Shinichi Takao

Rapid Detection of Norovirus from Fecal Specimens by Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay

ABSTRACT In this study, we developed a one-step, single-tube genogroup-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NoV) genomes targeting from the C terminus of the RNA-dependent RNA polymerase gene to the capsid N-terminal/shell domain region. This is the first report on the development of an RT-LAMP assay for the detection of NoV genomes. Because of the diversity of NoV genotypes, we used 9 and 13 specially designed primers containing mixed bases for genogroup I (GI) and II (GII), respectively. The RT-LAMP assay had the advantages of rapidity, simplicity, specificity, and selectively and could obtain results within 90 min, generally even within 60 min, under isothermal conditions at 62°C. The detection limits for NoV genomes were between 102 and 103 copies/tube for GI and GII with differentiation by genotype, and no cross-reactions among NoV GI and GII and other gastroenteritis viruses, such as sapovirus, human astrovirus, adenovirus type 40 and 41, and group A and C rotavirus, were found. In the evaluation tests with fecal specimens obtained from gastroenteritis outbreaks, the sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 94% for GI and 100 and 100% for GII, respectively. These findings establish that the RT-LAMP assay is potentially useful for the rapid detection of NoV genomes from fecal specimens in outbreaks of food-borne and person-to-person-transmitted gastroenteritis.

Japanese Encephalitis Virus in Meningitis Patients, Japan

Cerebrospinal fluid specimens from 57 patients diagnosed with meningitis were tested for Japanese encephalitis virus. Total RNA was extracted from the specimens and amplified. Two products had highest homology with Nakayama strain and 2 with Ishikawa strain. Results suggest that Japanese encephalitis virus causes some aseptic meningitis in Japan.

Immediate protection of mice from lethal wild-type Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, possessing homologous interfering capacity.

Protection of mice from lethal Sendai virus (HVJ) infections by a temperature-sensitive mutant, HVJpi, which was isolated from a carrier culture, was studied. HVJpi had a strong interfering capacity with the replication of virulent wild-type virus in LLCMK2 cells. When a high dose of HVJpi (3.0 x 10(7) CIU) was inoculated intranasally into mice, the mice showed neither illness nor lung lesions but gained significant resistance against the challenge of virulent wild-type virus (18 LD50) immediately after inoculation. In contrast, the mice inoculated with a lower dose of HVJpi (8.2 x 10(5) CIU) did not show the immediate resistance but became immune several days after inoculation. Time courses of the virus replication in the lung revealed that the replication of wild-type virus was strongly suppressed to about 1/1000 by the simultaneous infection with a high dose of HVJpi, thus resulting in minimizing the lung lesions and survival of all the mice infected. Neither interferon nor natural killer cells appeared to play a major role in the immediate immune status by HVJpi, since no difference was observed in protection of mice simultaneously infected with wild-type virus and HVJpi in spite of pretreatment of the mice with anti-interferon and anti-asialo GM1 antibodies as compared with that of the untreated doubly infected mice. On the other hand, it was suggested by analysis of viral polypeptides synthesized in the lung of infected mice by Western blotting that the early stage of replication of wild-type virus in the lung was inhibited mainly by the interfering capacity of HVJpi. These results indicate that HVJpi is an unique virus mutant which is capable of protecting mice from lethal Sendai virus infections by its interfering capacity immediately after inoculation and then by the induction of virus-specific immune responses.

Recombinant norovirus implicated in gastroenteritis outbreaks in Hiroshima prefecture, Japan

Norovirus (NoV) is a major etiological agent of acute gastroenteritis outbreaks worldwide. A total of 314 fecal specimens collected from patients of 39 NoV gastroenteritis outbreaks in Hiroshima Prefecture, Japan, between December 2001 and April 2006 were tested for the occurrence of recombinant NoVs. Sixteen genotypes (GI/1, GI/2, GI/4, GI/7, GI/8, GI/11, GI/14, GII/2, GII/3, GII/4, GII/5, GII/6, GII/8, GII/12, GII/14, and GII/untypeable) were detected in the 39 outbreaks based on capsid sequences and GII/4 was predominant recently. Twelve strains detected in 11 (28.2%) of the 39 outbreaks were suspected to be recombinants by using Simplot and Recco analyses and five recombinant genotypes, GII/4‐GII/12 (five strains), GIIb‐GII/3 (four strains), GII/4‐GII/2 (one strain), GII/4‐GII/14 (one strain), and GI/2‐GI/8 (one strain), were identified based on RNA‐dependent RNA polymerase and capsid sequences. None of the strains genotyped as GII/4 based on the capsid sequence was identified as a recombinant. The putative recombination points in the recombinant strains were placed either upstream or downstream of the open reading frame (ORF) 1 and ORF2 overlap. The present study indicates the following: (a) recombination among ORFs is common in nature, (b) the involvement of recombinant NoVs in gastroenteritis outbreaks is extensive even in a local area such as Hiroshima Prefecture, Japan, and (c) the conserved region (ORF1 and ORF2 overlap) has a meaningful function against the recombination event. J. Med. Virol. 80:921–928, 2008.

Expression of the HN, F, NP and M proteins of Sendai virus by recombinant vaccinia viruses and their contribution to protective immunity against Sendai virus infections in mice

Recombinant vaccinia viruses (RVVs) expressing each of the haemagglutinin-neuraminidase (HN), fusion (F), nucleocapsid (NP) and matrix (M) proteins of Sendai virus were constructed to investigate their capacities to induce protective immunity against Sendai virus infections. The proteins expressed in cultured cells appeared to be authentic with respect to their antigenicity, electrophoretic mobility, surface expression of the HN and F proteins and, in the case of the HN protein, biological activities. Mice inoculated intranasally with these RVVs developed serum antibodies to the respective Sendai virus proteins, suggesting their in vivo expression. In mice immunized with RVV carrying either the HN or the F gene, growth of the challenging Sendai virus was almost completely suppressed in the lung, indicating their capacities to induce effective protective immunity against Sendai virus infections. In contrast, in mice immunized with RVV carrying the NP or M gene, the challenging virus propagated as well as in the control mice, but the virus titres were significantly lower at the late stage of infection than those in the control mice, suggesting that they can also induce protective immunity especially at the late stage of the challenge infection.

Molecular epidemiology of subgenus F adenoviruses associated with pediatric gastroenteritis during eight years in Hiroshima Prefecture as a limited area.

Summary.We have studied the prevalence of the subgenus F adenoviruses and the molecular characteristics of adenovirus type 41 in Hiroshima Prefecture, Japan, as a limited area during the period of 1997–2004. Subgenus F adenoviruses were detected in 30 (3.4%) of 892 fecal specimens by enzyme immunoassay (EIA), and 80.0% (24 of 30) of positive patients were <36 months old. One (3.3%) and 29 (96.7%) of the 30 EIA-positive specimens were adenoviruses type 40 (Ad40) and 41 (Ad41), respectively. The genomes of Ad41 strains amplified by PCR were divided into two genomic type clusters (GTC1 and GTC2) based on the hexon gene as described by Li et al. (J Clin Microbiol 42: 4032–4039, 2004.). Twenty-one (95.5%) of 22 Ad41 strains detected between 2000 and 2004 belonged to GTC1, whereas all seven strains detected between 1997 and 1999 belonged to GTC2. These genomic typings were the same for the hexon and fiber genes except for one strain. This strain contained a hexon gene belonging to GTC1 and a fiber gene belonging to GTC2 and was considered to be a recombinant between adenoviruses of these types.

Transition of genotypes associated with norovirus gastroenteritis outbreaks in a limited area of Japan, Hiroshima Prefecture, during eight epidemic seasons

The transition of genotypes implicated in 102 NoV gastroenteritis outbreaks in Hiroshima Prefecture, Japan, during eight epidemic seasons was investigated. Eighteen genotypes were implicated in the outbreaks, with the chronological characteristics as in GII.3, GII.4, GII.5 and GII.12. In GII.4 variants, amino acid changes and positively selected sites were of note and significantly concentrated in the surface-exposed P2 subdomain of the VP1 protein. Notably, variant-specific epitopes at which positively selected sites are located may be significant for distinguishing a new GII.4 variant. The interaction of these genetic changes with developing immunity seems to influence NoV epidemics.

