Obtaining recombinant capsid protein VP60 of rabbit haemorrhagic disease virus and its antigenic and immunogenic activity study

Viral hemorrhagic disease of rabbits is an acute, highly contagious disease characterized by the phenomena of hemorrhagic diathesis in all organs, especially in the lungs and liver. The causative agent of viral haemorrhagic disease of rabbits is a virus of haemorrhagic disease of rabbits, belonging to the family Caliciviridae, genus Lagovirus. Currently, there are 4 genogroups of lagoviruses, two pathogenic: GI1 (GI1a-GI1d) and GI2, and two non-pathogenic: GI3 and GI4. The greatest danger to rabbits in the Russian Federation is posed by viruses of the GI1 genotype. The virulence of these viruses for rabbits is extremely high, the incubation period is 48-72 hours. Clinically, the disease is almost not manifested. Mortality can reach 100%. For the prevention of HBV in the Russian Federation, inactivated tissue vaccines are used, which are a suspension of the liver of rabbits infected with virulent strains of the rabbit hemorrhagic disease virus. Currently, in veterinary practice, subunit recombinant vaccines based on virus proteins obtained in the baculovirus gene expression system are increasingly used. The authors obtained the recombinant VP60 virus of rabbit hemorrhagic disease of the GI1 genotype in the baculovirus gene expression system and studied its antigenic and immunogenic activity for rabbits. It was found that the recombinant capsid protein VP60 of the hemorrhagic disease virus, administered to rabbits at a dose of 50 pg, causes the synthesis of specific antibodies in animals, detected by enzyme immunoassay and in the hemagglutination inhibition reaction, and protects 80% of animals during control infection with the virulent strain «Voronezh-87» at a dose of 1000 LD 50. These data indicate the possibility of using this protein as a specific component of a subunit vaccine against rabbit hemorrhagic disease caused by strains of the GI1 gene group.

118 A SINGLE INTRAUTERINE INFUSION OF SUSTAINED RELEASE RECOMBINANT BOVINE INTERFERON τ ON DAY 13 EXTENDS CORPUS LUTEUM LIFESPAN IN CYCLIC COWS

Bovine interferon (bIFN) τ has been implicated as a mediator of maternal recognition of pregnancy in cattle. (Geshi et al. 2001 Theriogenology 55, 325) reported that daily intrauterine infusion of recombinant (r) bIFNτ from Day 13 to 24 extended interestrous intervals in heifers. The objective of this study was to determine whether a single infusion of sustained release bIFNτ into the uterine horn would extend corpus luteum lifespan in cyclic cows. RbIFNτ was prepared from the Silkwarm-Baculovirus gene expression system (1 mg mL–1, 1 × 108 IU mg–1, Nagaya et al. 2004 J. Vet. Med. Sci. 66, 1395–1401). Two sustained release carriers, liposome and aluminum hydroxide (Al) gel were tested. Liposome encapsulated (lipo) BSA (control) and rbIFNτ were prepared from a lipid mixture solution contained 2 mg of each protein by the Bangham method including lyophilization and rehydration. The same amount of BSA or rbIFNτ was adsorbed to Al-gel containing 2.5 mg Al. Adsorption was allowed to proceed for 5 min at room temperature and unbound proteins were removed by centrifugation. Subsequently, lipo- or Al-gel adsorbed BSA and rbIFNτ were adjusted to 0.5 mL with saline and loaded into 0.5-mL AI straws. BIFNτ free from liposome or unbound to Al-gel was measured by RIA. Eight Japanese Black cows were used in this study. Their signs of oestrus were monitored twice a day. Cows were randomly assigned to four groups, receiving a single infusion of 1) lipo-BSA (n = 3); 2) lipo-rbIFN τ (n = 3); 3) BSA with Al-gel (n = 4); or 4) rbIFNτ with Al-gel (n = 4) on Day 13 (oestrus = Day 0). The BSA or rbIFNτ solution was introduced into the uterine horn ipsilateral to the corpus luteum by the cervical route. Blood samples were collected and rectal temperatures were recorded immediately preceding the infusion (0 h), thereafter every 3 h until 12 h, and 24 h after the infusion. Total white blood cells (WBC) were counted. Data were analysed by ANOVA using the STATVIEW program. Unbound bIFNτ in liposomal encapsulation and adsorption to Al-gel were estimated to be ∼90% and 2%, respectively. Corpus luteum lifespan was normal in controls (lipo-BSA; 21.6 ± 0.33 and BSA in Al-gel; 21.2 ± 0.25 days) but was extended in cows receiving rbIFNτ with Al-gel (27.0 ± 0.40 days; P < 0.01), whereas lipo-rbIFNτ was less effective. Rectal temperatures increased following rbIFNτ treatment with a peak at 6 h after infusion (P < 0.05). Correspondingly, total WBC was decreased following rbIFNτ treatment with a minimum at 9 h. Those changes were larger in liposomal encapsulation than in adsorbed to Al-gel. In conclusion, a single infusion of bIFNτ adsorbed to Al-gel can extend corpus luteum lifespan in cyclic cows.

Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation

ABSTRACT In this study, we characterized Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf76 (ac76), which is a highly conserved gene of unknown function in lepidopteran baculoviruses. Transcriptional analysis of ac76 revealed that transcription of multiple overlapping multicistronic transcripts initiates from a canonical TAAG late-transcription start motif but terminates at different 3′ ends at 24 h postinfection in AcMNPV-infected Sf9 cells. To investigate the role of ac76 in the baculovirus life cycle, an ac76-knockout virus was constructed using an AcMNPV bacmid system. Microscopy, titration assays, and Western blot analysis demonstrated that the resulting ac76-knockout virus was unable to produce budded viruses. Quantitative real-time PCR analysis demonstrated that ac76 deletion did not affect viral DNA synthesis. Electron microscopy showed that virus-induced intranuclear microvesicles as well as occlusion-derived virions were never observed in cells transfected with the ac76-knockout virus. Confocal microscopy analysis revealed that Ac76 was predominantly localized to the ring zone of nuclei during the late phase of infection. This suggests that ac76 plays a role in intranuclear microvesicle formation. To the best of our knowledge, this is the first baculovirus gene identified to be involved in intranuclear microvesicle formation.

Autographa californica Multiple Nucleopolyhedrovirus me53 (ac140) Is a Nonessential Gene Required for Efficient Budded-Virus Production

ABSTRACT me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (AcΔme53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with AcΔme53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.