Masafumi Sakata

Histidine at position 1042 of the p150 region of a KRT live attenuated rubella vaccine strain is responsible for the temperature sensitivity.

The Japanese live attenuated KRT rubella vaccine strain has a temperature sensitivity (ts) phenotype. The objective of this study is to identify the region responsible for this phenotype. Genomic sequences of the KRT strain and the wild-type strain (RVi/Matsue.JPN/68) with the non-ts phenotype were investigated and reverse genetic systems (RG) for these strains were developed. The ts phenotype of KRT varied drastically on replacement of the p150 gene (encoding a methyltransferase and a nonstructural protease). Analysis of four chimeric viruses showed the region responsible for the ts phenotype to be located between Bsm I and Nhe I sites (genome position 2803-3243). There were two amino acid differences at positions 1007 and 1042. Mutations were introduced into the KRT cDNA clone, designated G1007D, H1042Y and G1007D-H1042Y. H1042Y and G1007D-H1042Y grew well at a restrictive temperature with a 100-fold higher titer than G1007D and the KRT strain, but a 10-fold lower titer than RVi/Matsue.JPN/68. Since the growth of H1042Y was not completely the same as that of the wild-type strain at the restrictive temperature, we also assessed whether other genomic regions have an additive effect with H1042Y on the ts phenotype. H1042Y-RViM SP having structural proteins of RVi/Matsue.JPN/68 grew better than H1042Y, similar to RVi/Matsue.JPN/68. Thus, we concluded that one mutation, of the histidine at position 1042 of p150, was essential for the ts phenotype of the KRT strain, and structural proteins of KRT had an additive effect with H1042Y on the ts phenotype.

Mumps Virus Is Released from the Apical Surface of Polarized Epithelial Cells, and the Release Is Facilitated by a Rab11-Mediated Transport System

ABSTRACT Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients. In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection. IMPORTANCE Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV.

Short Self-interacting N-terminal Region of Rubella Virus Capsid Protein Is Essential for Cooperative Actions of Capsid and Nonstructural p150 Proteins

ABSTRACT Nucleocapsid formation is a primary function of the rubella virus capsid protein, which also promotes viral RNA synthesis via an unknown mechanism. The present study demonstrates that in infected cells, the capsid protein is associated with the nonstructural p150 protein via the short self-interacting N-terminal region of the capsid protein. Mutational analyses indicated that hydrophobic amino acids in this N-terminal region are essential for its N-terminal self-interaction, which is critical for the capsid-p150 association. An analysis based on a subgenomic replicon system demonstrated that the self-interacting N-terminal region of the capsid protein plays a key role in promoting viral gene expression. Analyses using a virus-like particle (VLP) system also showed that the self-interacting N-terminal region of the capsid protein is not essential for VLP production but is critical for VLP infectivity. These results demonstrate that the close cooperative actions of the capsid protein and p150 require the short self-interacting N-terminal region of the capsid protein during the life cycle of the rubella virus. IMPORTANCE The capsid protein of rubella virus promotes viral RNA replication via an unknown mechanism. This protein interacts with the nonstructural protein p150, but the importance of this interaction is unclear. In this study, we demonstrate that the short N-terminal region of the capsid protein forms a homo-oligomer that is critical for the capsid-p150 interaction. These interactions are required for the viral-gene-expression-promoting activity of the capsid protein, allowing efficient viral growth. These findings provide information about the mechanisms underlying the regulation of rubella virus RNA replication via the cooperative actions of the capsid protein and p150.

Protease and helicase domains are related to the temperature sensitivity of wild-type rubella viruses

Wild-type rubella viruses grow well at 39°C (non-temperature sensitivity: non-ts), while vaccine strains do not (temperature sensitivity: ts). Histidine at position 1042 of the p150 region of the KRT vaccine strain was found to be responsible for ts, while wild-type viruses had tyrosine at position 1042 (Vaccine 27; 234-42, 2009). The point-mutated virus (Y1042H) based on the wild-type unexpectedly showed little reduction in growth at 39°C. In this report, several recombinant viruses were characterized, and point-mutated Y1042H together with the p90 region of KRT significantly reduced virus growth, compared to the parental wild-type virus. There was one amino acid difference at position 1497 of the helicase domain in the p90 region. Double mutation involving both positions 1042 and 1497 markedly reduced virus growth at 39°C, but single substitution at 1497 did not. The other vaccine strain (TO-336vac) was investigated, and serine at position 1159 of the protease domain in p150 was a crucial amino acid for ts and non-ts characteristics among four amino acid substitutions between TO-336vac and the wild-type. Our results suggest that protease and helicase domains in non-structural protein were consistent with ts phenotype, possibly related to the attenuation process of wild-type viruses.

Development of an improved RT-LAMP assay for detection of currently circulating rubella viruses

Rubella virus is the causative agent of rubella. The symptoms are usually mild, and characterized by a maculopapular rash and fever. However, rubella infection in pregnant women sometimes can result in the birth of infants with congenital rubella syndrome (CRS). Global efforts have been made to reduce and eliminate CRS. Although a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of rubella virus has been reported, the primers contained several mismatched nucleotides with the genomes of currently circulating rubella virus strains. In the present study, a new RT-LAMP assay was established. The detection limit of this assay was 100-1000PFU/reaction of viruses for all rubella genotypes, except for genotype 2C, which is not commonly found in the current era. Therefore, the new RT-LAMP assay can successfully detect all current rubella virus genotypes, and does not require sophisticated devices like TaqMan real-time PCR systems. This assay should be a useful assay for laboratory diagnosis of rubella and CRS.

Molecular Epidemiology of Rubella Virus Strains Detected Around the Time of the 2012–2013 Epidemic in Japan

A nationwide rubella epidemic occurred from 2012 to 2013 in Japan, resulting in around 17,000 rubella cases and the birth of 45 infants with congenital rubella syndrome. The aim of this study was to genetically characterize the rubella viruses (RVs) circulating around the time of the epidemic in Japan. In total, 221 RV strains detected from 14 prefectures in Japan between 2010 and 2014 were sequenced in the 739 nucleotide-window region within the E1 gene. The virus strains were chronologically and geographically characterized into groups based on phylogenetic analysis. Among the 221 strains analyzed, 192 (87%), 26 (12%), and 3 (1%) strains were classified into genotypes 2B, 1E, and 1J, respectively. The majority (n = 184) of the genotype 2B strains belonged to lineage 2B-L1 and shared nucleotide homology with the strains detected in Southeast and East Asian countries. Phylogenetic analyses demonstrated that at least six distinct clusters of RV strains (clusters 1–6) induced outbreaks in Japan between 2010 and 2014. Among them, strains from clusters 3, 4, and 6 circulated almost simultaneously during 2012–2013. The cluster 3 strains circulated locally, whereas strains from cluster 4 spread nationwide. The findings suggest that RVs were introduced into Japan many times from neighboring countries. The 2012–2013 epidemic was a complex of outbreaks induced by at least three clusters of RV strains.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens

BACKGROUND An easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. OBJECTIVES To allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. STUDY DESIGN The detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. RESULTS The detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. CONCLUSIONS The TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination.

Analysis of VSV pseudotype virus infection mediated by rubella virus envelope proteins

Rubella virus (RV) generally causes a systemic infection in humans. Viral cell tropism is a key determinant of viral pathogenesis, but the tropism of RV is currently poorly understood. We analyzed various human cell lines and determined that RV only establishes an infection efficiently in particular non-immune cell lines. To establish an infection the host cells must be susceptible and permissible. To assess the susceptibility of individual cell lines, we generated a pseudotype vesicular stomatitis virus bearing RV envelope proteins (VSV-RV/CE2E1). VSV-RV/CE2E1 entered cells in an RV envelope protein-dependent manner, and thus the infection was neutralized completely by an RV-specific antibody. The infection was Ca2+-dependent and inhibited by endosomal acidification inhibitors, further confirming the dependency on RV envelope proteins for the VSV-RV/CE2E1 infection. Human non-immune cell lines were mostly susceptible to VSV-RV/CE2E1, while immune cell lines were much less susceptible than non-immune cell lines. However, susceptibility of immune cells to VSV-RV/CE2E1 was increased upon stimulation of these cells. Our data therefore suggest that immune cells are generally less susceptible to RV infection than non-immune cells, but the susceptibility of immune cells is enhanced upon stimulation.

Analysis of the temperature sensitivity of Japanese rubella vaccine strain TO-336.vac and its effect on immunogenicity in the guinea pig.

The marker of Japanese domestic rubella vaccines is their lack of immunogenicity in guinea pigs. This has long been thought to be related to the temperature sensitivity of the viruses, but supporting evidence has not been described. In this study, we generated infectious clones of TO-336.vac, a Japanese domestic vaccine, TO-336.GMK5, the parental virus of TO-336.vac, and their mutants, and determined the molecular bases of their temperature sensitivity and immunogenicity in guinea pigs. The results revealed that Ser(1159) in the non-structural protein-coding region was responsible for the temperature sensitivity of TO-336.vac dominantly, while the structural protein-coding region affected the temperature sensitivity subordinately. The findings further suggested that the temperature sensitivity of TO-336.vac affected the antibody induction in guinea pigs after subcutaneous inoculation.

Corrigendum to “Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens” [J. Clin. Virol. 80 (2016) 98–101]

orrigendum to “Evaluation of sensitivity of TaqMan RT-PCR for ubella virus detection in clinical specimens” J. Clin. Virol. 80 (2016) 98–101] iyoko Okamotoa, Yoshio Moria,∗, Rika Komagomeb, Hideki Naganob, Masahiro Miyoshib, otohiko Okanob, Yoko Aokic, Atsushi Ogurad, Chiemi Hottad, Tomoko Ogawad, iwako Saikusae, Hiroe Kodamaf, Yoshihiro Yasuig, Hiroko Minagawag, Takako Kuratah, aiki Kanbayashih, Tetsuo Kaseh, Sachiko Murata i, Komei Shirabe i, Mitsuhiro Hamasaki j, akashi Katok, Noriyuki Otsukia, Masafumi Sakataa, Katsuhiro Komasea, Makoto Takedaa