Dengue Virus Infection in Aedes albopictus during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan.

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed.

A summary of the imported cases of Chikungunya fever in Japan from 2006 to June 2016.

Background: Due to the huge 2-way human traffic between Japan and Chikungunya (CHIK) fever-endemic regions, 89 imported cases of CHIK fever were confirmed in Japan from January 2006 to June 2016. Fifty-four of 89 cases were confirmed virologically and serologically at the National Institute of Infectious Diseases, Japan and we present the demographic profiles of the patients and the phylogenetic features of 14 CHIK virus (CHIKV) isolates. Methods: Patients were diagnosed with CHIK fever by a combination of virus isolation, viral RNA amplification, IgM antibody-, IgG antibody-, and/or neutralizing antibody detection. The whole-genome sequences of the CHIKV isolates were determined by next-generation sequencing. Results: Prior to 2014, the source countries of the imported CHIK fever cases were limited to South and Southeast Asian countries. After 2014, when outbreaks occurred in the Pacific and Caribbean Islands and Latin American countries, there was an increase in the number of imported cases from these regions. A phylogenetic analysis of 14 isolates revealed that four isolates recovered from three patients who returned from Sri Lanka, Malaysia and Angola, belonged to the East/Central/South African genotype, while 10 isolates from 10 patients who returned from Indonesia, the Philippines, Tonga, the Commonwealth of Dominica, Colombia and Cuba, belonged to the Asian genotype. Conclusion: Through the phylogenetic analysis of the isolates, we could predict the situations of the CHIK fever epidemics in Indonesia, Angola and Cuba. Although Japan has not yet experienced an autochthonous outbreak of CHIK fever, the possibility of the future introduction of CHIKV through an imported case and subsequent local transmission should be considered, especially during the mosquito-active season. The monitoring and reporting of imported cases will be useful to understand the situation of the global epidemic, to increase awareness of and facilitate the diagnosis of CHIK fever, and to identify a future CHIK fever outbreak in Japan.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes.

We have successfully prepared a Japanese encephalitis virus (JEV) - Nakayama virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV) virus was determined in infected silkworm Bm-N cells by fluorescence and Western blot analysis. The recombinant was inoculated into silkworm pupae and the yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3 days. Additionally, in the peptide analysis of infected pupae homogenate, it appeared approximately 300-500 µg E protein/pupa were produced. When purified the above eluates on the discontinuous sucrose density gradient centrifugation, NVLP showed a strong hemagglutination (HA) activity by using chicken red blood cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The immune antisera against NVLP strain could efficiently neutralize the plaque formation of Nakayama, Beijing-1 and Muar strains, showing tendency of much higher reaction with heterologous Muar strain than homologous Nakayama strain. Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control in many endemic areas of Asian countries as a widely effective and less expensive JE vaccine.

Whole Genome Sequencing-Based Molecular Epidemiologic Analysis of Autochthonous Dengue Virus Type 1 Strains Circulating in Japan in 2014.

Cases of autochthonous infections of dengue virus type 1 (DENV-1) were detected in Japan after a 70-year period devoid of dengue outbreaks. We previously showed that E gene sequences are identical in 11 of the 12 DENV-1 strains autochthonous to Japan. However, the E sequence represents only 14% of the DENV-1 genome. In the present study, we have sequenced the entire genome of 6 autochthonous DENV-1 strains that were isolated from patients during the 2014 outbreak. Sequencing of 5 Yoyogi group strains with identical E sequences and 1 Shizuoka strain with a different E sequence revealed that the first Yoyogi group strain differed from the Shizuoka strain by 18 amino acid residues. Furthermore, 2 Yoyogi group strains had different genomic sequences while the other 3 had identical genomes. Phylogenetic analyses indicated that the Hyogo strain, a Yoyogi group strain, was the first to diverge from the other 4 Yoyogi group strains. The E gene sequence of the Yoyogi group strains exhibits the highest homology to those of the strains isolated in Malaysia and Singapore between 2013 and 2014. The patient infected with the Hyogo strain visited Malaysia before the onset of dengue fever, suggesting that this was a case of dengue infection imported from Malaysia.

Two different dengue virus strains in the Japanese epidemics of 2014.

