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Thirty-seven specimens of wild boar sera were collected from August 2016 to March 2018 in Ishikawa prefecture, Japan. Thirty-two specimens (86.5%) were positive for neutralizing antibodies against Japanese encephalitis virus (JEV). Eight specimens (21.6%) were positive for IgM antibodies against JEV. One sample was obtained from a wild boar captured in February during the winter season. Four other serum specimens obtained during the winter season were positive using a JEV gene-specific PCR assay. Based on IgM and PCR assays, wild boars were infected with JEV during the winter season, suggesting that the prevalence of JEV antibodies in wild boars in Ishikawa is high and JEV activity is possible during winter in this region. In addition, wild boars may play an important role in the infection cycle of JEV.

In July 2018, acute Q fever (AQF) was diagnosed in two Japanese individuals from the same family. They returned to Japan from Malawi, where the epidemiology of AQF is unknown. A child presented to the hospital with high-grade fever without any symptoms, and a mother presented with fever and dry cough. Paired serum antiphase Ⅱ IgM and IgG significantly elevated in the convalescent phase in both cases. Coxiella burnetii gene (IS1111) was detected from the mother’s blood sample. They had no reported direct animal contact, but the onset of symptoms coincided with the dry season in Malawi, which may have facilitated environmental dispersal. These cases may serve as an alert for high-risk people to possible AQF spread and underdiagnosis in Malawi.

We produced a Vero cell-derived inactivated Japanese encephalitis vaccine using a serum-free medium, as a substitute for the conventional mouse brain-derived Japanese encephalitis vaccine. The immunogenicity of this cell-derived vaccine was higher than that of the conventional mouse brain-derived vaccine. The results of a clinical study in humans also demonstrated higher immunogenicity of this cell-derived vaccine. No gene mutation was found in the viral structural proteins derived from Vero cells and mouse brain. So, we conducted a lectin blot analysis, assuming differing glycosylation as a cause of the higher immunogenicity in humans. The results demonstrated that vaccine reactivity varied with lectins, particularly with WGA, DBA, MAM, SSA, SBA, and GS-II. Thus, glycosylation differed with the vaccines, suggesting a possible cause of the differing immunogenicity between mice and humans.

We established a method of producing a Vero cell-derived Japanese encephalitis vaccine using serum-free medium, and tested its stability using various stabilizers during the inactivation process and storage at 4 degrees C and 28 degrees C. Similar to previously reported results of cell culture in serum-containing medium, Vero cells were cultured in a serum-free medium multiplied well, and the viral yield was successfully increased to about 10(9)PFU/ml. Following formalin-inactivation and purification via ethanol precipitation and sucrose density ultracentrifugation of the virus solution, the vaccine had the same quality as, and higher immunogenicity, the mouse brain-derived vaccine in current use. Testing of several stabilizers showed that the addition of 0.5% glycine during the virus inactivation process facilitated the maintenance of immunogenicity for a long period of time. Furthermore, the addition of 0.5% glycine and 1.0% sorbitol as vaccine stabilizers after purification led to the maintenance of immunogenicity for 1 year, not dependent on the storage temperature (4 degrees C or 28 degrees C). These results indicate that, in contrast to the current mouse brain-derived vaccine, the Vero cell-derived vaccine can be prepared using serum-free medium containing no animal-derived components, and that the vaccine can be stored at room temperature by adding stabilizers, suggesting the possibility of producing room temperature-stable vaccines.

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

The source of Q fever infection, was investigated by serological and polymerase chain reaction (PCR) analysis of specimens from humans and pets in an animal hospital. Two animal health technicians showed a positive serological reaction against Coxiella burnetii at the start. One of the two positive subjects remained PCR-positive for about 1 year and the other converted to PCR-negative, but the IgG antibody titer remained at 1 : 128 after minocycline treatment. Among animals housed in the hospital, two dogs were PCR-positive at the start, and the infection was transmitted to three cats about 5 months later. All these animals became negative for C. burnetii DNA after minocycline treatment. Furthermore, C. burnetii was isolated from the sera of the two humans and two dogs. C. burnetii isolates from the humans and dogs were analyzed, and it was found that the sequence homology of the com1 region was high, 99.9%, and the QpH1 plasmid was detected. These results suggest that these isolates were the same type, and it was considered that the infection was derived from the dogs, though the time of infection could not be confirmed.

