Detection of norovirus, sapovirus, and human astrovirus in fecal specimens using a multiplex reverse transcription-PCR with fluorescent dye-labeled primers.

We applied a multiplex reverse transcription-PCR with fluorescent dye-labeled primers (fluorescent multiplex RT-PCR) for noroviruses (NoV), sapovirus (SaV), and human astrovirus (HAstV) to diagnose 71 outbreaks of acute gastroenteritis during July 2007 and May 2010 in Hiroshima prefecture. In this assay, the green, red, yellow, and blue fluorescence for NoV genogroup I, NoV genogroup II, SaV, and HAstV, respectively, were indicated on an agarose gel under ultraviolet light. In 61 virus-positive outbreaks confirmed by fluorescent multiplex RT-PCR, detection rates of outbreaks for NoVs, SaV, and HAstV were 96.7%, 3.3%, and 0%, respectively.

Analysis of interaction of Sendai virus V protein and melanoma differentiation-associated gene 5.

Sendai virus (SeV), a pneumotropic virus of rodents, has an accessory protein, V, and the V protein has been shown to interact with MDA5, inhibiting IRF3 activation and interferon-ß production. In the present study, interaction of the V protein with various IRF3-activating proteins including MDA5 was investigated in a co-immunoprecipitation assay. We also investigated interaction of mutant V proteins from SeVs of low pathogenicity with MDA5. The V protein interacted with at least retinoic acid inducible gene I, inhibitor of κB kinase epsilon and IRF3 other than MDA5. However, only MDA5 interacted with the V protein dependently on the C-terminal V unique (Vu) region, inhibiting IRF3 reporter activation. The Vu region has been shown to be important for viral pathogenicity. We thus focused on interaction of the V protein with MDA5. Point mutations in the Vu region destabilized the V protein or abolished the interaction with MDA5 when the V protein was stable. The V-R320G protein was highly stable and interacted with MDA5, but did not inhibit activation of IRF3 induced by MDA5. Viral pathogenicity of SeV is related to the inhibitory effect of the V protein on MDA5, but is not always related to the binding of V protein with MDA5.

[Rapid detection of novel influenza A virus and seasonal influenza A (H1N1, H3N2) viruses by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)].

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus-had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63 degrees C for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.

[Evaluation of immunochromatography test for rapid detection of influenza A and B viruses using real-time PCR].

The sensitivity of rapid diagnostic kits to influenza B is lower than to influenza A. The cause-poor performance of the kit or the scarcity of viruses in type B specimens-has yet to be clarified. Using real-time PCR, we measured the amount of influenza viruses with nasopharyngeal aspirate fluid previously identified by virus isolation culture and passing the rapid diagnosis test by four types of kits, including the ESPLINE Influenza A&B-N (Fujirebio Corp., Japan). We classified the results of virus isolation and rapid diagnosis tests into three groups and examined them: group 1 (12 specimens, influenza B, all negative in tests using four types of kits); group 2 (57 specimens, influenza B, all positive in tests); and group 3 (36 specimens, AH3, all positive in tests). The average amount of viruses in group 1 (6.60 +/- 0.81 log10copies/mL) was significantly lower (p<0.0001) than that in group 2 (8.51 +/- 0.57 log10copies/mL) or group 3 (8.72 +/- 0.63 log10copies/mL). No significant difference was seen in the amount of viruses between groups 2 and 3. We concluded that the cause of low sensitivity in rapid diagnostic kits to influenza B are attributable to the scarcity of viruses in the specimen.

[Eleven cases of co-infection with influenza type A and type B suspected by use of a rapid diagnostic kit and confirmed by RT-PCR and virus isolation].

In the 2004/05 influenza season there were epidemics of influenza caused by several types of viruses (type B and A (H3) viruses, and type B, A (H3), and A (H1) viruses) in many areas of Japan. In such epidemics a single individual could be co-infected with several influenza viruses. From February to March in 2005, we examined 15 patients who were positive for influenza type A and B viruses when tested with a rapid diagnostic kit. The type A (H3) and B influenza virus genes were successfully amplified by RT-PCR in 10 of the 15 patients, confirming that they were co-infected with type A (H3) and B viruses. The type A (H1) and B virus genes were successfully amplified in another patient, confirming that the patient was co-infected with type A (H1) and B viruses. By contrast, 2 patients were clearly positive for type A and B viruses according to the rapid diagnostic kit, but positive for type B virus alone by RT-PCR. No influenza virus genes were detected by RT-PCR in the remaining 2 patients. To isolate one type from a mixture of two different types of influenza viruses in a specimen, we neutralized one of the types with type-specific antiserum, and isolated the other with MDCK (+) cells. The results obtained by virus isolation were identical to those obtained by RT-PCR. Influenza viruses corresponding to the results of RT-PCR were isolated from 9 of the 11 patients in which isolation was attempted. No viruses were isolated from the 2 patients in whom no virus genes were detectable by RT-PCR. Based on these results we concluded that 11 of 15 patients who were positive for type A and B viruses according to the rapid diagnostic kit were co-infected with type A (H3) or A (H1) and B virus. When several types of influenza viruses are prevalent, as in the 2004/05 influenza season, the possibility of a patient being co-infected with more than one type of influenza virus should be considered.

