Hirotaka Takagi

Development and application of a broadly reactive real-time reverse transcription-PCR assay for detection of murine noroviruses.

Murine norovirus (MNV) is a viral agent newly identified in laboratory mice and a large number of genetically diverse MNV strains have been reported to date. A broadly reactive TaqMan-based real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay was developed for MNVs. Novel primers and a TaqMan MGB probe were designed targeting highly conserved sequences among MNV strains, which are located in the open reading frames 1 (ORF1)-ORF2 junction region. The quantitative range of this assay was determined as 1.0 × 10(2)-1.0 × 10(8)copies/PCR tube based on a 10-fold serial dilution of plasmid DNA containing the target sequences. Viral RNA in eight murine stool specimens positive by nested RT-PCR assay was measured, and the highest viral RNA load was calculated at 4.7 × 10(6)copies/g-stool. MNV was inoculated into RAW 264.7 cells, and the viral RNA was monitored to validate assay sensitivity. MNV-RNA in the supernatant was detected during in vitro replication, which increased substantially from 5 to 30 h post-infection (hpi) and reached more than 1.0 × 10(10)copies/mL at 96 hpi. This real-time RT-PCR assay is a useful tool to detect and quantify MNV-RNA in in vivo and in vitro studies.

Amplification of all 11 RNA segments of group A rotaviruses based on reverse transcription polymerase chain reaction

Group A rotaviruses (RVA) are a major cause of acute infantile gastroenteritis. The viral genome comprises 11 double‐stranded RNA segments and the respective gene segments are classified into more than eight genotypes, according to the nucleotide sequence similarities. So far, it has been difficult to amplify full‐length sequences of long RNA segments of rotaviruses by one‐time only RT‐PCR (especially in the genes for the viral proteins VP1, VP2, VP3 and VP4). In this study, a set of universal primers to amplify all 11 segments of RVA was designed by aligning the nucleotide sequences of the typical rotavirus strains. Using these primers and a high‐fidelity and rapid DNA polymerase in a one‐step reverse transcription polymerase chain reaction, almost the entire length of all 11 segments of the seven rotavirus strains Wa, DS‐1, Hochi, 69M, WI61, M37 and SA11‐S1 were accurately and rapidly amplified. In addition, all 11 segments of rotavirus obtained from a fecal specimen were successfully amplified. In conclusion, the method described here will be useful as an RVA detection system and protocol for complete analysis of the 11 genome sequences.

Highly Conserved Configuration of Catalytic Amino Acid Residues among Calicivirus-Encoded Proteases

ABSTRACT A common feature of caliciviruses is the proteolytic processing of the viral polyprotein catalyzed by the viral 3C-like protease encoded in open reading frame 1 (ORF1). Here we report the identification and structural characterization of the protease domains and amino acid residues in sapovirus (SaV) and feline calicivirus (FCV). The in vitro expression and processing of a panel of truncated ORF1 polyproteins and corresponding mutant forms showed that the functional protease domain is 146 amino acids (aa) in SaV and 154 aa in FCV. Site-directed mutagenesis of the protease domains identified four amino acid residues essential to protease activities: H31, E52, C116, and H131 in SaV and H39, E60, C122, and H137 in FCV. A computer-assisted structural analysis showed that despite high levels of diversity in the primary structures of the protease domains in the family Caliciviridae, the configurations of the H, E, C, and H residues are highly conserved, with these residues positioned closely along the inner surface of the potential binding cleft for the substrate. These results strongly suggest that the H, E, C, and H residues are involved in the formation of a conserved catalytic surface of the SaV and FCV 3C-like proteases.

Protective Immunity Afforded by Inactivated H5N1 (NIBRG-14) Vaccine Requires Antibodies against Both Hemagglutinin and Neuraminidase in Mice

BACKGROUND Hemagglutination-inhibition (HI) antibody titers correlate with protective immunity to seasonal influenza viruses. However, inactivated H5N1 influenza vaccines from Vietnam 2004 strains afford protection without producing high or even detectable HI antibodies. METHODS BALB/c mice were immunized twice (at a 3-week interval) with inactivated whole-virus influenza vaccine produced using reverse genetics, with the internal genes of A/PR/8/34 (a high-yield strain) and the hemagglutinin (HA) and neuraminidase (NA) genes of A/Vietnam/1194/04 (H5N1) virus (NIBRG-14) adjuvanted with alum (5 microg of HA). Either HA- or NA-binding antibodies were absorbed from the immune serum. The protective efficacy of these antibodies was determined by injecting the absorbed serum into severe combined immunodeficiency mice, which were then challenged with highly pathogenic H5N1 virus (A/Vietnam/Jp1203/2004; Japanese isolate of A/Vietnam/1203/2004). RESULTS The NIBRG-14 vaccine elicited levels of anti-HA antibodies similar to levels elicited by the H1N1 vaccines, as well as levels of anti-NA antibodies higher than those elicited by the H1N1 vaccines. The absorption of either anti-HA or anti-NA antibody from immune serum samples obtained from NIBRG-14-vaccinated mice significantly reduced the protective efficacy of the serum. CONCLUSIONS For NIBRG-14 vaccines to confer protection to vaccinated hosts, both anti-HA and anti-NA antibodies are required. This finding implies that the measurement of both antibody levels may be required for accurate evaluation of vaccine efficacy.

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV.

Abstract In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.

Bioluminescence technologies to detect calicivirus protease activity in cell-free system and in infected cells

Abstract Feline calicivirus (FCV) is an important veterinary pathogen and causes respiratory disease in cats. Because it grows well in cell culture, FCV is often used as a model virus of non-culturable caliciviruses. In this study, a cell-free and two cell culture-based biosensor assay systems were established to detect FCV protease activity. The assays utilize luciferase sensor technology or second-generation bioluminescence resonance energy transfer (BRET2). A luciferase sensor was designed to contain an FCV protease cleavage motif within the permutated luciferase (GloSensor). The BRET2-based probe contained the same cleavage motif flanked by a renilla luciferase and a variant of green fluorescent protein. To confirm the specificity of these assay systems, GloSensor or a BRET2-based probe containing a mutation in the cleavage motif was also constructed. In a cell-free assay, GloSensor showed increased luminescence in proportion to the amount of FCV protease, while no signal change was observed when the construct harboring the mutant cleavage motif was used. A feline cell line stably expressing GloSensor or the BRET2-based probe was established. Increased levels of GloSensor luminescence, and decreased levels of BRET2 signals were observed according to input FCV titers. In contrast, no significant signal change was observed in the cells stably expressing the mutant cleavage motif. GloSensor and the BRET2-based probe were capable of detecting the inhibitory activity of ribavirin in FCV-infected cells. Our results demonstrate that these biosensors are useful to detect FCV protease activity induced in infected cells, and well worth consideration for screening of anti-FCV protease compounds in cell-free system as well as anti-FCV compounds in cultured cells.

Effect of hypochlorite-based disinfectants on inactivation of murine norovirus and attempt to eliminate or prevent infection in mice by addition to drinking water.

We evaluated the in vitro efficacy of weak acid hypochlorous solution (WAHS) against murine norovirus (MNV) by plaque assay and compared the efficacy with diluted NaOCl (Purelox) and 70% ethanol. WAHS was as effective as 70% ethanol and diluted Purelox for 0.5-min reactions. For 0.5-min reactions in the presence of mouse feces emulsion, the efficacy of WHAS and 1:600 diluted Purelox was decreased, reducing the virus titers by 2.3 and 2.6 log10, respectively, while 70% ethanol reduced the titer by more than 5 log10. However, WAHS showed more than 5 log10 reductions for the 5-min reaction even in the presence of feces emulsion. Since WAHS showed enough efficacy in inactivating MNV in vitro, we tried to eliminate MNV from MNV-infected mice by substituting WAHS for their drinking water. However, MNV was found to be positive in feces of mice drinking WAHS by an RT-nested PCR and plaque assay. To investigate whether hypochlorite-based disinfectants could prevent infection of a mouse with MNV, WAHS or 1:6,000 diluted Purelox was substituted for the drinking water of mice for 2 or 4 weeks, and then the mice were placed in a cage with an MNV-infected mouse. The supply of disinfectants was continued after cohabitation, but MNV was detected in the feces of all the mice at 1 week after cohabitation. In this study, we tried to eliminate and prevent MNV infection from mice by supplying hypochlorite-based disinfectants as an easy and low-cost method. Unfortunately, drinking disinfectants was ineffective, so it is important to keep the facility environment clean by use of effective disinfectants. Also, animals introduced into facilities should be tested as MNV free by quarantine and periodically confirmed as MNV free by microbiological monitoring.

Complete Genome Sequence of the Feline Calicivirus 2280 Strain from the American Tissue Culture Collection

ABSTRACT Feline calicivirus (FCV) is a highly contagious pathogen of cats that can be grown in cultured cells. FCV is used as a model to study nonculturable caliciviruses, such as noroviruses. We determined the complete genome sequence of the FCV 2280 strain from the American Tissue Culture Collection.

Antiviral effect of theaflavins against caliciviruses

Caliciviruses are contagious pathogens of humans and various animals. They are the most common cause of viral gastroenteritis in humans, and can cause lethal diseases in domestic animals such as cats, rabbits and immunocompromised mice. In this study, we conducted cytopathic effect-based screening of 2080 selected compounds from our in-house library to find antiviral compounds against three culturable caliciviruses: feline calicivirus, murine norovirus (MNV) and porcine sapovirus (PoSaV). We identified active six compounds, of which two compounds, both related to theaflavins, showed broad antiviral activities against all three caliciviruses; three compounds (abamectin, a mixture of avermectin B1a and B1b; avermectin B1a; and (−)-epigallocatechin gallate hydrate) were effective against PoSaV only; and a heterocyclic carboxamide derivative (BFTC) specifically inhibited MNV infectivity in cell cultures. Further studies of the antiviral mechanism and structure-activity relationship of theaflavins suggested the following: (1) theaflavins worked before the viral entry step; (2) the effect of theaflavins was time- and concentration-dependent; and (3) the hydroxyl groups of the benzocycloheptenone ring were probably important for the anti-calicivirus activity of theaflavins. Theaflavins could be used for the calicivirus research, and as potential disinfectants and antiviral reagents to prevent and control calicivirus infections in animals and humans.

Viral Population Changes during Murine Norovirus Propagation in RAW 264.7 Cells

Laboratory adaptation of viruses is an essential technique for basic virology research, including the generation of attenuated vaccine strains, although the principles of cell adaptation remain largely unknown. Deep sequencing of murine norovirus (MuNoV) S7 during serial passages in RAW264.7 cells showed that the frequencies of viral variants were altered more dynamically than previously reported. Serial passages of the virus following two different multiplicity of infections gave rise to distinct haplotypes, implying that multiple cell-adaptable sequences were present in the founder population. Nucleotide variants lost during passage were assembled into a viral genome representative of that prior to cell adaptation, which was unable to generate viral particles upon infection in cultured cells. In addition, presence of the reconstructed genome interfered with production of infectious particles from viruses that were fully adapted to in vitro culture. Although the key nucleotide changes dictating cell adaptation of MuNoV S7 viral infection are yet to be elucidated, our results revealed the elaborate interplay among selected sequences of viral variants better adapted to propagation in cell culture. Such knowledge will be instrumental in understanding the processes necessary for the laboratory adaptation of viruses, especially to those without relevant cell culture systems.