Tk-deleted pseudorabies virus retains high pathogenicity in rats

Introduction The pseudorabies virus (PRV) gene encoding thymidine kinase (tk) is an important virulence-associated factor. Attenuation of PRV in susceptible animals is a frequent result of tk deletion. The aim of the study was to assess the pathogenicity of tk-deleted PRV in rats. Material and Methods Sprague Dawley rats were infected with the tk-deleted PRV strain SuHV-1 ΔTK:247via intranasal or intramuscular inoculation. PRV loads in ten tissues from dead and euthanised rats were determined using real-time PCR. Results Infection with SuHV-1 ΔTK:247 could cause death in rats. The 50% lethal dose (LD50) of SuHV-1 ΔTK:247 via intranasal inoculation was 103.16 TCID50 in rats. Intramuscular inoculation required a higher dose of SuHV-1 ΔTK:247 (105.0 TCID50). A high SuHV-1 ΔTK:247 titre was observed in the trigeminal ganglia or spinal cord of dead rats. Conclusion The results of this study show that rats are highly susceptible to PRV infection, and tk deletion did not completely diminish the pathogenicity of PRV in rats.

Immunogenicity and efficacy of Heat Inactivated Whole-Cell Vaccine to increase murine survival after Extensively drug-resistant Acinetobacter baumannii infection.

Due to the rapid emergence of extensively drug-resistant (XDR) strains worldwide there is a necessity for greater consideration for the role of preventive vaccines to combat these pathogens. The study reported the effectiveness of a heat-inactivated whole-cell vaccine (HI-WCV) against A. baumannii to produce protective immunity with evaluation of the bactericidal antibody responses in mice after intramuscular inoculation with different XDR strains of A. baumannii Six XDR A. baumannii strains emulsified with complete freund's adjuvant (CFA) were used for intramuscular inoculation in the experimental group of mice (n=4) in three different phases on 14 days interval whereas placebo-controlled mice (n=4) received phosphate-buffered saline emulsified with CFA in same the schedule. Serum was collected from tail blood of each mouse on 10th day after each inoculation and by cardiac puncture on 14th day after lethal dose from the experimental group and after death of the placebo-controlled group. Mice inoculated with heat-inactivated whole-cell vaccine survived and showed a higher neutralizing (IgG) antibody response after 2nd (>7-fold titre) and 3rd inoculation (>12-fold titre) whereas all mice from placebo-controlled group did not survived. Sera from experimental group of mice collected (38th day) after 3rd inoculation resulted in higher serum bactericidal killing index (42.12) after three hours incubation with an XDR A. baumannii over sera collected after 1st and 2nd inoculation (50% cutoff value=14.5). These results suggest that intramuscular vaccination with heat-inactivated whole-cell vaccine stimulated IgG antibody responses that can increase murine survival after XDR A. baumannii infection.

The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host and tissue tropism and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule-A (JAM-A) and Nogo 66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease following inoculation of wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter replication of neurotropic reovirus strain T3SA- in the intestine and transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of both NgR1 and JAM-A also did not alter replication, neural tropism, and virulence of T3SA- following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. These results eliminate functions for JAM-A and NgR1 in shaping CNS tropism in mice and suggest that other receptors, yet to be identified, support this function.

Deletion of the L7L-L11L Genes Attenuates ASFV and Induces Protection against Homologous Challenge

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a major epidemic disease endangering the swine industry. Although a number of vaccine candidates have been reported, none are commercially available yet. To explore the effect of unknown genes on the biological characteristics of ASFV and the possibility of a gene-deleted isolate as a vaccine candidate, the strain SY18ΔL7-11, with deletions of L7L–L11L genes from ASFV SY18, was constructed, and its biological properties were analyzed. The results show that deletion of genes L7L-L11L did not affect replication of the virus in vitro. Virulence of SY18△L7-11 was significantly reduced, as 11 of the 12 pigs survived for 28 days after intramuscular inoculation with a low dose (103 TCID50) or a high dose (106 TCID50) of SY18ΔL7-11. All 11 surviving pigs were completely protected against challenge with the parental ASFV SY18 on 28 days postinoculation (dpi). Transient fever and/or irregularly low levels of genomic DNA in the blood were monitored in some pigs after inoculation. No ASF clinical signs or viremia were monitored after challenge. Antibodies to ASFV were induced in all pigs from 14 to 21 days postinoculation. IFN-γ was detected in most of the inoculated pigs, which is usually inhibited in ASFV-infected pigs. Overall, the results demonstrate that SY18ΔL7-11 is a candidate for further constructing safer vaccine(s), with better joint deletions of other gene(s) related to virulence.

Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis

ABSTRACTRabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level ofIfn-βgene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-β promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves.IMPORTANCEDespite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.