Genotype-Dependent Immunogenicity of Dengue Virus Type 2 Asian I and Asian/American Genotypes in Common Marmoset (Callithrix jacchus): Discrepancy in Neutralizing and Infection-Enhancing Antibody Levels between Genotypes.

Owing to genotype-specific neutralizing antibodies, analyzing differences in the immunogenic variation among dengue virus (DENV) genotypes is central to effective vaccine development. Herein, we characterized the viral kinetics and antibody response induced by DENV type 2 Asian I (AI) and Asian/American (AA) genotypes using marmosets (Callithrix jacchus) as models. Two groups of marmosets were inoculated with AI and AA genotypes, and serial plasma samples were collected. Viremia levels were determined using quantitative reverse transcription-PCR, plaque assays, and antigen enzyme-linked immunosorbent assay (ELISA). Anti-DENV immunoglobulin M and G antibodies, neutralizing antibody titer, and antibody-dependent enhancement (ADE) activity were determined using ELISA, plaque reduction neutralization test, and ADE assay, respectively. The AI genotype induced viremia for a longer duration, but the AA genotype induced higher levels of viremia. After four months, the neutralizing antibody titer induced by the AA genotype remained high, but that induced by the AI genotype waned. ADE activity toward Cosmopolitan genotypes was detected in marmosets inoculated with the AI genotype. These findings indicate discrepancies between heterologous genotypes that influence neutralizing antibodies and viremia in marmosets, a critical issue in vaccine development.

U.S.-Japan cooperative medical sciences program: 22nd International Conference on Emerging Infectious Diseases in the Pacific Rim.

This review summarizes the presentations given at the 22nd International conference on Emerging Infectious Diseases in the Pacific Rim. The purpose of this annual meeting is to foster international collaborations and address important public health issues in the Asia-Pacific region. This meeting was held in Bangkok in February 2020 and focused on emerging virus infections. Unexpectedly, the SARS-CoV-2 pandemic was in the initial stages leading to a special session on COVID-19 in addition to talks on dengue, influenza, hepatitis, AIDS, Zika, chikungunya, rabies, cervical cancer and nasopharyngeal carcinoma.

Dengue epidemic in Malaysia: urban versus rural comparison of dengue immunoglobulin G seroprevalence among Malaysian adults aged 35-74 years.

BACKGROUND: A periodic serosurvey of dengue seroprevalence is vital to determine the prevalence of dengue in countries where this disease is endemic. This study aimed to determine the prevalence of dengue immunoglobulin G (IgG) seropositivity among healthy Malaysian adults living in urban and rural areas. METHODS: A total of 2598 serum samples (1417 urban samples, 1181 rural samples) were randomly collected from adults ages 35-74 y. The presence of the dengue IgG antibody and neutralising antibodies to dengue virus (DENV) 1-4 was determined using enzyme-linked immunosorbent assay and the plaque reduction neutralisation test assay, respectively. RESULTS: The prevalence of dengue IgG seropositivity was 85.39% in urban areas and 83.48% in rural areas. The seropositivity increased with every 10-y increase in age. Ethnicity was associated with dengue seropositivity in urban areas but not in rural areas. The factors associated with dengue seropositivity were sex and working outdoors. In dengue IgG-positive serum samples, 98.39% of the samples had neutralising antibodies against DENV3, but only 70.97% of them had neutralising antibodies against DENV4. CONCLUSION: The high seroprevalence of dengue found in urban and rural areas suggests that both urban and rural communities are vital for establishing and sustaining DENV transmission in Malaysia.

Non-Human Primate Models of Dengue Virus Infection: A Comparison of Viremia Levels and Antibody Responses during Primary and Secondary Infection among Old World and New World Monkeys.