In late August 2014, dengue cases were reported in Japan, and a total of 162 cases were confirmed. In the present study, the envelope (E) gene sequences of 12 specimens from the dengue patients were determined. A dengue virus serotype 1 (DENV1) strain (D1/Hu/Shizuoka/NIID181/2014), which was clearly different from the first reported strain (D1/Hu/Saitama/NIID100/2014), was identified, although the other 11 specimens showed the same nucleotide sequences as D1/Hu/Saitama/NIID100/2014. The E gene sequences of two different strains were compared with those of nine DENV1 strains of imported cases in Japan in 2014. Phylogenetic analysis based on the E gene sequences showed that the D1/Hu/Saitama/NIID100/2014 strain was closely related to a strain isolated from an imported case from Singapore. Although no strain closely related to D1/Hu/Shizuoka/NIID181/2014 was found in these imported strains, the strain was closely related to isolates in Thailand and Taiwan in 2009. These data indicate that the dengue cases in Japan were caused by two different dengue virus strains that entered Japan through different means.

Zika fever imported from Thailand to Japan, and diagnosed by PCR in the urines.

In July 2014, a Japanese traveller returning from Thailand was investigated for fever, headache, rash and conjunctivitis. Zika virus RNA was detected in his urine sample by real-time reverse transcription polymerase chain reaction. Serological tests showed cross reactivity of IgM against the dengue virus. Zika fever could be misdiagnosed or missed and should be considered in febrile patients with a rash, especially those returning from Thailand.

Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan.

Among the tick-borne orbiviruses (genus Orbivirus, family Reoviridae), 36 serotypes are currently classified within a single virus species, Great Island virus. In this study, we report the first characterization of a tick-borne orbivirus isolated from the tick Ixodes turdus in Japan, which we identified as a new member of the species Great Island virus. The virus isolate, designated Muko virus (MUV), replicated and induced cytopathic effects in BHK-21, Vero E6, and CCL-141 cells and caused high mortality in suckling mice after intracerebral inoculation. Full genome sequence analysis showed that MUV shared the greatest phylogenetic similarity with Tribec virus in terms of the amino acid sequences of all viral proteins except for outer capsid protein 1 (OC1; VP4 of MUV). Analysis of genome segment 9 in MUV detected an uninterrupted open reading frame that overlaps with VP6 (Hel), which putatively encodes a molecular and functional equivalent of NS4 from Great Island virus. Our study provides new insights into the geographic distribution, genetic diversity, and evolutionary history of the members of the species Great Island virus.

In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain.

The characteristics of genotype V Japanese encephalitis virus (GV JEV) remain poorly understood as only two strains have been isolated to date. In this study, we examined the effects of the GV JEV Muar strain on in vitro growth and pathogenicity in mice; we also evaluated the efficacy of inactivated JEV vaccines against the Muar strain. Although growth of the Muar strain in mouse neuroblastoma N18 cells was clearly worse than that of the GIII Beijing-1 and GI Mie/41/2002 strains, neuroinvasiveness of the Muar strain was similar to that of the Beijing-1 strain and significantly higher than that of the Mie/41/2002 strain. The results of a plaque reduction neutralization test suggested that the neutralization ability of the JEV vaccines against the Muar strain was reduced compared with the GI and GIII strains. However, the protection potency of the JEV vaccine against the Muar strain was similar to that for the Beijing-1 strain in mice. Our data indicate that GV JEV has unique growth, virulence and antigenicity features.

Detecting Dengue Virus Nonstructural Protein 1 (NS1) in Urine Samples Using ELISA for the Diagnosis of Dengue Virus Infection.

Dengue virus (DENV) infection is a serious global health threat. For the surveillance and control of dengue, there is a need for robust diagnostic tools that are relatively easy to use and reliable in various clinical settings. We investigated the applicability of NS1 antigen detection in urine samples for the diagnosis of DENV. About 118 urine samples, obtained from 96 dengue patients at various phases of disease, were used for this study. NS1 antigen was detected by ELISA in the urine samples obtained from patients after 2-17 days of disease onset. Positive detection rates of NS1 antigen ranged between 13-43%. Based on real-time RT-PCR, positive detection rates of viral genome in the urine samples ranged between 20-33% on days 0 to ≥15. On days 11 to ≥15 after the disease onset, NS1 antigen was detected at similar rates in serum and urine samples. Additionally, NS1 antigen was detected in 2 urine samples, but not in the serum samples, on days 7 and 16 after the onset of the disease. The results confirm the applicability of NS1 antigen detection in urine samples using ELISA to diagnose acute DENV infection and suggests that the assay is potentially useful when only limited amounts of serum samples are available and in limited resource settings.

