Characterization of Japanese Encephalitis Virus Isolated from Persistently Infected Mouse Embryo Cells.

Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus Flavivirus of the family Flaviviridae. Persistent JEV infection was previously shown in pig blood cells, which act as a natural reservoir of this virus. We aimed to determine the pathogenicity factors involved in persistent JEV infection by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model. We established persistent JEV infections in cells by inoculating mouse fetus primary cell cultures with the Beijing-1 strain of JEV and then performing repeated infected cell passages, harvesting viruses after each passage while monitoring the plaque size over 100 generations. The virus growth rate was compared among Vero, C6/36, and Neuro-2a cells. The pathogenicity was examined in female ICR mice at several ages. Additionally, we determined the whole-genome sequences. The 134th Beijing-1-derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain but did not grow well in C6/36 or Neuro-2a cells. No differences were observed in pathogenicity after intracerebral inoculation in mice of different ages, but the survival time was extended in older mice. Mutations in the persistent virus genomes were found across all regions but were mainly focused in the NS3, NS4b, and 3'NCR regions, with a 34-base-pair deletion found in the variable region. The short deletion in the 3'NCR region appeared to be responsible for the reduced pathogenicity and growth efficiency.

An Advax-Adjuvanted Inactivated Cell-Culture Derived Japanese Encephalitis Vaccine Induces Broadly Neutralising Anti-Flavivirus Antibodies, Robust Cellular Immunity and Provides Single Dose Protection.

ccJE+Advax is an inactivated cell culture Japanese encephalitis (JE) vaccine formulated with Advax, a novel polysaccharide adjuvant based on delta inulin. This vaccine has previously shown promise in murine and equine studies and the current study sought to better understand its mechanism of action and assess the feasibility of single dose vaccine protection. Mice immunised with ccJE+Advax had higher serum neutralisation titres than those immunised with ccJE alone or with alum adjuvant. ccJE+Advax induced extraordinarily broad cross-neutralising antibodies against multiple flaviviruses including West Nile virus (WNV), Murray Valley encephalitis virus (MVEV), St Louis encephalitis virus (SLEV) and Dengue virus-1 and -2 (DENV-1 and -2). Notably, the DENV-2 cross-neutralising antibodies from ccJE+Advax immunised mice uniquely had no DENV-2 antibody-dependent infection enhancement (ADIE) activity, in contrast to high ADIE activity seen with DENV-1 cross-reactive antibodies induced by mbJE or ccJE alone or with alum adjuvant. JEV-stimulated splenocytes from ccJE+Advax immunised mice showed increased IL-17 and IFN-γ production, consistent with a mixed Th1 and Th17 response, whereas ccJE-alum was associated with production of mainly Th2 cytokines. In a mouse lethal challenge study against highly virulent JaTH160 JEV strain, ccJE+Advax conferred complete protection in a two-dose schedule with 50 ng of vaccine antigen and near complete protection after a single 200 ng dose of vaccine antigen. There is an ongoing lack of human vaccines against particular flaviviruses, including WNV, SLEV and MVEV. Given its ability to provide single-dose JEV protection and induce broadly neutralising antibodies devoid of ADIE activity, ccJE+Advax vaccine could be useful in situations where rapid protection is desirable, e.g., during a local outbreak or for use in travellers or armies requiring rapid deployment to JEV endemic regions.

First record of infestation of a pet sloth in Japan with the exotic tick Amblyomma geayi (Acari: Ixodidae).

On December 11, 2018, a single unengorged adult tick was found on the body surface of the trunk of an imported wild-caught Linnaeus's two-toed sloth (Choloepus didactylus) during a routine health check in an animal clinic in Tokyo, Japan. The tick was identified as Amblyomma geayi based on the morphological and molecular characteristics. This is the first case of the introduction of an Amblyomma species to Japan via an imported pet sloth. The present study highlights the current loopholes in Japan's regulatory system for animal imports.

Case Report: Two Cases of Acute Q Fever from the Same Family Who Returned from Malawi to Japan.

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.

Epidemiological study on Japanese encephalitis virus distribution in Ishikawa prefecture, Japan, by serological investigation using wild boar sera.

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.

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

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.

Survey of Coxiella burnetii in ticks collected from dogs in Japan.

For a survey of Coxiella burnetii, the Q fever agent, ticks infesting companion dogs were collected in Aomori, Tochigi, Gifu and Okinawa Prefectures, Japan. A total of 261 ticks were collected, and their species were identified morphologically. Five tick species were identified: Ixodes ovatus, Haemaphysalis concinna, H. flava, H. longicornis and Rhipicephalus sanguineus. Total DNA was extracted from them individually followed by real-time PCR to detect a C. burnetii-specific gene. The results of real-time PCR were all negative, which might suggest a low risk of C. burnetii infection via these ticks and their hosts in urban residential areas in Japan.

Comparison of viral glycosylation using lectin blotting with Vero cell-derived and mouse brain-derived Japanese encephalitis vaccines.

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.

An inactivated Vero cell-grown Japanese encephalitis vaccine formulated with Advax, a novel inulin-based adjuvant, induces protective neutralizing antibody against homologous and heterologous flaviviruses.

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.

Long-term stability of Vero cell-derived inactivated Japanese encephalitis vaccine prepared using serum-free medium.

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.

Prevalence of Coxiella burnetii antibodies in blood donors in Ankara, Central Anatolia, Turkey.

The aims of this study were to determine the prevalence of Coxiella burnetii antibodies among blood donors and to examine the epidemiological characteristics of C. bumetii infection in Ankara, Turkey. A total of 601 serum samples were collected from blood donors aged 18-61 years. Donor samples were stratified by age, sex, and residence (rural or urban). IgG and IgM antibodies to the C. bumetii phase II antigen were determined using a commercial ELISA. Blood samples reactive in the ELISA were also analysed using a commercial indirect immunofluorescence assay (IFA). The prevalence of anti-phase II IgG was 32.3%, and 17 (2.8%) were IgM positive. Seropositivity in men was higher than in women (33.2% vs. 21.7%, OR:1.88; 95% CI: 0.88-4.14) and the difference in seroprevalence rates between genders was not related to occupational exposure to domestic animals. 87.6% of seropositive donors reported no contact with farm animals. Our results revealed that C. burnetii infection is highly endemic in Ankara and that the majority of seropositive cases are not linked to specific occupational exposure in this area. In conclusion, the high rate of C. burnetii phase II antibodies among blood donors is a reflection of the high prevalence of Q fever in this area of Turkey and indicates the need for further studies, not only to determine the risk of transfusion-transmitted Q fever, but also to elucidate the epidemiology of Q fever in Turkey. These studies should be conducted through improved collaboration between the veterinary and medical services.

Pathology of canine oral malignant melanoma with cartilage and/or osteoid formation.

Of 197 cases of canine oral malignant melanoma, 29 cases with myxoid, cartilage, and osteoid formation were studied pathologically and immunohistochemically. Tumor tissues were classified into spindle cell type (13 cases), epithelioid cell type (1 case), and mixed type (15 cases). Myxoid matrixes (29 tumors) were formed mainly in the tissues of spindle cell type and were positive for Alcian blue (pH 2.5). Cartilaginous matrixes (12 tumors) were formed in the myxoid tumor tissues. The morphology of atrophied neoplastic cells, which were embedded in the cartilage cavities, significantly differed from that of spindle cells proliferating in surroundings. There were reticular areas in the process of transitioning from myxoid to cartilaginous matrixes. Osteoid matrixes were not continuous with myxoid or cartilaginous matrixes, and arose as eosinophilic trabeculae in the dense collagenous connective tissues. A calcified bone trabecula was present among the osteoid trabeculae in a case. Melanin-producing melanocytes were proliferating in the collagenous matrixes, while amelanotic cells were in the osteoid matrixes. Immunohistochemistry demonstrated proliferating neoplastic cells as melanocytes. All cells in/out of these three matrixes were positive for Melan-A, S-100 protein, NSE, and vimentin. From these results, it is suggested that cartilage and osteoid matrixes are produced by dedifferentiated melanocytes.

Japanese encephalitis virus production in Vero cells with serum-free medium using a novel oscillating bioreactor.

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.

Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification.

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.

Treatment of chronic fatigue syndrome with antibiotics: pilot study assessing the involvement of Coxiella burnetii infection.

