polymerase chain reaction were used to study virus reproduction daily for 10 days postinfection. The virus replication starts on day 4 after inoculation. The development of the cytopathic effect depended on the serum concentration in culture medium, whereas the virus replication did not depend on this factor. The replicative form (-RNA) of viral RNA appeared on day 4 postinoculation, as did the new genomic one (+RNA).
Subject(s)
Hepatitis E virus/physiology , Virus Replication , Animals , Cell Line , Cytopathogenic Effect, Viral , Genome, Viral , Hepatitis E virus/genetics , Hepatitis E virus/pathogenicity , Macaca mulatta , Polymerase Chain Reaction , RNA, Viral/geneticsABSTRACT
The morphogenetic pathway of hepatitis A virus (HAV), classified as a member of the enteroviruses within the Picornaviridae, still remains obscure and seems to differ considerably from that of poliovirus, the most studied representative of this genus. In order to elucidate the precursor/product relationship of HAV structural proteins, subviral particles, which represent more than 50% of the viral antigen produced in infected cells, were separated from mature virions and their polypeptide pattern was analyzed by polyacrylamide gel electrophoresis and immunoblotting using monospecific antisera. Whereas mature virions are composed of viral proteins VP1, VP2, and VP3, subviral particles contained VP0 and smaller polypeptides instead of VP2. Comparison of proteins of different strains of HAV showed that VP0 of strain HAS-15 migrated slower than that of strains MBB or GBM. During the course of the infectious cycle, VP0 accumulated and only small portions were converted to VP2 supporting earlier observations that encapsidation of RNA with concomitant cleavage of VP0 is rate-limiting, leaving a large amount of viral antigen in premature particles. Similar to VP0, accumulation of VP1 was observed and two immunologically related precursor proteins, p38 and p36, were found during the course of infection. Immunological characterization of p38 using antisera directed to the N-terminus of VP1 and to synthetic peptides located at the presumptive C- and N-termini of 2A suggests that p38 is VP1 delta 2A carrying 45 N-terminal amino acids of the P2-region.
Subject(s)
Hepatovirus/metabolism , Protein Precursors/metabolism , Viral Structural Proteins/metabolism , Animals , Cell Line , Electrophoresis, Polyacrylamide Gel , Hepatovirus/growth & development , Immunoblotting , Protein Precursors/isolation & purification , Viral Structural Proteins/isolation & purificationABSTRACT
Peptide (P6) representing amino acids 62-75 of HAV capsid protein VP3 was synthesized. Pure P6, octamer form of P6, and P6 conjugated to KLH were injected into rabbits. The sera against these preparations reacted with denaturized VP3 in immunoblotting tests; however, only anti-P6-KLH serum was capable of binding the native HAV.
Subject(s)
Antibodies, Viral/biosynthesis , Binding Sites, Antibody/drug effects , Hepatovirus/immunology , Peptide Fragments/immunology , Peptides/immunology , Viral Proteins/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/blood , Electrophoresis, Polyacrylamide Gel , Immune Sera/isolation & purification , Immunization/methods , Immunoblotting , Immunodominant Epitopes/immunology , Immunoenzyme Techniques , Molecular Sequence Data , Neutralization Tests , Peptide Fragments/chemical synthesis , Peptide Fragments/genetics , Peptides/chemical synthesis , Peptides/genetics , Rabbits , Viral Proteins/chemical synthesis , Viral Proteins/geneticsABSTRACT
The Western blot procedure with highly specific antipeptide antibody was applied to identify the electrophoretic mobility of hepatitis A virus capsid proteins. Polypeptides with molecular weights of 33, 29 and 27 kDa proved to be VP1, VP2, and VP3 proteins as they reacted with sera generated to VP1 recombinant protein and to synthetic oligopeptides 42-62 VP2 and 62-75 VP3, respectively.
Subject(s)
Hepatovirus/analysis , Peptide Mapping/methods , Viral Structural Proteins/analysis , Animals , Antibodies, Viral/blood , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Guinea Pigs , Hepatovirus/immunology , Humans , Immune Sera , Immunization , Immunoenzyme Techniques , Molecular Weight , Rabbits , Recombinant Proteins/analysis , Recombinant Proteins/immunology , Viral Structural Proteins/immunologyABSTRACT
A modified technique of protein staining with silver nitrate was employed in electrophoretic analysis of hepatitis A virus structural proteins. The modifications of the original technique, i.e. thorough washing of the gel, increased formaldehyde concentration and a more intensive lighting of the gel, have elevated the method sensitivity 10-fold permitting the detection of up to 0.1 ng protein. The modification has allowed detection of VP1, VP2, and VP3 structural proteins with molecular masses 33, 29, and 27 kD, respectively, in virus preparations of low concentrations.
