Shuetsu Fukushi

Broadly Reactive and Highly Sensitive Assay for Norwalk-Like Viruses Based on Real-Time Quantitative Reverse Transcription-PCR

ABSTRACT We have developed an assay for the detection of Norwalk-like viruses (NLVs) based on reverse transcription-PCR (RT-PCR) that is highly sensitive to a broad range of NLVs. We isolated virus from 71 NLV-positive stool specimens from 37 outbreaks of nonbacterial acute gastroenteritis and sequenced the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the NLV genome. The data were subjected to multiple-sequence alignment analysis and similarity plot analysis. We used the most conserved sequences that react with diverse NLVs to design primers and TaqMan probes for the respective genogroups of NLV, GI and GII, for use in a real-time quantitative RT-PCR assay. Our method detected NLV in 99% (80 of 81) of the stool specimens that were positive by electron microscopy, a better detection rate than with the two available RT-PCR methods. Furthermore, our new method also detected NLV in 20 of 28 stool specimens from the same NLV-related outbreaks that were negative for virus by electron microscopy. Our new assay is free from carryover DNA contamination and detects low copy numbers of NLV RNA. It can be used as a routine assay for diagnosis as well as for elucidation of the epidemiology of NLV infections.

Genogroup-specific PCR primers for detection of Norwalk-like viruses.

Norwalk-like viruses (NLV) are a major causative agent of nonbacterial gastroenteritis. There are still many NLV strains that are refractory to gene amplification by ordinary reverse transcription-polymerase chain reaction. This is due mainly to the genetic diversity among NLV, especially mismatches in the primer sequences, which limits this technique in clinical utility. In this study, improved primer sets based on the capsid region, to detect both genogroup I and II NLV by genogroup-specific manner, were developed. When stool specimens from gastroenteritis patients, that were positive for NLV by electron microscopy, were tested by this new primer set, all specimens were positive by RT-PCR. Primers described previously for RdRp and capsid protein were capable of amplifying the specimens by 31 and 77%, respectively. Therefore, new primer sets are extremely useful for the amplification and rapid diagnosis of nonbacterial gastroenteritis due to NLV as well as for epidemiological studies.

Coexistence of Multiple Genotypes, Including Newly Identified Genotypes, in Outbreaks of Gastroenteritis Due to Norovirus in Japan

ABSTRACT Norovirus (NV) (formerly called Norwalk-like virus) is the most common cause of acute nonbacterial gastroenteritis in humans. Recently, we reported an NV genotyping scheme based on variability in the capsid N-terminal/shell (N/S) domain gene (Katayama et al., Virology 299:225-239, 2002). We found 19 genotypes, including nine of genogroup I and 10 of genogroup II. In the present study, we investigated the molecular epidemiology of NV from 66 outbreaks that occurred in Saitama Prefecture, Japan, from 1997 to 2002. We screened 416 stool specimens by a real-time reverse transcription (RT)-PCR method (Kageyama et al., J. Clin. Microbiol. 41:1548-1557, 2003) and detected 156 NV-positive specimens, from which we amplified the capsid N/S domain gene by RT-PCR and then cloned the PCR products. After sequencing these clones, we obtained 368 sequence variants (strains). By applying our classification scheme to the strains from Saitama and other published strains, we identified a total of 31 genotypes, including an additional five genotypes for genogroup I and seven for genogroup II. Of the 31 genotypes, 26 were present in the Saitama area during that time period. These results provide additional evidence for the great diversity of human NV genotypes. Specimens from all shellfish-related infections contained multiple genotypes, including several new genotypes. On the other hand, single genotypes were observed mostly in outbreaks that originated in semiclosed communities. Thus, the number of NV genotypes in each outbreak depended on the route of transmission.

The First Identification and Retrospective Study of Severe Fever With Thrombocytopenia Syndrome in Japan

Abstract Background. Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. Methods. Virologic and pathologic examinations were performed on the patients samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. Results. A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. Conclusions. SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Phosphorylation of p38 MAPK and its downstream targets in SARS coronavirus-infected cells

Abstract Severe acute respiratory syndrome (SARS) has become a global public health emergency. Understanding the molecular mechanisms of SARS-induced cytopathic effects (CPEs) is a rational approach for the prevention of SARS, and an understanding of the cellular stress responses induced by viral infection is important for understanding the CPEs. Polyclonal antibodies, which recognized nucleocapsid (N) and membrane (M) proteins, detected viral N and M proteins in virus-infected Vero E6 cells at least 6 and 12h post-infection (h.p.i.), respectively. Furthermore, detection of DNA ladder and cleaved caspase-3 in the virus-infected cells at 24h.p.i. indicated that SARS-CoV infection induced apoptotic cell death. Phosphorylation of p38 MAPK was significantly up-regulated at 18h.p.i. in SARS-CoV-infected cells. The downstream targets of p38 MAPK, MAPKAPK-2, HSP-27, CREB, and eIF4E were phosphorylated in virus-infected cells. The p38 MAPK inhibitor, SB203580, inhibited effectively phosphorylation of HSP-27, CREB, and eIF4E in SARS-CoV-infected cells. However, viral protein synthesis was not affected by treatment of SB203580.

Reston Ebolavirus antibodies in bats, the Philippines.

