Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice.

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

Epitope Mapping of A Viral Propagation-Inhibiting Single-Chain Variable Fragment Against Rabies Lyssavirus Phosphoprotein.

Rabies is a highly neurotropic disease caused by rabies lyssavirus (RABV). Human rabies vaccines exist for pre- and postexposure prophylaxis; however, after clinical symptoms appear, the disease has an ∼100% mortality rate with no effective treatments available. In our previous study, mouse neuroblastoma cells transfected with a plasmid coding one clone of a single-chain variable fragment (scFv), scFv-P19, against RABV phosphoprotein (RABV-P) derived from an scFv phage-display library, before infection, exhibited reduced viral propagation after infection with the RABV-fixed strain, CVS11. In this study, we conducted epitope mapping of scFv-P19 through indirect fluorescent assay and Western blotting analysis against full-length and N- or C-terminal truncated RABV-P. Our results suggest that scFv-P19 targets a portion containing amino acids 47-52 at the N-terminus, which partially overlaps with the N-terminal nuclear export sequences. This provides insights into the underlying mechanism associated with inhibition of RABV by scFv-P19, while allowing for the design of additional scFv-based therapeutic studies for RABV by integrating appropriate delivery and application systems. Furthermore, the results of this study suggest that scFv-P19 may serve as an effective tool for investigating nuclear trafficking of RABV-P to explore the roles of RABV-P isoforms in rabies pathogenesis.

Severe Fever with Thrombocytopenia Syndrome Phlebovirus causes lethal viral hemorrhagic fever in cats.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by the SFTS phlebovirus (SFTSV). SFTS patients were first reported in China, followed by Japan and South Korea. In 2017, cats were diagnosed with SFTS for the first time, suggesting that these animals are susceptible to SFTSV. To confirm whether or not cats were indeed susceptible to SFTSV, animal subjects were experimentally infected with SFTSV. Four of the six cats infected with the SPL010 strain of SFTSV died, all showing similar or more severe symptoms than human SFTS patients, such as a fever, leukocytopenia, thrombocytopenia, weight loss, anorexia, jaundice and depression. High levels of SFTSV RNA loads were detected in the serum, eye swab, saliva, rectal swab and urine, indicating a risk of direct human infection from SFTS-infected animals. Histopathologically, acute necrotizing lymphadenitis and hemophagocytosis were prominent in the lymph nodes and spleen. Severe hemorrhaging was observed throughout the gastrointestinal tract. B cell lineage cells with MUM-1 and CD20, but not Pax-5 in the lesions were predominantly infected with SFTSV. The present study demonstrated that cats were highly susceptible to SFTSV. The risk of direct infection from SFTS-infected cats to humans should therefore be considered.

Association between RABV G Proteins Transported from the Perinuclear Space to the Cell Surface Membrane and N-Glycosylation of the Sequon Asn(204).

In this study, G proteins of the rabies virus (RABV) Kyoto strain were detected in the cytoplasm but not distributed at the cell membrane of mouse neuroblastoma (MNA) cells. G proteins of CVS-26 were detected in both the cell membrane and perinuclear space of MNA cells. We found that N-glycosylation of street RABV G protein by the insertion of the sequon Asn(204) induced the transfer of RABV G proteins to the cell surface membrane. Fixed RABV budding from the plasma membrane has been found to depend not only on G protein but also on other structural proteins such as M protein. However, the differing N-glycosylation of G protein could be associated with the distinct budding and antigenic features of RABV in street and fixed viruses. Our study of the association of N-glycan of G protein at Asn(204) with the transport of RABV G protein to the cell surface membrane contributes to the understanding of the evolution of fixed virus from street virus, which in turn would help for determine the mechanism underlying RABV budding and enhanced host immune responses.

Antigen capture ELISA system for henipaviruses using polyclonal antibodies obtained by DNA immunization.

A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.

Antimicrobial susceptibilities of Listeria monocytogenes isolated in Japan.

The antimicrobial susceptibility of 201 Listeria monocytogenes isolates from foods, environments, animals and human patients in Japan was determined. All isolates were susceptible to ampicillin, the first choice of drug for listeriosis treatment, chloramphenicol, dihydrostreptomycin, erythromycin, enrofloxacin, gentamicin, kanamycin, lincomycin, nosiheptide, salinomycin, vancomycin, and virginiamycin. A human strain was resistant to oxytetracycline. The Minimum Inhibitory Concentration (MIC) for 50% of the strains and the MIC for 90% of the strains were comparable in all the isolates. This is the first investigation to compare antibiotic resistances between isolates from foods and isolates from human patients in Japan. The result showed that most of the isolates were susceptible to antibiotics used in this study.

Effect of cellular cholesterol depletion on rabies virus infection.

Although there are several reports on candidates for rabies virus (RABV) receptor, possible roles played by these receptor candidates in determination of highly neurotropic nature of RABV have not been well understood. Since these candidate receptors for RABV were reported to be frequently associated with cholesterol-rich microdomains characterized by lipid rafts and caveolae structures, we attempted to determine whether the disturbance of microdomains caused by the cholesterol depletion showed any effects on RABV infection. When the cellular cholesterol was depleted by methyl-beta-cyclodextrin (MBCD) treatment, increase in RABV adsorption and infection, but not multiplication rather than suppression was observed in both BHK-21 and HEp-2 cells. These effects exerted by MBCD treatment on RABV infection could be reversed by cholesterol reconstitution. These results suggest that RABV enters BHK-21 or HEp-2 cells through ports of entry other than those located on cholesterol-rich microdomains and raise the possibility that RABV uses different mechanisms to enter the non-neuronal cells.