Due to the global burden of dengue disease, a vaccine is urgently needed. One of the key points in vaccine development is the development of a robust and reliable animal model of dengue virus infection. Characteristics including the ability to sustain viral replication, demonstration of clinical signs, and immune response that resemble those of human dengue virus infection are vital in animal models. Preclinical studies in vaccine development usually include parameters such as safety evaluation, induction of viremia and antigenemia, immunogenicity, and vaccine effectiveness. Although mice have been used as a model, non-human primates have an advantage over mice because of their relative similarity to humans in their genetic composition and immune responses. This review compares the viremia kinetics and antibody responses of cynomolgus macaques (Macaca fasicularis), common marmosets (Callithrix jacchus), and tamarins (Saguinus midas and Saguinus labitus) and summarize the perspectives and the usefulness along with challenges in dengue vaccine development.

Development and evaluation of human T-cell leukemia virus-1 and -2 multiplex quantitative PCR.

The diagnosis of human T -cell leukemia virus type 1 (HTLV-1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV-1 and/or HTLV-2 are used to confirm infection with HTLV-1 and/or HTLV-2 and are also used for the follow-up of HTLV-1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV-1 and HTLV-2, a multiplex quantitative polymerase chain reaction (qPCR) by large-scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV-1 provirus per 105 cells. Moreover, HTLV-1 provirus could be detected in 97.2% (205 of 211) of HTLV-1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV-2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV-1 and HTLV-2 provirus with extremely high sensitivity.

20th International Conference on Emerging Infectious Diseases in the Pacific Rim Organized by the United States-Japan Cooperative Medical Sciences Program (USJCMSP).

The 20th International Conference on Emerging Infectious Diseases in the Pacific Rim to3ok place in Shenzhen, China on January 8⁻9, 2018 followed by meetings of the acquired immunodeficiency syndrome (AIDS)/immunology, acute respiratory infections, cancer, hepatitis, and viral diseases panels on January 10⁻11. The conference was organized as part of the United States-Japan Cooperative Medical Sciences Program (USJCMSP) by the Japan Agency for Medical Research and Development (AMED) and the U.S. National Institutes of Health (NIH) and was locally hosted by the Shenzhen Third People's Hospital and the Chinese Academy of Sciences (CAS) Institute of Microbiology. The conference provides the basis for networking and fostering of collaboration opportunities between researchers in Southeast Asia and the United States based on the scientific and interactive platform of the USJCMSP and takes place in the region on an annual basis. This report summarizes the discussions and conclusions from the conference.

Correction to: A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

In the original publication of article [1], '20 × 101 copies', which is in the sentence 'As seen in Fig. 4, the sensitivity of the specimens containing equal to or more than 20 × 101 copies in 2 µL of extracted DNA (equivalent to ≥3.0 × 103 copies/mL CSF) was 100% (29/29)' changes to '2.0 × 101 copies' in results section. The publisher apologizes to the readers and authors for the inconvenience.

A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

BACKGROUND: JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required. METHODS: A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML. RESULTS: The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 100 to 3.2 × 106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay. CONCLUSIONS: The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.

Genotype-specific and cross-reactive neutralizing antibodies induced by dengue virus infection: detection of antibodies with different levels of neutralizing activities against homologous and heterologous genotypes of dengue virus type 2 in common marmosets (Callithrix jacchus).

BACKGROUND: A vaccine against all four dengue virus (DENV) serotypes includes the formulation of one genotype of each serotype. Although genetic similarities among genotypes within a serotype are higher as compared to those among serotypes, differences in the immunogenicity of the included genotypes would be a critical issue in maximizing successful dengue vaccine development. Thus, we determined the neutralizing antibody responses against three genotypes of dengue virus serotype 2 (DENV-2), namely Cosmopolitan, Asian I, and Asian/American, after primary and secondary inoculation with DENV-2 in a dengue animal model, the common marmoset (Callithrix jacchus). METHODS: A total of fifty-four plasma samples were obtained from thirty-four marmosets that were inoculated with clinically-isolated DENV strains or DENV candidate vaccines, were used in this study. Plasma samples were obtained from marmosets after primary inoculation with DENV-2 infection, secondary inoculation with homologous or heterologous genotypes, and tertiary inoculation with heterologous DENV. Neutralizing antibody titers against DENV-2 (Cosmopolitan, Asian I, and Asian/American genotypes) and DENV-1 were determined using a conventional plaque reduction neutralization assay. RESULTS: In marmosets that were inoculated with the Cosmopolitan genotype in primary infection, neutralizing antibody neutralized 3 genotypes, and the titers to Asian I genotype were significantly higher than those to homologous Cosmopolitan genotype. After secondary DENV-2 infection with heterologous genotype (Asian I in primary and Asian/American in secondary), neutralizing antibody titers to Asian/American genotype was significantly higher than those against Cosmopolitan and Asian I genotypes. Following tertiary infection with DENV-1 following DENV-2 Asian I and Cosmopolitan genotypes, neutralizing antibody titers to Asian/American were also significantly higher than those against Cosmopolitan and Asian I genotypes. CONCLUSION: The present study demonstrated that different levels of neutralizing antibodies were induced against variable DENV-2 genotypes after primary, secondary and tertiary infections, and that neutralizing antibody titers to some heterologous genotypes were higher than those to homologous genotypes within a serotype. The results indicate that heterogeneity and homogeneity of infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced in following infections. The results also suggest that certain genotypes may possess advantage in terms of breakthrough infections against vaccination.

Detection of Virus-Antibody Immune Complexes in Secondary Dengue Virus Infection.

It has been reported that virus-antibody immune complexes formed during secondary dengue virus infection are associated with increased disease severity. Here, we describe the details of a plaque titration method that uses FcγR -expressing BHK cells to detect and quantify infectious virus-immune complexes and a quantitative real-time PCR method for the quantification of virus genome in patients with secondary dengue infection. These methods detect both viruses in free-form and in immune complexes in patients with dengue infection, and are useful for determining viremia levels and patterns that better reflect in vivo conditions.

Marmosets (Callithrix jacchus) as a non-human primate model for evaluation of candidate dengue vaccines: induction and maintenance of specific protective immunity against challenges with clinical isolates.

Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.

A double-quadratic model for predicting Vibrio species in water environments of Japan.

Vibrio spp. are natural inhabitants of marine and estuarine environments. Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus are the major infectious agents for humans. Their densities are affected by environmental factors such as water temperature and salinity. The detailed contribution of each factor still remains to be elucidated. Here we conducted multi-coastal study in a 21-month period to examine relationships between environmental factors and V. cholerae, V. parahaemolyticus and V. vulnificus densities in sea surface water in eight coastal sites of four prefectures in Japan. Vibrio densities were measured by a most-probable-number with PCR method which is highly sensitive and quantitative (3/100 ml of detection limit). Vibrio densities were analyzed with environmental factors including water temperature, salinity, total dissolved substance, and pH, and their quadratics. A linear regression model suited best for prediction of V. cholerae density. A novel double-quadratic model suited best for the prediction of V. parahaemolyticus and V. vulnificus densities.

Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses.

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections.

Reduction of animal suffering in rabies vaccine potency testing by introduction of humane endpoints.

Potency controls of inactivated rabies vaccines for human use are confirmed by the National Institutes of Health challenge test in which lethal infection with severe neurological symptoms should be observed in approximately half of the mice inoculated with the rabies virus. Weight loss, decreased body temperature, and the presence of rabies-associated neurological signs have been proposed as humane endpoints. The potential for reduction of animal suffering by introducing humane endpoints in the potency test for inactivated rabies vaccine for human use was investigated. The clinical signs were scored and body weight was monitored. The average times to death following inoculation were 10.49 and 10.99 days post-inoculation (dpi) by the potency and challenge control tests, respectively, whereas the average times to showing Score-2 signs (paralysis, trembling, and coma) were 6.26 and 6.55 dpi, respectively. Body weight loss of more than 15% appeared at 5.82 and 6.42 dpi. The data provided here support the introduction of obvious neuronal signs combined with a body weight loss of ≥15% as a humane endpoint to reduce the time of animal suffering by approximately 4 days.

