Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Secretion of Truncated Recombinant Rabies Virus Glycoprotein with Preserved Antigenic Properties Using a Co-Expression System in Hansenula polymorpha: Weidong Qian , Frank Aguilar , Ting Wang , Bingsheng Qiu; J. Microbiol. 2013;51(2):234-240. Published online April 27, 2013; DOI: https://doi.org/10.1007/s12275-013-2337-0

Abstract: Rabies virus infection remains a serious public health threat in the developing world, where cost-concerns make widescale public health interventions impractical. The development of novel and inexpensive ELISA diagnostic antigens is critical in early detection and prevention of complications. The transmembrane glycoprotein (G) of rabies virus (RV) contains an external domain capable of inducing the synthesis of anti-rabies, virus-neutralizing antibodies, in infected or immunized hosts. In our study, the external G domain was synthesized and fused in-frame with a polyhistidine-tag coding sequence present in the expression plasmid. Soluble truncated recombinant G was secreted in Hansenula polymorpha (H. polymorpha) using H. polymorpha-derived calnexin (HpCNE1) overproduction and found to be correctly N-glycosylated. The truncated recombinant G was purified from cell culture supernatant by Ni-agarose affinity chromatography and when compared with the full-length glycoprotein, found to be similarly immunogenic in vaccinated rabbits. These results subsequently led us to explore the potential of truncated recombinant G as a diagnostic antigen in ELISA. Our results show that the truncated recombinant G can detect antibodies directed to both whole virion and native glycoprotein. More sophisticated applications of truncated recombinant G would profit from the correctly N-glycosylated and soluble monomer.

Molecular Survey of Latent Pseudorabies Virus Infection in Nervous Tissues of Slaughtered Pigs by Nested and Real-time PCR: Hyun A Yoon , Seong Kug Eo , Abi George Aleyas , Seong Ok Park , John Hwa Lee , Joon Seok Chae , Jeong Gon Cho , Hee Jong Song; J. Microbiol. 2005;43(5):430-436.; DOI: https://doi.org/2279 [pii]

Abstract: In this study, the prevalence and quantity of a latent pseudorabies virus (PrV) infection in the nervous tissues of randomly selected pigs was determined via nested and real-time PCR. The nervous tissues, including the trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS), were collected from the heads of 40 randomly selected pigs. The majority of the nervous tissues from the selected pigs evidenced a positively amplified band on nested PCR. In particular, nested PCR targeted to the PrV glycoprotein B (gB) gene yielded positive results in all of the BS samples. Nested PCR for either the gE or gG gene produced positive bands in a less number of nervous tissues (57.5% and 42.5%, respectively). Real-time PCR revealed that the examined tissues harbored large copy numbers of latent PrV DNA, ranging between 100.1 and 107.2 (1-1.58x107) copies per 1 g of genomic DNA. Real-time PCR targeted to the PrV gE gene exhibited an accumulated fluorescence of reporter dye at levels above threshold, thereby indicating a higher prevalence than was observed on the nested PCR (100% for BS, 92% for OB, and 85% for TG). These results indicate that a large number of farm-grown pigs are latently infected with a field PrV strain with a variety of copy numbers. This result is similar to what was found in association with the human herpes virus.

A Restrictive Virus Tropism, Latency and Reactivation of Pseudorabies Virus Following Irreversible Deletion of BsrI Restriction Site in the Thymidine-kinase Gene: Mohd Lila Mohd Azmi , Nazariah Allaudin Zeenathul , Abdel-Wahid Saeed Ali , Che Abdul Rahim Mohamed , Awang Isa Kamarudin; J. Microbiol. 2002;40(1):1-10.

Abstract: At the dose of 1000 p.f.u. per mouse, 100% mortality occurred in mice inoculated with wild-type pseudorabies virus (PrV). In contrast, upon stable deletion of 10 bp nucleotides at the BsrI site within the TK gene, PrV was rendered to be completely apathogenic. The deletion also caused the virus to be less capable of replicating in respiratory as well as in nervous system tissues. Although animals were exposed to high titers of TK-deleted PrVs, the virus failed to replicate to a high titer as compared to the pathogenic parental virus. In contrast to previous studies, the deletion in the TK gene did not prevent the virus from establishing latency. Upon immunosuppression, the latent virus, however, reactivated but replicated at low titers. Interestingly, TK-deleted virus established latency and reactivation, that are occurred only in trigeminal ganglia and the cerebrum, and no other tissues involved. Following reactivation, there was no indication of virus shedding in respiratory tissues as confirmed by virus isolation and polymerase chain reaction (PCR) technique targeting at the gB gene of PrV. The non-pathogenic virus with non-shedding characteristics, upon reactivation of the latent virus, would be the important feature of a live virus vaccine candidate.

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