Autochthonous dengue fever, Tokyo, Japan, 2014.

After 70 years with no confirmed autochthonous cases of dengue fever in Japan, 19 cases were reported during August-September 2014. Dengue virus serotype 1 was detected in 18 patients. Phylogenetic analysis of the envelope protein genome sequence from 3 patients revealed 100% identity with the strain from the first patient (2014) in Japan.

Identification and amplification of Japanese encephalitis virus and Getah virus propagated from a single porcine serum sample: a case of coinfection.

We detected two viruses, Japanese encephalitis virus (JEV)/Kochi/01/2005 and Getah virus (GETV)/Kochi/01/2005 in the same culture supernatant obtained by inoculation of Vero cells with a swine serum sample and subsequent passaging of the supernatant in Vero cells. Phylogenetic analysis using the nucleotide sequences of the complete genome and the E2 region of GETV indicated that GETV/Kochi/01/2005 is most similar to a Mongolian strain. In contrast, a partial sequence of the nsP1 protein coding region of GETV/Kochi/01/2005 showed that it was similar to Japanese strains isolated in the 1980s. Alignment of the nucleotide sequence of the E region of JEV showed that JEV/Kochi/01/2005 has the highest similarity to a Japanese strain. We also examined the changes in the amount of JEV/Kochi/01/2005 and GETV/Kochi/01/2005 present after passaging in Vero cells. The RNA copy number and infectious titer of JEV/Kochi/01/2005 decreased, whereas those of GETV/Kochi/01/2005 increased, following repeated passages in Vero cells. Our results provide evidence for coinfection with JEV and GETV in the Kochi/01/2005 pig. This is the first report of incidental confection with JEV and GETV in a domestic animal.

Immunogenicity of single-dose Vero cell-derived Japanese encephalitis vaccine in Japanese adults.

In Japan, intensive immunization against Japanese encephalitis (JE) was performed from 1967 to 1976, and regular JE immunization was performed thereafter. However, for Japanese adults facing JE risk, dates of vaccination with new inactivated Vero cell-derived JE vaccine are unavailable. This study investigated how a single dose of Vero cell-derived JE vaccine affects Japanese adults. Neutralizing antibodies were measured pre- and post-JE vaccination in 79 participants (age 40.7 ± 9.4 years), enrolled between October 2009 and March 2011, whose JE-vaccination data were gathered from vaccination records and history taking. Before vaccination, the participants' seroprotection rate (SPR) was 51.9%, whereas SPR after vaccination was 93.7%. The seroconversion rate (SCR), which measures seronegative cases that turn seropositive after vaccination, was 86.8%. The geometric mean titer (GMT) was 14.7 before vaccination and 70.1 after vaccination. Age was a significant difference between seroprotected (42.8 years) and non-seroprotected (38.7 years) groups before vaccination. Then the difference of age, SCR, pre-vaccination GMT, post-vaccination GMT and sex ratio were also significant in participants aged 25-39 years and ≥40 years, who represent generations born when Japan's JE-vaccination policy changed. SCR was 100% in participants aged 25-39 years with a vaccination recorded 55.6% in participants aged 25-39 without a vaccination record, and 96.0% in participants aged ≥40 years. Thus, more participants aged 25-39 years were seroprotected before vaccination, but SCR was higher in those aged ≥40 years. Most Japanese adults can be protected after one-dose vaccination, but this may be insufficient for people aged 25-39 years without recorded JE vaccination.

The effect of precipitation on the transmission of Japanese encephalitis (JE) virus in nature: a complex effect on antibody-positive rate to JE virus in sentinel pigs.

Japanese encephalitis (JE) is one of the most important mosquito-borne viral diseases in Asia. Pigs are a natural host and the amplifier of JE virus. The sero-conversion rate to JE virus in sentinel pigs reflects the activity of JE virus in the region. We analyzed whether precipitation has any effect on the sero-conversion rate to JE virus in sentinel pigs. Linear regression analysis was performed to determine the correlations between the levels of precipitation and sero-conversion rates to JE virus, in the entire year and during summertime over the period of 32 years from 1969 to 2000. The levels of the annual and summertime precipitation demonstrated statistically significant positive correlations with sero-conversion rates for the whole of the country and for some regions in Japan. The levels of the summertime precipitation, on the other hand, demonstrated statistically significant inverse correlations with the sero-conversion rates in other regions. Further, the levels of precipitation during preceding 10-day periods from days 1-40 before blood collection showed inverse correlation with antibody-positive rates in some regions. The results indicate that the relationship between the annual and summertime precipitation, and the sero-conversion rate to JE virus is complex; both positive and inverse effects are demonstrated depending on the regions.

Detection of dengue virus nonstructural protein 1 (NS1) by using ELISA as a useful laboratory diagnostic method for dengue virus infection of international travelers.

BACKGROUND: Dengue virus ( DENV) nonstructural protein 1 ( NS1) has been used as a novel diagnostic marker during the early phase of DENV infection. METHODS: Presence of NS1 antigen was examined using 336 serum samples obtained from 276 travelers returning to Japan from Asia, Central and South America, Pacific Islands, and Africa with dengue. Assay specificity was evaluated using 148 non-dengue samples. RESULTS: Positive rates among four DENV serotypes were 68%-89%. NS1 antigen positive rates were at similar levels between primary infection and secondary infection. NS1 antigen positive rates were 88%-96% on days 1-5, 75%-100% on days 6-10, and 36-60% on ≥ day 11. Positive rates using real-time polymerase chain reaction (RT-PCR) were over 70% on days 1-5, but decreased thereafter. CONCLUSIONS: The results indicate that NS1 antigen positive rates were higher than those of RT-PCR during longer period of early phase in DENV infection. Thus, NS1 antigen ELISA is a useful tool for confirming DENV infection in international travelers, when it is used in combination with anti-DENV IgM ELISA.

Characterization of a serine-to-asparagine substitution at position 123 in the Japanese encephalitis virus E protein.

Amino acid position 123 in the E protein of Japanese encephalitis virus (JEV) determines viral growth properties and pathogenicity. The majority of JEV strains have a serine residue at this position (E(123S)); however, JEV with an asparagine residue (E(123N)) has also been isolated. To compare the growth properties and pathogenicity of E(123S) and E(123N) JEV, we produced recombinant JEV with a serine-to-asparagine substitution at position 123 (rJEV-Mie41-E(S123N)) in the E(123S)-type strain Mie/41/2002 background. The growth rate of rJEV-Mie41-E(S123N) was similar to that of Mie/41/2002 in mammalian and mosquito cell lines. Mouse challenge experiments showed that there was only a slight difference in neuroinvasiveness between the parent strain (Mie/41/2002) and rJEV-Mie41-E(S123N). Thus, our results indicate that the Ser-to-Asn substitution in the JEV E protein has weak impact on viral growth properties in vitro or on pathogenicity in vivo.

Detection of dengue virus genome in urine by real-time reverse transcriptase PCR: a laboratory diagnostic method useful after disappearance of the genome in serum.

The reemergence of dengue virus (DENV) infection has created a requirement for improved laboratory diagnostic procedures. In this study, DENV genome detection in urine was evaluated as a diagnostic method. The DENV genome was detected by real-time reverse transcriptase PCR (RT-PCR) in urine and serum of dengue patients. The detection rate of DENV genome in urine was 25% (2/8) on disease days 0 to 3 and 32% (7/22) on days 4 to 5. The rate was 50% or higher on days 6 to 16, 52% (11/21) on days 6 to 7, 78% (7/9) on days 8 to 9, 80% (4/5) on days 10 to 11, 50% (2/4) on days 12 to 13, and 60% (3/5) on days 14 to 16. The last positive urine sample was on day 16. The detection rates in serum were highest on days 0 to 3 and were greater than 50% on days 0 to 7. Detection rates decreased thereafter, and the last positive detection was on day 11. These results indicate that the time frames for positive detection differ between urine and serum samples, whereby detection rates of 50% or higher are evident between days 6 to 16 for urine samples and days 0 to 7 for serum samples. Nucleotide sequences of PCR products were identical between urine and serum samples. The detection of DENV genome in urine samples by real-time RT-PCR is useful to confirm DENV infection, particularly after viremia disappears.

