Membrane fusion, potential threats, and natural antiviral drugs of pseudorabies virus.

Pseudorabies virus (PrV) can infect several animals and causes severe economic losses in the swine industry. Recently, human encephalitis or endophthalmitis caused by PrV infection has been frequently reported in China. Thus, PrV can infect animals and is becoming a potential threat to human health. Although vaccines and drugs are the main strategies to prevent and treat PrV outbreaks, there is no specific drug, and the emergence of new PrV variants has reduced the effectiveness of classical vaccines. Therefore, it is challenging to eradicate PrV. In the present review, the membrane fusion process of PrV entering target cells, which is conducive to revealing new therapeutic and vaccine strategies for PrV, is presented and discussed. The current and potential PrV pathways of infection in humans are analyzed, and it is hypothesized that PrV may become a zoonotic agent. The efficacy of chemically synthesized drugs for treating PrV infections in animals and humans is unsatisfactory. In contrast, multiple extracts of traditional Chinese medicine (TCM) have shown anti-PRV activity, exerting its effects in different phases of the PrV life-cycle and suggesting that TCM compounds may have great potential against PrV. Overall, this review provides insights into developing effective anti-PrV drugs and emphasizes that human PrV infection should receive more attention.

PRRS virus receptors and an alternative pathway for viral invasion.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a highly restricted cell tropism, which is closely related to the specific receptors associated with PRRSV infection. At least nine cellular molecules have been identified as putative receptors for PRRSV, including CD163, a cysteine-rich scavenger receptor. With the participation of the CD163 receptor and other cofactors, PRRSV invades cells via low pH-dependent clathrin-mediated endocytosis. In addition, PRRSV utilizes viral apoptotic mimicry to infect cells though macropinocytosis as an alternative pathway. In this review, we discuss recent advances in the studies on receptors and pathways that play an important role in PRRSV invasion, and simultaneously explore the use of specific antibodies, small molecules, and blockers targeting receptor-ligand interactions, as a potential strategy for controlling PRRSV infection. Novel antiviral strategies against PRRSV could be developed by identifying the interaction between receptors and ligands.

Construction of a novel porcine circovirus type 2 infectious clone as a basis for the development of a PCV2 iDNA vaccine.

Porcine circovirus-associated disease is a highly contagious disease that has significant economic consequences. The disease is prevalent in many countries and regions. To generate a genetic marker strain of PCV2, a Sal I restriction enzyme site was inserted into the PCV2 clone as a genetic marker by applying iDNA infectious clone technology. The iDNA represents plasmids that encode the full-length DNA genome of PCV2 assembled in a pcDNA3.1-based vectors. The mutant PCV2 was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA). The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). Kunming mice were inoculated with the PCV2 infectious clone or rescued virus via intranasal and intraperitoneal routes. Seroconversion to PCV2-specific antibody appeared in the majority of mice from the two inoculated groups at 7 days postinoculation (DPI), and the specific antibody level was steady for at least 42 days. Viraemia, beginning at 7 DPI and lasting 4 weeks, was detected in the majority of the pigs from the two inoculated groups. The animal experiments revealed that the PCV2 infectious clone and rescued virus both could replicate in mice and induce mice to generate anti-PCV2 antibodies. The infectious clones of PCV2 will be useful for further research investigating a potential tractable iDNA vaccine by reverse genetics technology for attenuated virulance.

Establishment and application of a multiplex PCR for rapid and simultaneous detection of six viruses in swine.

A multiplex PCR assay was developed and evaluated subsequently for its effectiveness in simultaneously detecting mixed viral infections of swine. Specific primers were designed and used for testing the six swine viruses: three DNA viruses, including pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus type 2 (PCV2); three common RNA viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV). This technique has shown to be highly sensitive in that the minimum detection amounts of nucleic acids from PRV, PPV, PCV2, PRRSV, CSFV, and JEV were 6.6, 96, 12.9, 10.5, 51, and 46 pg, respectively. It also was effective for detecting one or multiple viruses in the specimens, such as the lungs, spleens, lymph nodes, and tonsils collected from clinically ill pigs. The multiplex PCR method can detect simultaneously not only infection of the six viruses, but also other swine DNA and RNA viruses. Given its rapidity, specificity, and sensitivity, the multiplex PCR is a useful tool for diagnosing clinically the mixed infections of swine DNA and RNA viruses.

Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine.

The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA- ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNA- IL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4(+) and CD8(+) T lymphocytes, proliferation indices, and interferon-g expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4(+) and CD8(+) T lymphocytes, and significantly higher IFN-γ production than the other inoculated pigs (p < 0.05).