A Single Vaccination of Nonhuman Primates with Highly Attenuated Smallpox Vaccine, LC16m8, Provides Long-term Protection against Monkeypox.

Monkeypox virus (MPXV) causes human monkeypox (human MPX), which is a similar disease to smallpox in humans. A previous study showed that a single vaccination of monkeys with LC16m8, a highly attenuated smallpox vaccine, protected them from MPX from 4-5 weeks post-vaccination. In this study, we evaluated the long-term efficacy of a single vaccination with LC16m8 in a nonhuman primate model of MPXV infection. The monkeys were inoculated with either LC16m8, Lister (parental strain of LC16m8), or a mock-up vaccine, and then challenged with MPXV via a subcutaneous route, at 6 and 12 months after vaccination, which we compared with either Lister or the mock-up vaccination. The LC16m8 monkeys exhibited almost no MPX-associated symptoms, whereas most of the naïve monkeys died. LC16m8 generated the protective memory immune response against MPXV, as suggested by the immediate viremia reduction and the response of the IgG antibody. The results demonstrated that the vaccination of monkeys with a single dose of LC16m8 provided durable protection against MPXV for longer than one year after immunization. The results suggest that the vaccination of humans with LC16m8 could induce long-term protection against MPXV infection.

Neutralizing and enhancing antibody responses to five genotypes of dengue virus type 1 (DENV-1) in DENV-1 patients.

Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.

Dengue Virus Isolation in Mosquito Aedes albopictus Captured During an Outbreak in Tokyo, 2014, by a Method Relying on Antibody-Dependent Enhancement Mechanism Using FcγR-Expressing BHK Cells.

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field.

Japanese encephalitis vaccine-facilitated dengue virus infection-enhancement antibody in adults.

BACKGROUND: Dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the genus Flavivirus, and infection with a virus within this genus induces antibodies that are cross-reactive to other flaviviruses. Particularly in DENV infection, antibodies to DENV possess two competing activities: neutralizing activity and infection-enhancing activity. These antibody activities are considered central in modulating clinical outcomes of DENV infection. Here, we determined the neutralizing and infection-enhancing activity of DENV cross-reactive antibodies in adults before and after JE vaccination. METHODS: Participants were 77 Japanese adults who had received a single dose of inactivated Vero cell-derived JE vaccine. A total of 154 serum samples were obtained either before or approximately a month after a single dose of JE vaccination. The antibody-dependent enhancement (ADE) activity to each of four DENV serotypes and the neutralizing activities to DENV and to JEV were determined in each of the serum samples by using baby hamster kidney (BHK) cells and FcγR-expressing BHK cells. RESULTS: A total of 18 post-JE immunization samples demonstrated cross-reactivity to DENV in an anti-DENV IgG ELISA. DENV neutralizing antibodies were not detected after JE vaccination in this study. However, undiluted post-JE vaccination serum samples from 26 participants demonstrated monotypic and heterotypic ADE activity to DENV. ADE activity was also observed in 1:10-diluted samples from 35 of the JE vaccine recipients (35/77, 45 %). CONCLUSION: In summary, JE vaccination induced DENV cross-reactive antibodies, and at sub-neutralizing levels, these DENV cross-reactive antibodies possess DENV infection-enhancement activity. The results also indicate that cross-reactivity to DENV is associated with high levels of JEV neutralizing antibodies and, the DENV cross-reactivity is further facilitated by JE vaccination.

Development of a novel dengue virus serotype-specific multiplex real-time reverse transcription-polymerase chain reaction assay for blood screening.

BACKGROUND: Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS: Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS: A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION: This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection.