Pseudorabies virus (PRV), an alpha-herpesvirus, causes substantial economic losses in the swine industry and is currently the focus of eradication and control programs. Some of these programs rely on the ability of veterinarians to differentiate animals exposed to virulent strains of PRV from animals exposed to avirulent vaccine strains of PRV on the basis of a serologic response to nonessential glycoproteins that are deleted in some vaccine strains of PRV. Genetic recombination resulting in the creation of virulent strains of PRV with the same negative immunologic markers as vaccine strains could disrupt these programs. Two strains of PRV were coinoculated either into tissue culture or into sheep to facilitate recombination. Progeny viruses were selected to detect a specific recombinant phenotype. We were able to detect genetic recombination between vaccine strains of PRV following in vitro or in vivo coinoculation of 2 strains of PRV. The selected recombinants had marker-deleted phenotypes in strains with restored virulence genes. Increased virulence was observed in sheep after coinoculation of 2 avirulent vaccine strains of PRV.

A recombinant pseudorabies virus that is defective in the early protein 0 (EP0) and large latency transcript (LLT) genes was constructed. A portion of the EP0 and LLT genes was replaced by the lacZ gene of Escherichia coli that had been placed under the control of the pseudorabies virus gX gene promoter. This recombinant virus produces smaller size plaques and yields less virus than does the parent virus on Madin-Darby bovine kidney cells. Although the time course of virus replication and release into the medium of the recombinant and parent viruses are similar, the recombinant virus did not reach as high a titer. Similar to the parent virus, the recombinant virus replicates in the upper segment of the respiratory tract of swine, but the amount of progeny viruses produced is significantly reduced. The data indicated that the EP0 and LLT genes of pseudorabies virus are nonessential for replication. Virus that lacks these 2 genes has impaired growth in tissue culture and is attenuated for swine, compared with the parent virus.

Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wildtype or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.

Genetic recombination between field strains and vaccine strains of pseudorabies virus (PRV) has been suggested as a scenario that might arise from use of deletion-mutant modified-live vaccine strains, particularly those strains attenuated by deletions within the thymidine kinase (TK-) gene locus. To address this hypothesis experimentally, it is necessary to screen large numbers of PRV isolates for their TK genotype. Techniques to detect the native TK genotype are routinely used in molecular virology laboratories, but are time-consuming. We adapted the polymerase chain reaction to define the genotypic status of PRV isolates with regard to the presence or absence of deletions in the TK gene locus. Used in tandem with the existing glycoprotein-specific ELISA that discriminate between PRV-vaccinated and field strain-infected swine populations, the described technique may help to clarify whether vaccine-derived recombinants are generated under natural conditions and after normal vaccine usage.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.

Experiments concerned with the immunogenicity, pathogenicity, and transmissibility of a recombinant vaccinia:Sindbis virus were conducted. The WR strain of the recombinant vaccinia:Sindbis virus was found to be infective for calves and mildly pathogenic, resulting in local tissue reaction. It was not transmissible to other calves. Also, it was found to be immunogenic when inoculated intradermally into calves, and antibody was produced against the parent vector virus (vaccinia) and the Sindbis antigen. Recombinant virus given IV to calves induced no detectable clinical signs, nor did the calves develop neutralizing antibodies. Furthermore, second-passage lesion material containing up to 10(7) tissue culture infective doses of the recombinant virus failed to induce development of lesions or illness in intradermally inoculated calves, and virus could not be recovered from the inoculation sites. In this series of experiments, this vaccinia recombinant given intradermally was immunogenic, mildly pathogenic at the local injection site only, and was not transmissible to contact animals, thus demonstrating the potential efficacy and safety of the WR strain of vaccinia virus when used as a live vector system in cattle.

Hantavirus is the causative agent of hemorrhagic fever with renal syndrome (HFRS). Heilongjiang Province is experiencing an epidemic of HFRS, the main causative agent is a variant of hantavirus called Seoul virus (SEOV). In this study, the entire genome of one SEOV, the DN2 strain, was sequenced and analyzed. The alignment analysis of the sequences indicated that the DN2 strain shares the highest homology with the SEOV-LYO852 strain. The nucleotide identity is 97.6% for the S segment, 97.7% for the M segment, and 98.0% for the L segment. The corresponding amino acid sequence homologies are 99.1%, 98.9% and 99.8%. The phylogenetic analysis of the segments suggests that the DN2 strain has a high genetic relationship with SEOV strains and no genetic recombination occurs.

We report here genetic recombination between 2USDA-licensed vaccine strains of pseudorabies virus co-inoculated into swine. The vaccine strains, one of which was a conventionally attenuated strain and the other, a genetically engineered deleted strain containing a negative immunologic marker, had complementary genomes. Coinoculation resulted in the creation of novel strains of pseudorabies virus containing negative immunologic markers with restored virulence genes. Plaque-purified recombinant progeny viruses were found in 2 litters of pigs in which both strains were co-inoculated IM, a litter in which both strains were co-inoculated oronasally, and a litter in which the conventionally attenuated strain was inoculated oronasally and the genetically engineered strain was inoculated IM. Recombinant phenotypes and recombinant restriction fragment patterns were observed. The creation, spread, and potential misdiagnosis of these types of recombinant strains could disrupt control and eradication programs that are based on the serologic identification of swine infected with potentially virulent strains of pseudorabies virus.

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 inhibiting 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.

Cerebrospinal fluid obtained from clinically normal free-ranging raccoons was analyzed and compared with CSF obtained from raccoons vaccinated orally with vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine and subsequently challenged peripherally with street rabies virus, and CSF from naive, rabies virus challenge-exposed control raccoons. Significant differences were not found in CSF of free-ranging or V-RG recombinant virus vaccine recipient raccoons, and there was no evidence of CNS invasion by V-RG virus. The CSF of naive, rabies challenge-exposed control raccoons contained high numbers of lymphocytes and monocytes, compatible with rabies virus encephalitis. Although V-RG orally vaccinated challenge-exposed raccoons were protected from lethal rabies virus infection, mild lymphocytic pleocytosis was evident at 90 days after challenge exposure.