Advax is a polysaccharide-based adjuvant that potently stimulates vaccine immunogenicity without the increased reactogenicity seen with other adjuvants. This study investigated the immunogenicity of a novel Advax-adjuvanted Vero cell culture candidate vaccine against Japanese encephalitis virus (JEV) in mice and horses. The results showed that, in mice, a two-immunization, low-dose (50 ng JEV antigen) regimen with adjuvanted vaccine produced solid neutralizing immunity comparable to that elicited with live ChimeriVax-JE immunization and superior to that elicited with tenfold higher doses of a traditional non-adjuvanted JEV vaccine (JE-VAX; Biken Institute) or a newly approved alum-adjuvanted vaccine (Jespect; Novartis). Mice vaccinated with the Advax-adjuvanted, but not the unadjuvanted vaccine, were protected against live JEV challenge. Equine immunizations against JEV with Advax-formulated vaccine similarly showed enhanced vaccine immunogenicity, confirming that the adjuvant effects of Advax are not restricted to rodent models. Advax-adjuvanted JEV vaccine elicited a balanced T-helper 1 (Th1)/Th2 immune response against JEV with protective levels of cross-neutralizing antibody against other viruses belonging to the JEV serocomplex, including Murray Valley encephalitis virus (MVEV). The adjuvanted JEV vaccine was well tolerated with minimal reactogenicity and no systemic toxicity in immunized animals. The cessation of manufacture of traditional mouse brain-derived unadjuvanted JEV vaccine in Japan has resulted in a JEV vaccine shortage internationally. There is also an ongoing lack of human vaccines against other JEV serocomplex flaviviruses, such as MVEV, making this adjuvanted, cell culture-grown JEV vaccine a promising candidate to address both needs with one vaccine.

A novel oscillating bioreactor, BelloCell, was successfully applied for the cultivation of Vero cells using serum-free medium, and the production of Japanese encephalitis virus. The BelloCell requires no air sparging, pumping, or agitation, and thus provides a low shear environment. Owing to its simple design, BelloCell is extremely easy to handle and operate. Using this BelloCell (500 ml culture), Vero cells reached a maximum number of 2.8 x 10(9) cells and the Japanese encephalitis virus yield reached 6.91 x 10(11) PFU, versus 9.0 x 10(8) cells and 2.98 x 10(11) PFU using a spinner flask (500 ml) with microcarriers. The cell yield and virus production using BelloCell were markedly higher than with microcarrier culture. The neutralizing capacity of the Japanese encephalitis virus produced using BelloCell was equal to that using a microcarrier system. Therefore, these benefits should enable BelloCell to be adopted as a simple system for high population density cell culture and virus production.

We established a rapid, quantitative real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the envelope gene of Japanese encephalitis virus. The RT-LAMP enabled us to detect the target product within 1 hr by only reacting reverse transcriptase and Bst DNA polymerase in a single tube at an isothermal temperature. The detection sensitivity of the RT-LAMP for Japanese encephalitis virus was 1 PFU, similar to that of conventional reverse transcription-polymerase chain reaction (RT-PCR). Flaviviruses of the Japanese encephalitis virus group, such as Dengue virus and West Nile virus, could not be detected. This confirmed the specificity of the RT-LAMP assay for Japanese encephalitis virus. A standard curve was constructed by plotting viral titer against the time for virus detection by the RT-LAMP, validating the quantitative accuracy of the assay. In addition, the amount of virus estimated by RT-LAMP was strongly correlated (r = 0.902) with that determined by plaque assay, a conventional method for virus quantification. These results indicate that the RT-LAMP assay established in this study is specific for Japanese encephalitis virus, and allows more rapid detection and quantification of the virus.