Phylogenetic and Geographic Relationships of Severe Fever With Thrombocytopenia Syndrome Virus in China, South Korea, and Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne acute infectious disease caused by the SFTS virus (SFTSV). SFTS has been reported in China, South Korea, and Japan as a novel Bunyavirus. Although several molecular epidemiology and phylogenetic studies have been performed, the information obtained was limited, because the analyses included no or only a small number of SFTSV strains from Japan. METHODS: The nucleotide sequences of 75 SFTSV samples in Japan were newly determined directly from the patients' serum samples. In addition, the sequences of 7 strains isolated in vitro were determined and compared with those in the patients' serum samples. More than 90 strains that were identified in China, 1 strain in South Korea, and 50 strains in Japan were phylogenetically analyzed. RESULTS: The viruses were clustered into 2 clades, which were consistent with the geographic distribution. Three strains identified in Japan were clustered in the Chinese clade, and 4 strains identified in China and 26 in South Korea were clustered in the Japanese clade. CONCLUSIONS: Two clades of SFTSV may have evolved separately over time. On rare occasions, the viruses were transmitted overseas to the region in which viruses of the other clade were prevalent.

Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.

Japanese spotted fever with post-infectious encephalitis.

Japanese spotted fever (JSF) is a rickettsial disease caused by Rickettsia japonica. To the best of our knowledge, there have only been five reported cases of JSF involving the central nervous system. A 74-year-old man was admitted after 1 week of fever and maculopapular rash. JSF was definitively diagnosed by PCR; however, the patient showed mental disturbance and abnormal behavior. After intravenous immunoglobulin, his mental state and behavior improved. The findings of cerebrospinal fluid analysis, electroencephalography, and 99 mTcHM-PAO single photon computed emission tomography suggested post-infectious encephalitis. JSF causes post-infectious encephalitis and early treatment is recommended.

Molecular characterization of OP354-like P[8] (P[8]b subtype) human rotaviruses A species isolated in Japan.

OP354-like P[8] (P[8]b subtype) species A rotaviruses (RVAs) were isolated first time in Japan during a RVA survey in Okayama Prefecture between 2006 and 2009. Two of 236 RVA-positive samples were identified as G1P[8]b by reverse transcription polymerase chain reaction. P[8]b strains (RVA/human-wt/JPN/OH1998/2008/G1P[8]b and RVA/human-wt/JPN/OH2024/2008/G1P[8]b) were isolated only in May, 2008 and both patients infected with P[8]b viruses lived in the same city, suggesting that the prevalence of P[8]b RVAs is limited considerably in Okayama Prefecture. Molecular analysis of four genes (VP4, VP6, VP7, and NSP4 genes) of Japanese P[8]b strains revealed that the VP4 genes of these strains were related closely to those of Southeast Asian and Indian P[8]b strains. In contrast, the VP6, VP7, and NSP4 genes of Japanese P[8]b strains were highly homologous to G1P[8]a strains prevalent in the same area. These results suggest that the Japanese P[8]b strain may be a result of reassortment events between Japanese G1P[8]a viruses and unidentified Asian viruses possessing the P[8]b VP4 gene.

Extremely Low Genomic Diversity of Rickettsia japonica Distributed in Japan.

Rickettsiae are obligate intracellular bacteria that have small genomes as a result of reductive evolution. Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fevers". The life cycle of SFG rickettsiae is closely associated with that of the tick, which is generally thought to act as a bacterial vector and reservoir that maintains the bacterium through transstadial and transovarial transmission. Each SFG member is thought to have adapted to a specific tick species, thus restricting the bacterial distribution to a relatively limited geographic region. These unique features of SFG rickettsiae allow investigation of how the genomes of such biologically and ecologically specialized bacteria evolve after genome reduction and the types of population structures that are generated. Here, we performed a nationwide, high-resolution phylogenetic analysis of Rickettsia japonica, an etiological agent of Japanese spotted fever that is distributed in Japan and Korea. The comparison of complete or nearly complete sequences obtained from 31 R. japonica strains isolated from various sources in Japan over the past 30 years demonstrated an extremely low level of genomic diversity. In particular, only 34 single nucleotide polymorphisms were identified among the 27 strains of the major lineage containing all clinical isolates and tick isolates from the three tick species. Our data provide novel insights into the biology and genome evolution of R. japonica, including the possibilities of recent clonal expansion and a long generation time in nature due to the long dormant phase associated with tick life cycles.

Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350-1220 TCID50/100 µl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>105 copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV. CONCLUSIONS: The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia.