A-type Inclusion Body Research Articles

A comparison of the genome organization of capripoxvirus with that of the orthopoxviruses

Comprehensive comparisons of genome organizations for poxviruses of different genera have not previously been reported. Here we have made such a comparison by cross-hybridizing genome fragments from capripoxvirus KS-1 and vaccinia virus WR (VV). This showed that a 100- to 115-kilobase (kb) centrally placed section is essentially colinear in organization in the two viruses and that a small region has translocated between the ends of one or other of the genomes during their divergence. No cross-hybridization could be detected between VV DNA and the respective left- and right-hand terminal 8 and 25 kb of capripoxvirus DNA or between capripoxvirus DNA and the respective left- and right-hand terminal 38 and 35 kb of VV DNA. By using the cross-hybridization data, a 4-kb fragment of KS-1 DNA was identified, which corresponds to the regions of the cowpox virus and VV genomes containing genes for the orthopoxvirus A-type inclusion body protein ("ATI"). The sequence of the KS-1 DNA fragment contains homologs of genes which are on either side of the orthopoxvirus ATI genes but contains no homolog of the ATI gene itself. Overall, these results show that the pattern of genomic conservation and variation between two poxvirus genera reflects the pattern within the orthopoxvirus genus but that, as observed previously, individual genes may not be present in genomic regions which are otherwise conserved in organization.

Development of heat-stable recombinant rinderpest vaccine.

Recombinant vaccinia virus (RVV) containing the full-length cDNA of rinderpest virus (RV)-haemagglutinin (H) gene was constructed. The H gene was inserted into the attenuated vaccine strain of vaccinia virus (VV), Le 16 m0, with two different promoters, namely cowpox virus A-type inclusion body (ATI) promoter or VV 7.5 kilodalton (P7.5) promoter. These RVVs produced the same sized fully glycosylated RV-H protein in RK 13 cells as that of the authentic RV-H. Their heat stability in the lyophylized state was similar to that of the parental VV. All rabbits immunized with these RVVs produced virus neutralizing (VN) antibody to RV as well as anti RV-H antibody. Four weeks after immunization, these animals were challenged with RV intravenously. None of the RVV-immunized rabbits developed any clinical signs of RV infection except one which was immunized with RVV containing the ATI promoter and developed low VN titer. These results indicate the possibility of developing a heat-stable recombinant vaccine for the eradication of rinderpest in tropical countries without cold storage systems.

High-level synthesis of biologically active reovirus protein σ1 in a mammalian expression vector system

The reovirus cell-attachment protein, which modulates tissue tropism and the nature of the antiviral immune response, is protein σ1. This protein is present in reovirus particles in the form of 12 tetramers anchored in the projections or spikes. It is formed in infected cells in very small amounts only. In order to produce larger amounts of it that are necessary for studying its properties in detail, we have cloned it into a mammalian expression vector system that consists of the very strong cowpox virus A-type inclusion body protein gene promoter inserted into the vaccinia virus genome. In cells infected with the resultant recombinant vaccina virus, protein σ1 is formed in large amounts (2–3 mg/10 9 cells). We have isolated the native protein and shown that it exists as tetramers that possess cell-binding activity, hemagglutinating activity, and functional epitopes recognized by both polyvalent antisera and monoclonal antibodies.

Cloning and characterization of the gene encoding the major protein of the A-type inclusion body of cowpox virus.

We have mapped and determined the nucleotide sequence of the cowpox virus (CPV) gene coding for the protein of A-type inclusion bodies (ATI). The ATI gene was mapped to the central part of the largest HindIII restriction fragment of the CPV genome. Vaccinia virus (VV) also contained a similar sequence at the equivalent position in its genome, which even under stringent conditions hybridized with the CPV ATI gene. Nucleotide sequence analysis revealed that the upstream region of the coding frame contained a sequence closely resembling the VV late promoter consensus sequence and that the gene encoded a protein of Mr 150,000.

Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5′-terminal poly(A) sequences.

We have identified and characterized one of the most strongly-expressed genes of cowpox virus (CPV). This is the gene encoding the major protein component of the A-type inclusion bodies produced by this virus. This gene (designated the 160K gene) is transcribed late during the infection. Analyses of its mRNAs showed that these late RNAs, unlike all other characterized late mRNAs of poxviruses, are uniform in length. However, the most remarkable feature of the mRNAs of the 160K gene is the structure of their 5'-termini. Most of these mRNAs have 5'-terminal poly(A) sequences containing 5-21 residues. Furthermore, these 5'-terminal poly(A) sequences are not complementary to the corresponding region of the template strand of the viral DNA. Instead, the nucleotide sequences of the mRNA and the viral DNA diverge at the site of the three As in the sequence 5'-TAAATG-3' containing the gene's initiation codon. Consequently, the poly(A) provides the leader sequences of these mRNAs. These unusual 5'-terminal structures suggest that the late mRNAs of pox-virus genes are generated by a novel process.

Cross-reactivity among cowpox, ectromelia and vaccinia viruses with monoclonal antibodies recognizing distinct antigenic determinants in A-type inclusion bodies

Several monoclonal antibodies recognizing distinct antigenic determinants in A-type inclusion bodies (ATIB) induced by cowpox virus (CPV) were obtained to examine the cross-reactivity among various strains of poxviridae, comprising CPV, ectromelia virus (EV), vaccinia virus (VV) and Shope fibroma virus (SFV). The monoclonal antibodies were classified into at least 3 groups on the basis of the results of an immunofluorescence test and immunoblotting; i. e., strain-specific, CPV and EV-specific and Orthopoxvirus (CPV, EV and VV)-specific antibodies. Differences were found between the antigenic determinants of ATIB of LB strains (LB red and LB white) and other strains (Amsterdam, 53, 58 and 60) of CPV and also between those of ATIB of CPV and EV. An interesting finding was that VV also produces the antigen analogous to that associated with ATIB in CPV- and EV-infected cells despite the absence of morphologically defined ATIB.

Polypeptide analysis with monoclonal antibodies of A type inclusion bodies induced by cowpox virus.

The antigen in A type inclusion bodies (ATIB) induced by cowpox virus (CPV) was examined by immunofluorescent staining with monoclonal antibodies and polypeptide analysis. In the immunofluorescence (IF) test, these monoclonal antibodies reacted only with cytoplasmic inclusion bodies in cells infected with CPV. The fluorescence became detectable in the cells 6-9 hours after infection with CPV. No fluorescence was detectable in cells infected with CPV in the presence of cytosine-I-beta-D-arabinofuranosyl-HCl (Ara C) or in cells infected with other poxviruses, such as vaccinia virus (VV) or Shope fibroma virus (SFV). On Western blotting and immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), only one component with a molecular weight of about 160,000 (160 K) was detected in CPV-infected cells. This 160 K polypeptide was first detectable 12 hours after infection of cells with CPV, and was not detectable in infected cells in the presence of Ara C. The 160 K polypeptide was not detected in cells infected with VV or SFV, or in virions purified from CPV-, VV- or SFV-infected cells.

The relationship between poxvirus and A-type inclusion body during double infection

The A-type inclusion body characteristic of the poxvirus infection differs with respect to virions, which in one group appear independently of the inclusion body (V −) and in the other are enclosed within or on it (V +). This character of Marchal (ectromelia virus) as well as Downie bodies (cowpox virus) is genetically stable. Simultaneous infection of L or HeLa cells with two poxviruses was studied, employing characterization of the A-type inclusion body and plaque morphology as markers. Inclusion bodies appearing in doubly infected cells contained progeny virions with both V + and V − characters similar to those of the parent virions. It can be concluded that the V + character is predominant over the V − type. Furthermore, double infection with a combination of the virus with V + character and the vaccinia virus IHD, deficient in formation of the A-type inclusion body, resulted in the appearance of only V + inclusion bodies. It becomes apparent that the V character can be separated into two markers: the capability to include mature progeny virions within or on the inclusion body and the formation of ground substance of the inclusion body.