Hiromichi Mitake

Involvement of the Rabies Virus Phosphoprotein Gene in Neuroinvasiveness

ABSTRACT Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-β) gene (Ifn-β) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis

ABSTRACT Rabies 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 of Ifn-β gene 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. IMPORTANCE Despite 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.

Genome Sequences of Rotavirus A Strains Ty-1 and Ty-3, Isolated from Turkeys in Ireland in 1979

ABSTRACT To obtain complete genome sequences of turkey rotavirus A strains Ty-1 and Ty-3, we sequenced the gene segments that had not been decoded previously. The genotype constellations of the respective strains were determined to be G17-P[38]-I4-R4-C4-M4-A16-N4-T4-E4-H4 and G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H14. Notably, their VP4 and NSP5 genes were classified into novel genotypes.

Defect of rabies virus phosphoprotein in its interferon-antagonist activity negatively affects viral replication in muscle cells

Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-β promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-β-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.

Isolation and characterization of a novel type of rotavirus species A in sugar gliders (Petaurus breviceps).

To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.

Isolation of a sp. nov. Ljungan virus from wild birds in Japan.

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

Detection of avian-like rotavirus A VP4 from a calf in Japan

A total of 568 normal feces from calves on a beef farm in Fukui Prefecture, Japan, in 2011–2012 were examined by RT-semi-nested PCR for rotavirus A (RVA) VP4 genes. Through partial sequencing and BLAST analyses of 84 VP4-positive specimens, we identified an avian-like RVA strain, N2342, which shares highest nucleotide identity (80.0%) with known avian-like bovine strain 993/83, in one specimen. Phylogenetic analysis also revealed a close genetic relationship between N2342 and avian RVAs, suggesting bird-to-cattle transmission. We observed frequent contact of wild birds with calves in the farm, suggesting that these birds were the source of the virus.

Persistence of the rotavirus A genome in mesenteric lymph nodes of cattle raised on farms.

Previous studies revealed that rotavirus A (RVA) is present in not only the small intestine but also various organs. It was reported that RVA persisted in mesenteric lymph nodes (MLNs) in experimental models. However, there have been no reports focused on RVA in MLNs of animals under natural conditions. In this study, in order to investigate the persistence of the RVA genome in MLNs in cattle under natural conditions, reverse transcription-semi-nested PCR was carried out to detect RVA genomes in the MLNs from 17 calves that had been subjected to autopsy examinations. RVA genomes were detected in MLNs from 10 (˜60  %) of the 17 autopsied calves. MLNs from 170 healthy adult cattle that had been slaughtered were also examined; 15 (∼10  %) of the 170 cattle had RVA genomes in their MLNs, indicating that RNA genomes are found frequently in MLNs of cattle under natural conditions. Genetic analyses revealed that RVAs in MLNs were classified as G and/or P genotypes generally prevalent in bovines. Basically, the strains in intestinal contents were genetically identical to those in MLNs from individual cattle, suggesting that bovine RVAs have the ability to spread from the intestine to MLNs. Furthermore, amongst RVA-positive cattle, six of 10 autopsied calves and 12 of 15 healthy adult cattle were negative for the virus in the intestinal contents, indicating that bovine RVA genomes can persist in MLNs after viral clearance in the digestive tract.

Generation of a novel live rabies vaccine strain with a high level of safety by introducing attenuating mutations in the nucleoprotein and glycoprotein

The current live rabies vaccine SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333). This fact generates a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain highly and stably attenuated by multiple mutations. However, the information on attenuating mutations other than that at G333 is very limited. We previously reported that amino acids at positions 273 and 394 in the nucleoprotein (N273/394) (Leu and His, respectively) of fixed rabies virus Ni-CE are responsible for the attenuated phenotype by enhancing interferon (IFN)/chemokine gene expressions in infected neural cells. In this study, we found that amino acid substitutions at N273/394 (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 attenuated by the combination of the above attenuating mutations at G333 and N273/394, and checked its safety. Similar to the ERA-G333, which is attenuated by only the mutation at G333, ERA-N273/394-G333 did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of ERA-N273/394-G333. Further examination revealed that infection with ERA-N273/394-G333 induces IFN-β and CXCL10 mRNA expressions more strongly than ERA-G333 infection in a neuroblastoma cell line. Importantly, we found that the ERA-N273/394-G333 stain has a lower risk for emergence of a pathogenic revertant than does the ERA-G333. These results indicate that ERA-N273/394-G333 has a potential to be a promising candidate for a live rabies vaccine strain with a high level of safety.