A-type Inclusion Research Articles

Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses.

We constructed A-type inclusion body (ATI) hybrid promoters, that is, late ATI promoters followed by tandemly repeated early regions of the promoter for the 7.5-kDa protein (the 7.5-kDa promoter). The repetition of the whole early promoter sequence of the 7.5-kDa gene, including the upstream consensus sequence and initiation region, efficiently increased the early expression of the bacterial chloramphenicol acetyltransferase gene in recombinant vaccinia virus. Recombinant vaccinia virus could express influenza virus hemagglutinin via the hybrid promoter more efficiently, induced higher levels of neutralizing antibody and cytotoxic T lymphocytes, and consequently protected mice more efficiently against challenge with influenza virus than did recombinant vaccinia virus containing the widely used 7.5-kDa promoter.

Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence

Different passages of the vaccinia virus strain Ankara (CVA wild-type) during attenuation to MVA (modified vaccinia virus Ankara) have been analysed to detect alterations in the genome. Physical maps for the restriction enzymes HindIII and XhoI have been established. Six major deletions relative to the wild-type strain CVA could be localized. They reduce the size of the entire genome from 208 kb (CVA wild-type) to 177 kb for the MVA strain. Four deletions occurred during the first 382 passages and the resulting variant (CVA 382) displays an attenuated phenotype similar to that of the MVA strain. The deletions are located in both terminal fragments, affect two-thirds of the host range gene K1L and eliminate 3.5 kb of a highly conserved region in the HindIII A fragment. During the next 190 passages leading to MVA two additional deletions appeared. Again, one is located in the left terminal fragment, and the other includes the A-type inclusion body gene. Neither of the deletions appear to participate in further attenuation of the virus. Rescue of the partially deleted host range region with the corresponding wild-type DNA restored the ability of the attenuated strains MVA and CVA 382 to grow in some non-permissive tissue cultures. Nevertheless, the complete host range of the wild-type strain was not recovered. Also, plaque-forming behaviour and reduced virulence were not influenced. From the data presented it may be concluded that the partially deleted host range gene is not solely responsible for attenuation.

Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection

The bovine leukaemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into the HA locus of vaccinia virus (Copenhagen strain), downstream of the vaccinia virus early-late promoter, H6, or a triple promoter element consisting of the promoter for the vaccinia virus H6 gene, the promoter for the cowpox virus A-type inclusion (AT1) gene and the promoter for the vaccinia virus HA gene. Inoculation of rabbits or sheep with the recombinant vaccinia virus coding for gp51 and gp30 or an uncleaved env precursor induced neutralizing antibodies to BLV. These antibodies competed with monoclonal antibodies directed against gp51 epitopes F, G, and H previously shown to be of crucial importance for BLV infection. Seven out of eight sheep vaccinated with the vaccinia recombinants resisted a drastic challenge (1.5 × 10 3 sheep infectious doses) with BLV-infected lymphocytes. These results show that vaccination with BLV env vaccinia recombinants protects sheep against infection with extremely high doses of BLV-infected heterologous lymphocytes.

Induction of neutralizing antibodies against bovine leukosis virus in rabbits by vaccination with recombinant vaccinia virus expressing bovine leukosis virus envelope glycoprotein

SUMMARY Three kinds of recombinant vaccinia virus (rvv)—mO-ha/ati, LO1-ha/ati and mO-ha/7.5kD—expressing bovine leukosis virus (blv) envelope glycoprotein (gp60) were constructed. The blv envelope gene of rvv mO-ha/ati and LO1-ha/ati or of rvv mO-ha/7.5kD was expressed under control of the promoter of A-type inclusion body (ati) protein gene of cow-pox virus or vaccinia virus 7.5-kD protein gene, respectively. The vaccinia virus strain, LC16mO, was used as vector for rvv mO-ha/ati and mO-ha/7.5kD, and strain LO-1 was used for rvv LO1-ha/ati. Strains LC16mO and LO-1 are attenuated vaccine virus strains originating from the Lister original vaccinia virus. All 3 kinds of constructed rvv expressed gp60 in cultured rabbit kidney cells after infection; mO-ha/ati expressed more antigen than did mO-ha/7.5kD. Rabbits vaccinated with rvv produced considerable antibody capable of inihibiting syncytium formation, as well as antibody with virion-binding ability. The rvv that used ati promoter induced higher antibody titer than did the rvv that used 7.5-kD promoter. Results indicate that blv gp60 is responsible for induction of neutralizing antibodies that suppress in vitro formation of syncytia among blv-infected cells. Applicability of rvv, especially those using ati promoter, was evaluated in a vaccine against bovine leukosis.