Three-Year Study of Viral Etiology and Features of Febrile Respiratory Tract Infections in Japanese Pediatric Outpatients

Background: For most febrile respiratory tract infections (RTIs) in children, the causative pathogen is never identified. We sought to identify the causative pathogen in individual cases of pediatric outpatient with RTIs and to determine whether particular clinical features of RTIs are associated with particular viruses. Methods: Over 3 years, we prospectively collected nasopharyngeal aspirate specimens from individual pediatric outpatients with an RTI accompanied by persistent fever (>3 days, ≥38.0°C) and peak temperature ≥39.0°C. Two methods—(1) viral culture for respiratory viruses and (2) real-time polymerase chain reaction (PCR) assays identifying 9 different respiratory viruses and 2 respiratory bacteria—were used to test specimens. Results: For 495 specimens, viral culture and real-time PCR assays together identified at least 1 pathogen in 83.0% and ≥1 viruses alone in 79.4%. These 2 methods identified 138 children with respiratory syncytial virus, 66 with human metapneumovirus, 73 with parainfluenza viruses, 124 with adenovirus, 23 with rhinovirus, 38 with enterovirus, 11 with influenza type C virus, 15 with Mycoplasma pneumoniae and 3 with Chlamydophila pneumoniae; the coinfection rate was 19.7% among all infections. Among the patients with single-pathogen infections, the rate of lower RTI was 37.6% for respiratory syncytial virus, 40.7% for human metapneumovirus, 18.2% for parainfluenza viruses and 2.2% for adenovirus (P < 0.01). Conclusions: Viral culture and real-time PCR assays were used together to identify causative pathogens in 83% of febrile outpatient children with RTI; specific viruses were associated with particular clinical diagnoses.

Evaluation of Three Immunochromatographic Kits for Rapid Detection of Influenza Virus A and B

Background: Clinically, it is difficult to distinguish influenza infection from febrile infections caused by respiratory syncytial virus, adenovirus, rhinovirus, and human metapneumovirus without the use of rapid diagnostic tests. Methods: Nasopharyngeal aspirates (NPAs) were collected from 494 pediatric patients and were tested for the presence of influenza A and B virus antigen using 3 rapid diagnotic kits: ESPLINE Influenza A and B-N, Directigen EZ Flu A and B, and Binax NOW Influenza A and B. Results: Fifty-three specimens positive by viral culture were positive for influenza A by all 3 kits. Of the 270 influenza B virus cellculture-positive specimens, the sensitivities of ESPLINE, EZ, and NOW were 86%, 76%, and 78%, respectively. Conclusion: Under well-controlled conditions, influenza A virus sensitivities for all 3 kits can be 100% with specificities ranging from 98% to 100%.

本邦において初めて流行が確認された小児のhuman metapneumovirus感染症の臨床的, 疫学的解析

Human metapneumovirus (hMPV) was newly discovered as a pathogen in 2001 and is thought to be associated with respiratory disease. To elucidate the prevalence and clinical significance of hMPV among children, we investigated the positive cases of hMPV-RNA by reverse transcription-polymerase chain reaction (RT-PCR) in their nasopharyngeal specimens collected from January to August 2003 in Hiroshima Prefecture, Japan. Our prospective study revealed 77 hMPV-positive cases among 377 children with acute respiratory diseases. Clinical diagnoses of 77 hMPV-positive cases were as follows; bronchitis (33.8%), pneumonia (24.7%), acute respiratory illness (19.5%), asthmatic bronchitis (11.7%) and bronchiolitis (5.2%). The most common symptoms were cough (97.4%), high fever (94.8%) and rhinorrhea (76.6%). Most of the hMPV-positive cases were identified in the spring (between March and May), indicating the presence of an epidemic of hMPV infection in Hiroshima Prefecture. Phylogenetic analysis of the amplified F gene of hMPV isolates revealed that hMPV strains were divided into two genotypes and that their simultaneous circulation occurred within the same epidemic area of Hiroshima Prefecture.