OBJECTIVE: To examine whether Coxiella burnetii (C. burnetii) is involved in chronic fatigue syndrome (CFS), we administered tetracycline antibiotics to subjects with CFS, and followed changes in clinical symptoms, PCR findings, and C. burnetii antibody titers. PATIENTS AND METHODS: The subjects were 8 patients with CFS and 213 with nonspecific complaints such as chronic fatigue and low-grade fever for several months or longer but not meeting the diagnostic criteria for CFS. All were examined for C. burnetii infection by nested PCR and the indirect immunofluorescence test (IF). RESULTS: Four CFS patients (the CFS group) and 54 controls [the post-Q fever fatigue syndrome (QFS) group] positive for C. burnetii were treated mainly with minocycline or doxycycline (100 mg/day) for 3 months. After treatment, all 58 patients tested negative for C. burnetii infection. In the CFS group, no significant difference was noted between the mean pre- and post-treatment temperatures or headache scores. Similarly, there was no significant improvement in performance status (PS) scores. In the QFS group, however, mean temperatures and headache scores were significantly decreased after treatment (p<0.001). PS scores were also improved. CONCLUSION: These results suggest the possibility of direct involvement of C. burnetii in the pathological state of CFS to be low, despite the C. burnetii infection rate being high in CFS patients. This is a pilot study and further larger investigations are necessary to confirm our preliminary results.

[A case of Q fever infection causing acute exacerbation of chronic respiratory failure].

The authors report a case of Q fever infection that caused acute exacerbation of chronic respiratory failure, which had developed as a sequela of pulmonary tuberculosis. This case was found on wide-ranging serological screening for respiratory infection performed in order to investigate the prevalence of Q fever in Japan. A 73-year-old man who had been treated for hypertension and sequelae of pulmonary tuberculosis was admitted to our hospital because of fever, productive cough, and dyspnea on effort. Hypoxia and right heart failure were detected on arterial blood analysis and ultrasonography. The acute exacerbation was triggered by respiratory infection and although the infection improved on azithromycin treatment after admission, respiratory failure continued for the period of admission. Home oxygen therapy was required for the management of chronic respiratory failure on discharge. Paired serum samples were tested for antibodies against Coxiella burnetii by indirect immunofluorescence, showing an elevated antibody titer in the convalescent phase. We believe that Q fever infection caused acute exacerbation of chronic respiratory failure, and that C. burnetii is an agent that might influence the clinical course of chronic respiratory failure.

Improvement of chronic nonspecific symptoms by long-term minocycline treatment in Japanese patients with Coxiella burnetii infection considered to have post-Q fever fatigue syndrome.

OBJECTIVE: To address the presence of post-Q fever fatigue syndrome (post-QFS) in Japan, and to evaluate the efficacy of minocycline for this condition. PATIENTS AND METHODS: In 20 Coxiella burnetii (C. burnetii) seropositive patients with persistent nonspecific symptoms including general fatigue, low-grade fever, myalgia and arthralgia, changes in subjective symptoms, C. burnetii antibody titers and C. burnetii DNA were evaluated after antibiotic treatment. RESULTS: After treatment mainly with minocycline (100 mg/day for 3 months), the clinical picture improved in all 20 patients as evidenced by decreases in body temperature (13/17), general fatigue (20/20) and headache (9/12). The mean performance status (PS) score improved from 5.0 to 1.8 (p<0.01). All 7 who had been positive for C. burnetii DNA, became negative together with an improvement in subjective symptoms. Indirect immunofluorescence tests demonstrated 6 of the 20 patients to be positive for C. burnetii IgM antibody to phase II antigen (1:32), and 18 to be positive for IgG antibody (1:128, 1:256). Antibody titers of both IgM (6/6, 1:16) and IgG (18/18, 1:16) decreased markedly after treatment. CONCLUSION: These results of an open label study in Japan suggest that minocycline administration is useful for improving chronic nonspecific symptoms considered to be post-Q fever fatigue syndrome caused by C. burnetii infection.

Seroprevalence of Coxiella burnetii infections among cats in different living environments.

The seroprevalence of Coxiella burnetii infection among pet cats in Japan and Korea and stray cats in Japan was investigated by an indirect fluorescent antibody technique and PCR test. Forty-four (14.2%) of 310 pet cats in Japan were seropositive, as were 15 (41.7%) of 36 stray cats in Japan and 10 (8.6%) of 116 pet cats in Korea. The antibody positive rate in stray cats was significantly higher than that in pet cats, but there was no correlation between the rates in Japanese and Korean pet cats. In this study, the prevalence of C. burnetii infection among cats in different living environments was found and it is difficult to deny that stray cats would be one of the important sources of infection for human Q fever.