Identification and characterization of the short variable region of the Japanese encephalitis virus 3' NTR.

Since the 1980s, the Japanese encephalitis virus (JEV) variants with slightly short variable regions (VR) of the 3' non-translated region (NTR) have been found; however, the implications of these short VR remain unclear. We recently identified two novel types of short VR (5 and 9 nt shorter than that of major group of genotype I JEV strains) of genotype I JEV isolates. To elucidate the impact of these short VR on the replication and virulence of JEV, we generated five recombinant JEV viruses: M41-d5 and M41-d9 have deletions in the VR that correspond to those observed in some recent JEV isolates, M41-d5d9 has both the 5- and 9-nt deletions in the VR, M41-d27 has a large deletion that encompasses both the 5- and 9-nt deletion regions, and M41-a13 has a 13-nt sequence insertion of the genotype III JEV strain Beijing-1 into the parent genotype I JEV strain Mie/41/2002 genome. The recombinant viruses and the parent virus, except for the M41-d27 mutant, showed similar growth properties in mammalian and mosquito cell lines. Mouse challenge experiments indicated that no significant differences among the recombinant viruses M41-d5d9, M41-d27, M41-a13, and the parent virus. Our results suggest that the short VR in JEV 3' NTR do not affect its growth in vitro or its pathogenicity in mice.

Dengue virus isolation relying on antibody-dependent enhancement mechanism using FcγR-expressing BHK cells and a monoclonal antibody with infection-enhancing capacity.

BACKGROUND: Virus isolation is the most reliable evidence of dengue virus (DENV) infection. However, conventional virus isolation methods generally posses lower sensitivity and are time consuming as compared to other diagnostic methods such as detection of viral genome by RT-PCR, and determination of NS1 antigen and anti-DENV antibody by ELISA. OBJECTIVES: A virus isolation method relying on the antibody-dependent enhancement mechanism was established and the assay's efficacy in DENV isolation was confirmed. STUDY DESIGN: FcγR-expressing BHK cells were used for DENV isolation from patient serum samples in the presence of a flavivirus-group reactive monoclonal antibody, mAb4G2, which possesses DENV infection-enhancement activity. DENV genome copy numbers in the culture supernatant fluids of FcγR-expressing BHK cells were assessed and compared to those of parent BHK cells and C6/36 mosquito cells, a cell line commonly used for DENV isolation. RESULTS: The virus titer levels were higher in the culture supernatant fluid of FcγR-expressing BHK cells in the presence of enhancing antibody in comparison with other cell lines using laboratory-established strains and some clinical samples. DENV was isolated from 7 of 16 serum samples by using FcγR-expressing BHK cells in the presence of mAb4G2, but not by using cell lines commonly employed in conventional isolation assays, the FcγR-negative BHK cells and C6/36 cell lines. CONCLUSIONS: The results demonstrate that FcγR-expressing BHK cell line in the presence of antibodies, which possess antibody dependent enhancement (ADE) activity, is a useful tool for DENV isolation.

Detection of higher levels of dengue viremia using FcγR-expressing BHK-21 cells than FcγR-negative cells in secondary infection but not in primary infection.

It has been reported that levels of viremia reflect the severity of illness in dengue virus infection. We assessed the levels of viremia in patients with primary and secondary infections, using 2 cell lines: FcγR-expressing BHK cells and FcγR-negative cells. In primary infection, virus titers were at similar levels between FcγR-expressing and FcγR-negative cells. In secondary infection, however, virus titers were ∼10 times higher in FcγR-expressing cells on days 1-6 when compared with FcγR-negative cells, indicating discrepancy in viremia titers between FcγR-expressing and FcγR-negative cells. The results suggest that dengue virus-antibody complexes with infectious capacity exist in patients with secondary infection, and these immune complexes can be detected by using FcγR-expressing cells. As it has been reported that principal target cells of dengue virus infection are FcγR-positive, monocyte/macrophage lineage cells, virus titers determined using FcγR-expressing cells may better reflect the actual viremic conditions in vivo.