[Comparison of four rapid diagnostic kits using immunochromatography to detect influenza B viruses].

We compared the usefulness of 4 rapid influenza diagnostic 1-device kits using immunochromatography, which facilitate type differentiation, i.e. ESPLINE Influenza A&B-N (Fujirebio Corp., Japan: ESPLINE), POCTEM INFLUENZA A/B (Sysmex Corp., Japan: POCTEM), Quick Vue Rapid SP influ (Quidel Corp., U.S.A.: Quick Vue), and Capilia Flu A + B (TAUNS Corp., Japan: Capilia), in 278 children in whom influenza infection was suspected in 2004 and 2005. Nasopharyngeal aspirates were diluted for virus isolation and residual samples were centrifuged. Using the supernatant, we conducted rapid diagnosis testing. Influenza virus AH3 was isolated from 40 children, and influenza B virus from 163. Of the 40 children, the sensitivity and specificity of ESPLINE, POCTEM, Quick Vue, and Capilia were 100%/100%, 95%/100%, 98%/96%, and 98%/96%. In the 163 children, the sensitivity and specificity were 89%/100%, 87%/100%, 88%/97%, and 86%/98%. ESPLINE showed the highest sensitivity and specificity to influenza viruses AH3 and B. All kits were less sensitive to influenza B virus than to influenza A virus, however. The specificity of Quick Vue and Capilia was low; so these kits must be improved.

[The clinical features about 5 cases of Japanese encephalitis reported in Japan, 2002].

We discussed the clinical features of 5 Japanese encephalitis (JE) cases which we experienced in 2002. Today there are few opportunities for a clinician to see JE patients. Until the 1950s, the number of JE patients was more than 2000 in Japan, but the annual cases of JE are decreasing remarkably due to the extermination of mosquitoes, thorough vaccination and improvement of environmental sanitation. However, even today the disease still has a high fatality rate. In fact 4 in 5 cases we experienced had poor prognosis and one of them resulted in death despite the relatively early diagnosis. It shows the difficulty of diagnosis and treatment. When we see elderly patients with high fever, headache, and impaired consciousness in late summer and autumn, the important thing is to recognize the possibility of JE. Moreover it turned out that brain MRI and detecting serologic JE virus antibodies was very helpful for diagnosis and treatment. Nowadays we clinicians tend to consider JE as a disease of the past in Japan, however, this experience taught us that it is necessary for us to study JE again and to continue educating the public about it.

Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers.

We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining.

Simultaneous detection and genogroup-screening test for norovirus genogroups I and II from fecal specimens in single tube by reverse transcription- loop-mediated isothermal amplification assay.

We developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of noroviruses (NoVs) in a genogroup-specific manner in a previous study. In this study, to detect NoVs more easily and simply, we have developed an RT-LAMP assay for the simultaneous detection of NoV genogroup I (GI) and II (GII) genomes in a single tube. The genogrouping was achieved by using fluorescence-labeled primers, and the green and red colors for GI and GII, respectively, sometimes with yellow color for GI and GII mixture, were indicated on the agarose gel under UV light at 312 nm.

Molecular epidemiological analyses of Japanese encephalitis virus isolates from swine in Japan from 2002 to 2004.

To characterize Japanese encephalitis virus (JEV) strains recently prevalent in Japan, JEV surveillance was performed in pigs from 2002 to 2004. Eleven new JEV isolates were obtained and compared with previous isolates from Japan and other Asian countries. All of the isolates were classified into genotype 1 by nucleotide sequence analysis of the E gene. Two new isolates with different levels of neurovirulence and neuroinvasiveness, but with only one nucleotide difference in the E gene, Sw/Mie/34/2004 and Sw/Mie/40/2004, were isolated at the same farm on the same day. Sw/Mie/40/2004 displayed higher neurovirulence and neuroinvasiveness in mice than the other four new isolates. Another new isolate, Sw/Hiroshima/25/2002, was neutralized by antiserum to Beijing-1 at a level similar to the homologous Beijing-1 strain, whilst seven other new isolates were neutralized at 10-fold-lower titres. However, there were no amino acid differences in the E protein among these eight isolates. The present study indicated that the 11 new JEV isolates were genetically similar, but biologically and serologically heterogeneous.

Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay.

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 degrees C. The detection limits for NoV genomes were between 10(2) and 10(3) 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.

Cell-specific inhibition of paramyxovirus maturation by proteasome inhibitors.

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)-infected LLC-MK2 cells with 50 microM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose-dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV-like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC-MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell-specific and partly virus-specific.

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.

Paramyxovirus Sendai virus-like particle formation by expression of multiple viral proteins and acceleration of its release by C protein.

Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein-protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding.