Takahiro Maeki

Genotype IV Japanese encephalitis (JE) virus (GIV JEV) is the least common and most neglected genotype in JEV. We evaluated the growth and pathogenic potential of the GIV strain 19CxBa-83-Cv, which was isolated from a mosquito pool in Bali, Indonesia, in 2019, and serological analyses were also conducted. The growth ability of 19CxBa-83-Cv in Vero cells was intermediate between that of the genotype I (GI) strain Mie/41/2002 and the genotype V (GV) strain Muar, whereas 19CxBa-83-Cv and Mie/41/2002 grew faster than Muar in mouse neuroblastoma cells. The neuroinvasiveness of 19CxBa-83-Cv in mice was higher than that of Mie/41/2002 but lower than that of Muar; however, there were no significant differences in neurovirulence in mice among the three strains. The neutralizing titers of sera from 19CxBa-83-Cv- and Mie/41/2002-inoculated mice against 19CxBa-83-Cv and Mie/41/2002 were similar, whereas the titers against Muar were lower than those of the other two viruses. The neutralizing titers of JE vaccine-inoculated mouse pool serum against 19CxBa-83-Cv and Muar were significantly lower than those against Mie/41/2002. The neutralizing titers against the three viruses were similar in three out of the five serum samples from GI-infected JE patients, although the titers against Mie/41/2002 were higher than those against 19CxBa-83-Cv and Muar in the remaining two sera samples. In summary, we identified the basic characteristics of 19CxBa-83-Cv, but further studies are needed to better understand GIV JEV.

Heartland bandavirus (HRTV) is an emerging tick-borne virus that is distributed in the United States and that causes febrile illness with thrombocytopenia and leukocytopenia. It is genetically close to Dabie bandavirus, which is well known as severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV). The mortality rate of human HRTV infection is approximately 10%; however, neither approved anti-HRTV agents nor vaccines exist. An appropriate animal model should be developed to evaluate the efficacy of antiviral agents and vaccines against HRTV. The susceptibility of IFNAR^−/− mice with HRTV infection was evaluated using subcutaneous, intraperitoneal, and retro-orbital inoculation routes. IFNAR^−/− mice intraperitoneally infected with HRTV showed the most severe clinical signs, and the 50% lethal dose was 3.2 × 10⁶ TCID₅₀. Furthermore, to evaluate the utility of a novel lethal IFNAR^−/− mice model, IFNAR^−/− mice were orally administered favipiravir, ribavirin, or a solvent for 5 days immediately after a lethal dose of HRTV inoculation. The survival rates of the favipiravir-, ribavirin-, and solvent-administered mice were 100, 33, and 0%, respectively. The changes in bodyweights and HRTV RNA loads in the blood of favipiravir-treated IFNAR^−/− mice were the lowest among the three groups, which suggests that favipiravir is a promising drug candidate for the treatment of patients with HRTV infection.

Genotype V (GV) Japanese encephalitis virus (JEV) has emerged in Korea and China since 2009. Recent findings suggest that current Japanese encephalitis (JE) vaccines may reduce the ability to induce neutralizing antibodies against GV JEV compared to other genotypes. This study sought to produce a novel live attenuated JE vaccine with a high efficacy against GV JEV. Genotype I (GI)-GV intertypic recombinant strain rJEV-E^XZ0934-M41 (E^XZ0934), in which the E region of the GI Mie/41/2002 strain was replaced with that of GV strain XZ0934, was introduced with the same 10 attenuation substitutions in the E region found in the live attenuated JE vaccine strain SA 14-14-2 to produce a novel mutant virus rJEV-E^XZ/SA14142m-M41 (E^XZ/SA14142m). In addition, another mutant rJEV-E^M41/SA14142m-M41 (E^M41/SA14142m), which has the same substitutions in the Mie/41/2002, was also produced. The neuroinvasiveness and neurovirulence of the two mutant viruses were significantly reduced in mice. The mutant viruses induced neutralizing antibodies against GV JEV in mice. The growth of E^XZ/SA14142m was lower than that of E^M41/SA14142m. In mouse challenge tests, a single inoculation with a high dose of the mutants blocked lethal GV JEV infections; however, the protective efficacy of E^XZ/SA14142m was weaker than that of E^M41/SA14142m in low-dose inoculations. The lower protection potency of E^XZ/SA14142m may be ascribed to the reduced growth ability caused by the attenuation mutations.

Lymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus. The function of untranslated regions (UTRs) of the LCMV genome has not been well studied except for the extreme 19 nucleotide residues of both the 5′ and 3′ termini. There are internal UTRs composed of 58 and 41 nucleotide residues in the 5′ and 3′ UTRs, respectively, in the LCMV S segment. Their functional roles have yet to be elucidated. In this study, reverse genetics and minigenome systems were established for LCMV strain WE and the function of these regions were analyzed. It was revealed that nucleotides 20–40 and 20–38 located downstream of the 19 nucleotides in the 5′ and 3′ termini, respectively, were involved in viral genome replication and transcription. Furthermore, it was revealed that the other internal UTRs (nucleotides 41–77 and 39–60 in the 5′ and 3′ termini, respectively) in the S segment were involved in virulence in vivo, even though these regions did not affect viral growth capacity in Vero cells. The introduction of LCMV with mutations in these regions attenuates the virus and may enable the production of LCMV vaccine candidates.

We previously showed that the growth ability of the Japanese encephalitis virus (JEV) genotype V (GV) strain Muar is clearly lower than that of the genotype I (GI) JEV strain Mie/41/2002 in murine neuroblastoma cells. Here, we sought to identify the region in GV JEV that is involved in its low growth potential in cultured cells. An intertypic virus containing the NS1-3 region of Muar in the Mie/41/2002 backbone (NS1-3^Muar) exhibited a markedly diminished growth ability in murine neuroblastoma cells. Moreover, the growth rate of a Muar NS2A-bearing intertypic virus (NS2A^Muar) was also similar to that of Muar in these cells, indicating that NS2A of Muar is one of the regions responsible for the Muar-specific growth ability in murine neuroblastoma cells. Sequencing analysis of murine neuroblastoma Neuro-2a cell-adapted NS1-3^Muar virus clones revealed that His-to-Tyr mutation at position 166 of NS2A (NS2A¹⁶⁶) could rescue the low replication ability of NS1-3^Muar in Neuro-2a cells. Notably, a virus harboring a Tyr-to-His substitution at NS2A¹⁶⁶ (NS2A^Y166H) showed a decreased growth ability relative to that of the parental virus Mie/41/2002, whereas an NS2A^Muar-based mutant virus, NS2A^Muar-H166Y, showed a higher growth ability than NS2A^Muar in Neuro-2a cells. Thus, these results indicate that the NS2A¹⁶⁶ amino acid in JEV is critical for the growth and tissue tropism of JEV in vitro.

Lymphocytic choriomeningitis virus (LCMV) is a prototypic arenavirus. The viral genome consists of two RNA segments, L and S. The 5’- and 3’-termini of both L and S segments are highly conserved among arenaviruses. These regions consist of 19 complementary base pairs and are essential for viral genome replication and transcription. In addition to these 19 nucleotides in the 5’- and 3’-termini, there are untranslated regions (UTRs) composed of 58 and 41 nucleotide residues in the 5’ and 3’ UTRs, respectively, in the LCMV S segment. Their functional roles, however, have yet to be elucidated. In this study, a reverse genetics and a minigenome system for the LCMV strain WE were established and used to analyze the function of these regions. The results obtained from these analyses, plus RNA secondary structure prediction, revealed that not only these 19 nucleotides but also the 20th–40th and 20th–38th nucleotides located downstream of the 19 nucleotides in the 5’- and 3’-termini, respectively, are heavily involved in viral genome replication and transcription. Furthermore, the introduction of mutations in these regions depressed viral propagation in vitro and enhanced attenuation in vivo . Conversely, recombinant LCMVs (rLCMVs), which had various deletions in the other UTRs, propagated as well as wild-type LCMV in vitro but were attenuated in vivo . Most mice previously infected with rLCMVs with mutated UTRs, when further infected with a lethal dose of wild-type LCMV, survived. These results suggest that rLCMVs with mutated UTRs could be candidates for an LCMV vaccine. IMPORTANCE: The function of untranslated regions (UTRs) of the arenavirus genome has not well been studied except for the 19 nucleotides of the 5’- and 3’-termini. In this study the function of the UTRs of the LCMV S segment was analyzed. It was found that not only the 19 nucleotides of the 5’- and 3’-termini but also the 20th–40th and 20th–38th nucleotides located downstream of the 19 nucleotides in the 5’- and 3’-termini, respectively, were involved in viral genome replication and transcription. Furthermore, other UTRs in the S segment were involved in virulence in vivo . The introduction of mutations to these regions makes it possible to establish attenuated LCMV and potentially develop LCMV vaccine candidates.

Japanese encephalitis (JE) is an acute viral disease caused by the Japanese encephalitis virus (JEV). JEV strains are classified into 5 genotypes (I–V). JEV genotype V strains have never been detected in Japan to date, but they were recently detected in South Korea. In the present analysis, we tried to determine if a JEV genotype V strain caused any JE case in Japan in 2016. Serum and cerebrospinal fluid samples were collected from 10 JE patients reported in Japan in 2016. JEV RNA was not detected in any of the samples. Although JEV is a single-serotype virus, it can be expected that the neutralizing antibody titers against JEV genotype V strains are higher than those against genotype I and III strains in the serum of patients with JE in Japan whose causative JEV was the genotype V strain. The neutralizing antibody titers against the JEV genotype V strain were not higher than those against the genotype I or III strain in any serum samples. Therefore, the evidence that the JEV genotype V strain caused any JE case in Japan in 2016 was absent.