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.

Mechanism of virus occlusion into A-type inclusion during poxvirus infection

The A-type inclusion (ATI), a product as a late function of some poxviruses, has different strain-specific properties, some containing mature viruses (V + ATI) and some without (V − ATI). The properties of virus-occluding factor (VO factor) responsible for the V + trait were examined either employing a cell fusion technique by inactivated Sendai virus or under the cell-free condition. The VO factor was synthesized late in the (V − virus-infection corresponding to the time of ATI formation. A proportion of the VO factor synthesized may rapidly attach to the surface of mature V+ viruses (vaccinia) so as to acquire the ability of occlusion within ATI, and a residual proportion of the VO factor released throughout the cytoplasm may attach similarly to that of (V − viruses carried in by cell fusion or added in vitro. Neither protein synthesis nor energy was required to occlude mature viruses into ATIs, and the V + trait occurred in the presence of rifampicin. Comparison of the electrophoresis distribution of proteins constituting V + and (V − virions revealed that the VO factor was identical with the VP 4c polypeptide located at the surface of vaccinia virion. The polypeptide showing the same mobility on polyacrylamide gel electrophoresis was also found in the postviral supernatant fraction prepared from V + virus-infected cells.

ラメラ・テアの発生におよぼす継手の拘束度の影響(第2報)

In the previous paper, multirun RRC (Rigid Restraint Cracking) test and Modified Cranfield test were carried out for evaluating the susceptibility of lamellar tearing of SM41 and HT50. As the result, the phenomena of lamellar tearing was deeply dependent on the distribution of non-metallic inclusion (mainly Mn-S of A-type inclusion) and the restraint condition of joint, with discussing the relation between the susceptibility of lamellar tearing in the both testing methods and through-thickness tensile properties of steels.In this report, for the application to an actual structure, it is examined to get a standard for selection of plate material in the point of view of the prevention of lamellar tearing by a H-type restraint cracking test with an interposed plate.Conclusions obtained are as follows.(1) Higher sulphur content and restraint intensity (K) increase crack susceptibility. Also, good correlation between the susceptibility of lamellar tearing and through-thickness properties, such as the reduction of area at Z-direction, the ratio of transverse shrinkage by Brodeau-type test, or Inclusion Shape Factor in microstructure. Therefore, these may be available as an acceptance criteria for lamellar tearing.(2) The changes of leg length and groove geometry influence to the critical value of crack initiation in the above tests.(3) As for the susceptibility for lamellar tearing, good correlation between crack sensitivity parameters in Modified Cranfield test and H-type restraint cracking test is obtained. So, the susceptibility of lamellar tearing may be evaluated with the parameter obtained by Modified Cranfield test, which is simple and effective in reproducible, by clarifying the above correlation.(4) The susceptibility of lamellar tearing at interposed structure, as an example of the application of an actual structure in shipbuilding, is determined by calculating the value of restraint intensity of joint. As a result, grade of steel plate adopted may be selected by the above mentioned criteria for the prevention of lamellar tearing.

痘瘡群ウイルスの遺伝学的解析

It was previously suggested that the character of the A-type inclusion can be separated into two markers: the capability to include mature progeny virions within or on the inclusion body (V), and the formation of ground substance of the inclusion body (In). In addition, another difference in the susceptibility to the L cells was found between the vaccinia IHD strain virus and the CPR-Cl cowpox virus (L). The low growth rate of CPR-Cl virus calculated about 3 logs can be rescued by vaccinia IHD virus in mixedly infected L cells.The recombinants among these markers were found in variable percentages of the progeny viruses of mixed infection with a combination of vaccinia IHD and cowpox CPR-Cl viruses, using the plaque types as control markers. Especially our prediction that the phenotypes of the A-type inclusion is controled by two viral genes was proved by the appearance of V+In+ recombinants of 1.8%. Concerning the plaque types being of polygenic nature, morphological features of cytopathic effect in HeLa cells were coincided with plaque types of all recombinant clones.

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.

Studies on the nature of marchal bodies (A-type inclusion) during ectromelia virus infection

Marchal body development regularly followed the appearance of mature ectromelia virus. Isolated Marchal bodies consisted mainly of protein, the antigenicity of which was quite different from that of NP antigen, soluble antigen, or hemagglutinin. The protein became soluble at pH 11.5 and reprecipitated at pH 5.2. There was no significant difference in these properties between the Hampstead and Ishibashi strains. Considerable variation with respect to plaque size was found between the two strains.