Recombination in adenovirus: crossover sites in intertypic recombinants are located in regions of homology

Wild-type recombinants have been selected from intertypic genetic crosses between Ad2 and Ad5 ts mutants, with lesions within or close to the hexon gene, and the structure of the recombinant genomes has been examined by restriction endonuclease analysis. The recombinants with a crossing over within or in the vicinity of the hexon gene were subjected to a more detailed analysis, using restriction enzyme maps of the HindIII A fragment (map coordinates, 50.1–72.8) constructed for endonucleases that cut the DNA many times, viz. HaeII, HindII, and TaqI. These analyses indicated that crossover sites are confined to regions of relatively high DNA homology. More detailed mapping within the hexon region of Ad2 and Ad5, using a large battery of enzymes, confirmed this and allowed subdivision into three zones on the basis of the distribution of restriction endonuclease recognition sites; at the left-hand amino terminus there is almost complete homology between Ad2 and Ad5, the right-hand half of the gene displays partial homology as judged by the even distribution of common and unique sites, while a central segment of the hexon gene has no common restriction sites. This segment, defined by unique sites, coincides with the region of the Ad2 hexon polypeptide implicated in the trypsin sensitivity of the native hexon, and probably also determines the type-specific antigenicity and electrophoretic mobility of the hexon.     

Recombination in adenovirus: DNA sequence analysis of crossover sites in intertypic recombinants

The nucleotide sequence of the adenovirus type 5 genome has been determined for a 620-bp region that spans the C terminus of the pVI gene and the N terminus of the hexon gene, and compared to the adenovirus type 2 DNA sequence: 25 base changes have been identified, most of which do not lead to alterations in the amino acid sequence and regulatory signals in the region. Crossover sites in three intertypic recombinants have been previously located in this region of the genome by fine restriction mapping. A sequence determination for the three recombinants, and the four ts mutants used in generating the ts+ recombinants, was carried out. The crossovers were in each case located in a small region of complete sequence homology (from 45 to 156 nucleotides long) flanked on either side by sequences derived from each parent. These structures are compatible with a reciprocal crossing over model of generalised recombination, where a recombinant joint has resolved in a region of high DNA homology. For the recombinants considered here, this region abutts onto a neighbouring region of much lower sequence homology, and it is possible that the position of the crossover is determined at least in part by the termination of branch migration at a heterologous boundary.     

Disabled infectious single-cycle herpes simplex virus as an oncolytic vector for immunotherapy of colorectal cancer

New modalities of treatment for colorectal cancer are required to support and improve those currently available. One such approach is immunotherapy by transfer of immunostimulatory genes to tumor cells. Here, we report the use of a herpes simplex virus (HSV) vector that is capable of a single round of infection (disabled infectious single-cycle [DISC]-HSV) as a gene transfer vehicle for colorectal cancer. This vector has potential advantages over other vectors for cancer immunotherapy in that it lyses infected tumor cells. Infection with DISC-HSV inhibited tumor cell growth both in vitro and in vivo. In addition, DISC-HSV-mediated cell killing occurs by both apoptotic and necrotic mechanisms. A range of colorectal tumor cell lines could be rapidly transduced with DISC-HSV/lacZ (14-90% in 4 hr). Both tumor prevention and tumor therapy protocols showed clear antitumor effects with DISC-HSV/mGM-CSF. In the prophylactic approach, an infected/irradiated whole cell vaccine protected up to 80% of mice from rechallenge. In addition, intratumoral injection of established tumors with DISC-HSV/GM-CSF caused rejection in 40% of mice and generated some protection from subsequent rechallenge. In both cases, however, it is clear that a dominant therapeutic effect of the DISC-HSV vector derives from its oncolytic properties, irrespective of the transduced gene. As a prelude to taking these studies forward to human clinical trials, we demonstrate that tumor cells could be successfully grown from freshly obtained human colorectal cancer resections (within 1 week of surgery), were transduced with DISC-HSV/hGM-CSF, and secreted the cytokine. This study provides the preclinical basis for trials of immunotherapy of colorectal cancer using DISC-HSV.     

Analysis of the fowlpoxvirus gene encoding the 4b core polypeptide and demonstration that it possesses efficient promoter sequences

The gene encoding the fowlpox virus 4b core polypeptide has been identified by analogy with the vaccinia 4b gene. It has been cloned, and its nucleotide sequence determined. The gene, which is 1971 nucleotides long, can encode a protein of 75,200 Da (75.2K polypeptide), slightly longer than its vaccinia counterpart with which it shares 52% identity. Sequences upstream of the fowlpox virus 4b gene correspond to the consensus sequence determined for vaccinia late promoters, suggesting that late promoter signals may be shared by the different genera of poxviruses. Upstream sequences have been cloned into a beta-galactosidase translational fusion vector and shown to promote the efficient expression of beta-galactosidase in a transient assay system. This expression was abolished in the presence of araC, an inhibitor of DNA replication which blocks late gene expression in poxviruses. The fowlpoxvirus 4b promoter should be a useful component of genetically engineered fowlpox virus vaccines.     

Sequence analysis of the leader RNA of two porcine coronaviruses: Transmissible gastroenteritis virus and porcine respiratory coronavirus

The leader RNA sequence was determined for two pig coronaviruses, tranmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV). Primer extension, of a synthetic oligonucleotide complementary to the 5 end of the nucleoprotein gene of TGEV was used to produce a single-stranded DNA copy of the leader RNA from the nucleoprotein mRNA species from TGEV and PRCV, the sequences of which were determined by Maxam and Gilbert cleavage. Northern blot analysis, using a synthetic oligonucleotide complementary to the leader RNA, showed that the leader RNA sequence was present on all of the subgenomic mRNA species. The porcine coronavirus leader RNA sequences were compared to each other and to published coronavirus leader RNA sequences. Sequence homologies and secondary structure similarities were identified that may play a role in the biological function of these RNA sequences.The nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases and have been assigned the accession numbers X52157, X52668.     

High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H.

Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.     

Studies with marked antisera: III. Interchange between unmarked antibody and 131I-marked antibody in combination with cellular antigens

Specific interchange takes place between ¹³¹I-marked antibody combined with cellular antigens and the free unmarked antiserum antibody in three systems studied: (A) bovine cells with (i) human infectious mononucleosis serum and (ii) rabbit anti-bovine serum and (B) human Rh D-positive cells with Rh incomplete anti-D serum. The rate of interchange is much slower than the initial combination of an unmarked antibody with an unoccupied antigenic site, and also is slower in the Rh incomplete system than in the other systems studied. Rabbit antibovine red cell antiserum partly interchanges with ¹³¹I-marked infectious antibody on bovine cells, but the reverse interaction does not apparently take place at all. Possible reasons are put forward to explain this. The infectious mononucleosis interchange reaction has a large temperature coefficient (about 4.9) which corresponds to a heat of reaction of 29,000 cal.     

The use of a random priming procedure to generate cDNA libraries of infectious bronchitis virus, a large RNA virus

An efficient method for the generation of gene banks from large RNA viruses is described using infectious bronchitis virus as an example. Randomly primed clones have been characterized and found to be representative of the viral genome including 5' leader sequences.     

Confirmation of the presence of avian infectious bronchitis virus (IBV) using cloned DNA complementary to the 3' terminus of the IBV genome

A hybridisation test has been developed to aid the identification of virus isolates as avian infectious bronchitis virus (IBV) when serum neutralisation tests have proved inadequate. In this test nucleic acid prepared from four recent virus isolates suspected of being IBV hybridised with a 32P-radiolabelled cloned cDNA (complementary DNA) probe complementary to RNA from IBV strain Beaudette. Since these strains represent one or more new serotypes they had not been identified by neutralisation tests using antisera to known IBV serotypes. Polyacrylamide gel electro-phoresis of the isolates radiolabelled in de-embryonated eggs showed that the viruses had proteins characteristic of IBV. Nine other IBV strains, representing five serotypes, also hybridised with the probe.