To the Editor: Filoviruses cause highly lethal hemorrhagic fever in humans and nonhuman primates, except for Reston Ebolavirus (REBOV), which causes severe hemorrhagic fever in macaques (1,2). REBOV epizootics among cynomolgus macaques occurred in 1989, 1990, 1992, and 1996 (2) and among swine in 2008 (3). African fruit bats have been suggested to be natural reservoirs for Zaire Ebolavirus and Marburg virus (4–6). However, the natural reservoir of REBOV in the Philippines is unknown. Thus, we determined the prevalence of REBOV antibody–positive bats in the Philippines. Permission for this study was obtained from the Department of Environment and Natural Resources, the Philippines, before collecting bat specimens. Serum specimens from 141 wild-caught bats were collected at several locations during 2008–2009. The bat species tested are summarized in the Table. Captured bats were humanely killed and various tissues were obtained. Carcasses were then provided to the Department of Environment and Natural Resources for issuance of a transport permit. Table REBOV-specific IgG in Rousettus amplexicaudatus bats and other bats, the Philippines* We used immunoglobulin (Ig) G ELISAs with recombinant nucleoprotein (NP) and glycoprotein (GP) of REBOV (7) to determine REBOV antibody prevalence. REBOV NP and GP were expressed and purified from Tn5 cells infected with recombinant baculoviruses AcResNP and AcResGPDTM, which express NP and the ectodomain of GP with the histidine tag at its C-terminus. We also used histidine-tagged recombinant Crimean-Congo hemorrhagic fever virus NP as a negative control antigen in the IgG ELISA to confirm specificity of reactivity. In IgG ELISAs for bat specimens, positive results were detected by using rabbit anti-bat IgG and horseradish peroxidase–conjugated anti-rabbit IgG. Anti-bat (Rousettus aegyptiacus) rabbit IgG strongly cross-reacts with IgGs of other bat species, including insectivorous bats (8). Bat serum samples were 4-fold serially diluted (1:100–1:6,400) and tested by using IgG ELISAs. Results of IgG ELISAs were the sum of optical densities at serum dilutions of 1:100, 1:400, 1:1,600, and 1:6,400. Cutoff values (0.82 for both IgG ELISAs) were determined by using serum specimens from REBOV antibody–negative bats. Among 16 serum samples from R. amplexicaudatus bats, 5 (31%) captured at either the forest of Diliman (14°38′N, 121°2′E) or the forest of Quezon (14°10′N, 121°50′E) had positive results in the IgG ELISA for REBOV NP, and 5 (31%) captured at the forest of Quezon had positive results in the IgG ELISA for REBOV GP. The REBOV NP antibody–positive bats serum samples were confirmed to be NP antibody positive in the IgG ELISA by using glutathione-S-transferase–tagged partial REBOV NP antigen (9). Three samples had positive results in both IgG ELISAs (Table). Serum samples from other bat species had negative results in IgG ELISAs. All bat serum samples were also tested by indirect immunofluorescence assays (IFAs) that used HeLa cells expressing NP and GP (10). In the IFAs, 2 samples from R. amplexicaudatus bats captured at the forest of Diliman and the forest of Quezon had high titers (1,280 and 640, respectively) of NP-specific antibodies, and 1 sample from an R. amplexicaudatus bat captured at the forest of Quezon had a positive result in the GP-specific IFA (titer 20). All IFA-positive samples were also positive in the IgG ELISA (Table). The forest of Diliman is ≈30 km from the monkey facility and the Bulacan farm where REBOV infections in monkeys and swine, respectively, were detected. The forest of Quezon is ≈60 km from the monkey facility. Samples from other bat species had negative results in IFAs. We also performed heminested reverse transcription PCR specific for the REBOV NP gene with spleen specimens from all 16 R. amplexicaudatus bats but failed to detect any REBOV-specific amplicons. REBOV-specific antibodies were detected only in R. amplexicaudatus bats, a common species of fruit bat, in the Philippines. In Africa, R. aegyptiacus bats, which are genetically similar to R. amplexicaudatus bats, have been shown to be naturally infected with Zaire Ebolavirus and Marburg virus. Thus, R. amplexicaudatus bats are a possible natural reservoir of REBOV. However, only 16 specimens of R. amplexicaudatus bats were available in this study, and it will be necessary to investigate more specimens of this species to detect the REBOV genome or antigens to conclude the bat is a natural reservoir for REBOV. We have shown that R. amplexicaudatus bats are putatively infected with REBOV or closely related viruses in the Philippines. Antibody-positive bats were captured at the sites near the study areas, where REBOV infections in cynomolgus monkeys and swine have been identified. Thus, bats are a possible natural reservoir of REBOV. Further analysis to demonstrate the REBOV genome in bats is necessary to conclude that the bat is a reservoir of REBOV.

LC16m8, a Highly Attenuated Vaccinia Virus Vaccine Lacking Expression of the Membrane Protein B5R, Protects Monkeys from Monkeypox

ABSTRACT The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.

Lethal Canine Distemper Virus Outbreak in Cynomolgus Monkeys in Japan in 2008

ABSTRACT Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.

Bat Coronaviruses and Experimental Infection of Bats, the Philippines

Virus-infected fruit bats showed no signs of clinical infection.

Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells

Abstract Severe acute respiratory syndrome (SARS) is an acute respiratory tract infectious disease that is associated with a new coronavirus (SARS-CoV). Our recent study indicated that SARS-CoV infection induces activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and the p38 MAPK inhibitor partially inhibited its cytopathic effect in Vero E6 cells. The results of the present study indicated that before cell death, Akt, which is an inhibitor of apoptosis, was also activated in response to viral replication. Phosphorylation of a serine residue on Akt was detected at least 8 h postinfection (hpi), which declined after 18 hpi. Thus, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in virus-infected Vero E6 cells. However, a threonine residue was not phosphorylated. A downstream target of Akt, glycogen synthase kinase 3β (GSK-3β), was slightly phosphorylated, indicating that the level of activation of Akt was very low. PKCζ, which is downstream of the PI3K pathway, was also phosphorylated in virus-infected cells. These results suggested that weak activation of Akt cannot prevent apoptosis induced by SARS-CoV infection in Vero E6 cells.