Futoshi Hasebe

Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus

ABSTRACT A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 104 PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 102 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.

Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

ABSTRACT Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

West Nile virus-induced bax-dependent apoptosis

The mechanism of cell death induced by West Nile virus (WNV), a causative agent of human febrile syndrome and encephalitis, was investigated. WNV‐infected K562 and Neuro‐2a cells manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation and subdiploid DNA content by flow cytometry. DNA fragmentation into nucleosomal size and changes in outer cell membrane phospholipid composition were also observed in K562 cells. UV‐inactivated virus failed to induce the above‐mentioned characteristics, suggesting that viral replication may be required for the induction of apoptosis by WNV. Additionally, signals involved in WNV‐induced apoptosis are associated with the up‐regulation of bax gene expression.

Distribution of three arbovirus antibodies among monkeys (Macaca fascicularis) in the Philippines

Serum samples from 54 monkeys were collected from healthy individuals in a monkey farm in Luzon island, Philippines, in 1999, and examined by IgM‐capture ELISA and indirect IgG ELISA for the presence of dengue (DEN), Japanese encephalitis (JE) and chikungunya (CHIK) viruses. The positive rates for IgM ELISA were 3.7, 35.2 and 14.8% against DEN, JE and CHIK, respectively. Higher positive rates were obtained when indirect IgG ELISA was used: 100% against flaviviruses (JE or DEN) and 59.3% against CHIK virus. The results indicate a high prevalence of flavivirus infections such as JE and DEN, and a lesser prevalence of CHIK virus infections, among monkeys in the Philippines. These findings suggest possible sylvatic transmission cycles of these viruses.

Bla NDM-1-positive Klebsiella pneumoniae from environment, Vietnam

To the Editor: The blaNDM-1 gene, which produces the New Delhi metallo-β-lactamase (NDM-1) enzyme, confers resistance to the carbapenem class of antimicrobial drugs and can be transferred among different types of bacteria. NDM-1 was identified in 2008 in Sweden from a patient from India who had been hospitalized in New Delhi (1). Since that report, blaNDM-1–positive bacteria have been identified from patients in several countries; most of these patients had a direct link with the Indian subcontinent (2). The spread of blaNDM-1 among bacterial pathogens is of concern not only because of resistance to carbapenems but also because such pathogens typically are resistant to multiple antimicrobial drug classes, which leaves few treatment choices available (3–5). In 2011, spread of blaNDM-1–positive bacteria in an environmental setting in New Delhi was reported (6). The possible appearance of bacteria harboring blaNDM-1 in Vietnam is of concern because cultural and economic links between Vietnam and India are strongly established, including extensive person-to-person exchanges that could enable easy exchange of pathogens. In addition, Vietnam faces a serious problem of antimicrobial drug resistance because drugs are freely available and used in an indiscriminate fashion. Thus, once blaNDM-1–positive bacteria colonize persons in Vietnam, they would be able to spread easily and pose a serious public health threat. During September 2011, we collected paired swab samples (1 for PCR, 1 for culture) of seepage water from 20 sites (rivers, lakes, and water pools in streets) within a 10-km radius of central Hanoi, Vietnam. Samples were transported in Transystem (COPAN Italia S.p.A, Brescia, Italy) to preserve bacteria and DNA. The 20 PCR swab specimens were squeezed out into 0.5-mL volumes of sterile water and centrifuged at 3,000 × g for 30 seconds; 1 μL of the resulting suspension was then used as PCR template to detect blaNDM-1 as described (7). Two samples were positive for blaNDM-1; these 2 samples were collected from the same river (Kim Nguu River) but at sites 3 km apart. To isolate and identify the phenotype and genotype of blaNDM-1–positive bacteria, we repeatedly spread the 20 culture swab specimens onto Muller-Hinton agar (Nissui, Tokyo, Japan) containing 100 mg/L vancomycin (Nakalai, Kyoto, Japan) plus 0.5 mg/L meropenem (LKT Laboratories, St. Paul, MN, USA) until single colonies were obtained. Each colony was then subcultured by plating onto MacConkey agar (Nihon Seiyaku, Tokyo, Japan) containing 0.5 mg/L meropenem to ensure culture purity; colonies were identified by using API 20E strips (bioMerieux, Basingstoke, UK). MICs of these isolates for 13 antimicrobial drugs were calculated by using Etest (bioMerieux), and susceptibility data were interpreted by using Clinical and Laboratory Standards Institute guidelines (www.clsi.org). We harvested several species of bacteria from the 2 seepage samples positive for blaNDM-1: Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. fluorescens/putida, and P. luteola. These isolates were placed onto media containing 0.5 mg/L meropenem, and bacterial DNA was extracted and used for the template for PCR analysis to detect blaNDM-1 as described (7). blaNDM-1 was detected in 3 K. pneumoniae isolates from each of the 2 positive samples (6 isolates total); this result was confirmed by sequencing. All 6 isolates were highly resistant to all β-lactam antimicrobial drugs, including carbapenems (Table). To detect another β-lactamase, multiplex PCRs were carried out as described (8); genetic variants blaTEM, blaSHV, blaOXA, blaCTX-M, blaIMP, blaVIM, and blaKPC were not detected in any of the isolates other than K. pneumoniae. All 6 K. pneumoniae isolates were positive for blaTEM and blaCTX-M variants by PCR; these variants were confirmed as blaTEM-1 and blaCTX-M-3 by sequencing. Table Resistance to 13 antimicrobial drugs of blaNDM-1–positive Klebsiella pneumoniae isolates from the Kim Nguu River, Hanoi, Vietnam* Aminoglycosides are often used in the management of severe infectious diseases caused by gram-negative pathogens. 16S rRNA methylases were found to confer high levels of resistance to aminoglycosides such as amikacin, tobramycin, and gentamicin. The 6 K. pneumoniae isolates we found were highly resistant to gentamicin (MIC >1,024 mg/L) and tobramycin (MIC 256–>1,024 mg/L) (Table). Therefore, we screened genetic elements of 16S rRNA methylases (rmtB, rmtC, and armA) by PCR and detected rmtB in all 6 isolates (9). Multilocus sequence typing was applied for these 6 isolates; all were identified as K. pneumoniae sequence type 283 (10), which had not been reported as harboring blaNDM-1. The azide-resistant Escherichia coli strain J53 has been used as recipient for conjugation assay, which had been reported previously (6), but we found no transconjugant strain with blaNDM-1 on MacConkey agar containing 100 mg/L sodium azide and 0.5 mg/L meropenem. Our results show that blaNDM-1–positive K. pneumoniae sequence type 283 is present in the Kim Nguu River, which flows through the central part of Hanoi at 2 sites. The isolates we obtained were also positive for 2 other β-lactamases, blaTEM-1 and blaCTX-M-3, were highly resistant to aminoglycosides related to rmtB, and showed mild elevation of MIC against ciprofloxacin up to 1.5 mg/L. Wide-scale surveillance of environmental and clinical samples in Vietnam and establishment of a strategy to prevent further spread of blaNDM-1 are urgently needed.

Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein

The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His‐tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp‐285‐to‐Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C‐terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.

Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses

Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.

Evidence of frequent introductions of Japanese encephalitis virus from south-east Asia and continental east Asia to Japan

The Japanese encephalitis virus (JEV) circulating in Japan consists of viruses with multiple phylogenetic origins. Phylogenetic analysis revealed that some JEV strains have recently migrated from south-east and continental east Asian countries. One phylogenetic subcluster of the JEV strains circulating in Japan was closely related to viruses isolated in Vietnam and Chinas inland region while other JEV subclusters were related to viruses isolated in Shanghai, China. One virus subcluster, however, was isolated solely in Japan and was not found in any other Asian country. Therefore, our data suggests that the JEVs that have remained or are circulating in Japan include a mixture of viruses that have previously migrated from south-east and continental east Asian countries.

Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system

In order to localize denaturation-resistant neutralizing epitope(s) in the C-terminal 180 amino acids of Japanese encephalitis (JE) virus E-protein, four recombinant clones encoding different or overlapping nucleotide sequences were constructed by PCR from a recombinant plasmid pS22. The amplified fragments were cloned into PCR 1000 vector, and then transferred into Escherichia coli expression vector pRIT2T. The inserted genes were expressed as fusion proteins with protein-A and examined for their antigenicity and immunogenicity by Western blotting and mouse immunization, respectively. Among the four recombinant fusion proteins, the highest neutralizing antibody titre was obtained by the one expressed by the recombinant clone pRIT2T-B3, which carried the coding sequence of amino acid number 373-399 of JE virus E protein. The results indicated that this short region of 27 amino acids sequence near the C-terminal of JE virus E protein possesses neutralizing epitope(s). These data should assist in the design of an efficient subunit vaccine against JE virus infection in future.