The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.     

Conserved cysteine-rich domain of paramyxovirus simian virus 5 V protein plays an important role in blocking apoptosis

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5VDeltaC) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5VDeltaC induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5VDeltaC, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5VDeltaC induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5VDeltaC was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5VDeltaC was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5VDeltaC-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5VDeltaC infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5VDeltaC can trigger cell death by inducing ER stress.     

Naturally occurring substitutions in the P/V gene convert the noncytopathic paramyxovirus simian virus 5 into a virus that induces alpha/beta interferon synthesis and cell death

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-alpha/beta) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI-) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-alpha/beta signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI- had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI- were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI- mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI- but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-beta promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI- but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI- mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI- growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI- by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI- induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-alpha/beta synthesis, and virus-induced apoptosis.     

Discrimination of mumps virus small hydrophobic gene deletion effects from gene translation effects on virus virulence

Deletion of the small hydrophobic (SH) protein of certain paramyxoviruses has been found to result in attenuation, suggesting that the SH protein is a virulence factor. To investigate the role of the mumps virus (MuV) SH protein in virulence, multiple stop codons were introduced into the open reading frame (ORF) of a MuV molecular clone (r88-1961(SHstop)), preserving genome structure but precluding production of the SH protein. No differences in neurovirulence were seen between the wild-type and the SH(stop) viruses. In contrast, upon deletion of the SH gene, significant neuroattenuation was observed. These data indicate that the MuV SH protein is not a neurovirulence factor and highlight the importance of distinguishing gene deletion effects from protein-specific effects.     

Function of the small hydrophobic protein of J paramyxovirus

At 18,954 nucleotides, the J paramyxovirus (JPV) genome is one of the largest in the family Paramyxoviridae, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. To study the function of novel paramyxovirus genes in JPV, a plasmid containing a full-length cDNA clone of the genome of JPV was constructed. In this study, the function of the small hydrophobic (SH) protein of JPV was examined by generating a recombinant JPV lacking the coding sequence of the SH protein (rJPVΔSH). rJPVΔSH was viable and had no growth defect in tissue culture cells. However, more tumor necrosis factor alpha (TNF-α) was produced during rJPVΔSH infection, suggesting that SH plays a role in inhibiting TNF-α production. rJPVΔSH induced more apoptosis in tissue culture cells than rJPV. Virus-induced apoptosis was inhibited by neutralizing antibody against TNF-α, suggesting that TNF-α contributes to JPV-induced apoptosis in vitro. The expression of JPV SH protein inhibited TNF-α-induced NF-κB activation in a reporter gene assay, suggesting that JPV SH protein can inhibit TNF-α signaling in vitro. Furthermore, infection of mice with rJPVΔSH induced more TNF-α expression, indicating that SH plays a role in blocking TNF-α expression in vivo.     

A simian virus 5 (SV5) P/V mutant is less cytopathic than wild-type SV5 in human dendritic cells and is a more effective activator of dendritic cell maturation and function

Human epithelial cells infected with the parainfluenza virus simian virus 5 (SV5) show minimal activation of host cell interferon (IFN), cytokine, and cell death pathways. In contrast, a recombinant SV5 P/V gene mutant (rSV5-P/V-CPI-) overexpresses viral gene products and is a potent inducer of IFN, proinflammatory cytokines, and apoptosis in these cells. In this study, we have compared the outcomes of wild-type (WT) SV5 and rSV5-P/V-CPI- infections of primary human dendritic cells (DC), important antigen-presenting cells for initiating adaptive immune responses. We have tested the hypothesis that a P/V mutant which activates host antiviral responses will be a more potent inducer of DC maturation and function than WT rSV5, which suppresses host cell responses. Infection of peripheral blood mononuclear cell-derived immature DC with WT rSV5 resulted in high levels of viral protein and progeny virus but very little increase in cell surface costimulatory molecules or secretion of IFN and proinflammatory cytokines. In contrast, immature DC infected with the rSV5-P/V-CPI- mutant produced only low levels of viral protein and progeny virus, but these infected cells were induced to secrete IFN-alpha and other cytokines and showed elevated levels of maturation markers. Unexpectedly, DC infected with WT rSV5 showed extensive cytopathic effects and increased levels of active caspase-3, while infection of DC with the P/V mutant was largely noncytopathic. In mixed-culture assays, WT rSV5-infected DC were impaired in the ability to stimulate proliferation of autologous CD4+ T cells, whereas DC infected with the P/V mutant were very effective at activating T-cell proliferation. The addition of a pancaspase inhibitor to DC infected with WT rSV5 reduced cytopathic effects and resulted in higher surface expression levels of maturation markers. Our finding that the SV5 P/V mutant has both a reduced cytopathic effect in human DC compared to WT SV5 and an enhanced ability to induce DC function has implications for the rational design of novel recombinant paramyxovirus vectors based on engineered mutations in the viral P/V gene.     

Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.     

Gene therapy using SV40-derived vectors: what does the future hold?

Effective genetic therapy requires both a fragment of genetic material to be used therapeutically and a means to deliver it. We began to study simian virus-40 (SV40) as a vector for gene transfer because available gene delivery vehicles did not provide for the full range of therapeutic uses. Other vectors are variably limited by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc. In theory recombinant viral vectors based on SV40 (rSV40) should not, on the other hand, be similarly constrained. rSV40 vectors are easily manipulated and produced at very high titer, stable, lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in both resting and dividing cells. The principle limitation of SV40-derived vectors is the size of the packageable insert (相似文献   

Simian virus-40 as a gene therapy vector

Simian virus-40 (SV40), an icosahedral papovavirus, has recently been modified to serve as a gene delivery vector. Recombinant SV40 vectors (rSV40) are good candidates for gene transfer, as they display some unique features: SV40 is a well-known virus, nonreplicative vectors are easy-to-make, and can be produced in titers of 10(12) IU/ml. They also efficiently transduce both resting and dividing cells, deliver persistent transgene expression to a wide range of cell types, and are nonimmunogenic. Present disadvantages of rSV40 vectors for gene therapy are a small cloning capacity and the possible risks related to random integration of the viral genome into the host genome. Considerable efforts have been devoted to modifing this virus and setting up protocols for viral production. Preliminary therapeutic results obtained both in tissue culture cells and in animal models for heritable and acquired diseases indicate that rSV40 vectors are promising gene transfer vehicles. This article reviews the work performed with SV40 viruses as recombinant vectors for gene transfer. A summary of the structure, genomic organization, and life cycle of wild-type SV40 viruses is presented. Furthermore, the strategies utilized for the development, production, and titering of rSV40 vectors are discussed. Last, the therapeutic applications developed to date are highlighted.     

Long-term gene expression in dividing and nondividing cells using SV40-derived vectors

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tag-deleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.     

Recombinant human Metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate

Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The DeltaSH, DeltaG, and DeltaSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the DeltaG and DeltaSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the DeltaG and DeltaSH/G viruses represent promising vaccine candidates. In contrast, DeltaSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.     

The F gene of rodent brain-adapted mumps virus is a major determinant of neurovirulence

Prior to the introduction of live-attenuated vaccines, mumps virus (MuV) was the leading cause of virus-induced meningitis. Although vaccination has been effective at controlling the disease, the use of insufficiently attenuated strains has been associated with high rates of aseptic meningitis in vaccinees. The molecular basis of MuV attenuation is poorly understood, and no reliable molecular markers of virulence have been identified. In this study, reverse genetics has been used to identify molecular determinants of MuV neuropathogenesis. Recombinant viruses, containing the envelope-associated genes from the Kilham (MuV(KH)) rodent brain-adapted strain of MuV, were generated in the Jeryl Lynn 5 (MuV(JL5)) vaccine strain background. The syncytium phenotypes of the recombinant viruses on Vero cells differed depending on the source of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, with heterologous combinations showing either an increase or a decrease in the level of cell fusion compared to that of the homologous parental combinations. This was confirmed by transiently cotransfecting eukaryotic F and HN glycoprotein expression constructs. A Lewis rat model that discriminates between neurovirulent and nonneurovirulent MuV strains based on the extent of hydrocephalus induced in the rat brain after intracerebral inoculation was used to assess the phenotype of the recombinant viruses. Expression of the matrix (M), small hydrophobic (SH), or HN gene in isolation did not confer a neurovirulent phenotype. Expression of the F gene of the neurovirulent strain alone was sufficient to induce significant levels of hydrocephalus. Coexpression of the homologous HN gene led to a marginal increase in the level of hydrocephalus.     

Delivering genes to the organ-localized immune system: long-term results of direct intramarrow transduction

We studied the distribution of transgene-expressing cells after direct gene transfer into the bone marrow (BM). Rats received direct injection into the femoral BM of SV(Nef-FLAG), a Tag-deleted recombinant SV40 carrying a marker gene (FLAG epitope). Controls received an unrelated rSV40 or saline. Blood cells (5%) and femoral marrow cells (25%) expressed FLAG throughout. FLAG expression was assessed in different organs at 1, 4 and 16 months. FLAG+ macrophages were seen throughout the body, and were prominent in the spleen. FLAG+ cells were common in pulmonary alveoli. The former included alveolar macrophages and type II pneumocytes. These cells were not detected at 1 month, occasional at 4 months and common at 16 months after intramarrow injection. Rare liver cells were positive for both FLAG and ferritin, indicating that some hepatocytes also expressed this BM-delivered transgene. Control animals were negative. Thus: (a) fixed tissue phagocytes may be accessible to gene delivery by intramarrow transduction of their progenitors; (b) transduced BM-resident cells or their derivatives may migrate to other organs (lungs) and may differentiate into epithelial cells; and (c) intramarrow injection of rSV40s does not detectably transduce parenchymal cells of other organs.     

EGF-dependent association of phospholipase C-gamma1 with c-Cbl

The structure of phospholipase Cgamma1 (PLC-gamma1) contains two SH2 domains and one SH3 domain. While the function of the SH2 domains in PLC-gamma1 are well described, to date no growth factor-dependent function for the SH3 domain has been presented. To assess SH3 domain function in the context of the full-length PLC-gamma1, this domain was deleted and the mutant was stably expressed in Plcg1 null mouse embryonic fibroblasts. Following EGF treatment of cells, the PLC-gamma1DeltaSH3 mutant displayed the same increased level of tyrosine phosphorylation and association with EGF receptor as wild-type PLC-gamma1. Also, the SH3 mutant demonstrated membrane translocation and mediated the mobilization of intracellular Ca(2+) in response to EGF. c-Cbl is shown to associate with tyrosine phosphorylated PLC-gamma1 in an EGF-dependent manner, but no association was detected with the PLC-gamma1DeltaSH3 mutant. Interestingly, PDGF, which also tyrosine phosphorylates PLC-gamma1, failed to induce c-Cbl association with PLC-gamma1 and also provoked no c-Cbl tyrosine phosphorylation. This suggests that c-Cbl tyrosine phosphorylation is necessary for its interaction with PLC-gamma1. Evidence of a direct association of c-Cbl with PLC-gamma1 was provided by pull-down and overlay experiments, using glutathione S-transferase fusion proteins that contain the SH3 domain of PLC-gamma1. The data, therefore, show an EGF-inducible direct association of PLC-gamma1 with c-Cbl in vivo that is mediated by the SH3 domain of PLC-gamma1.     

Genotyping of Mumps viruses based on SH gene: Development of a server using alignment-free and alignment-based methods

Mumps is an acute infectious childhood disease caused by mumps virus (MuV), a member of genus Rubu-lavirus, family Paramyxoviridae. Based on the genetic variability in small hydrophobic (SH) genes, currently MuVs have been divided into twelve confirmed genotypes designated as A-L and one proposed genotype, M. Despite successful vaccination program, a few genotypes are observed to co-circulate amongst vaccinated population. Furthermore, lack of cross protection between different genotypes is reported and hence, as a part of epidemiological surveillance, WHO has recommended genotyping of MuV. Currently genotyping is carried out using molecular phylogeny analysis (MPA) of SH genes and no genotyping server is available for MuV. The present study reports development of a genotyping server for the same, which employs three independent methods. The server uses two conventional methods viz., BLAST, MPA and a novel method based on Return Time Distribution (RTD), which is developed in-house. A server for genotyping of mumps virus is developed and made available at http://bioinfo.net.in/muv/homepage.html. RTD-based alignment-free method was initially developed for MPA and is applied for genotyping of MuV for the first time. It is found to have 98.95% of accuracy when measured using leave-one-out cross validation method on reference and test datasets. In addition to RTD, the server also imple-ments BLAST and MPA for genotyping of MuV. All the three methods were found to be highly reliable as evident from consensus predictions. A server for genotyping of MuV, which implements sequence-based bioinformatics approaches is developed and validated using SH gene sequences of known genotypes. This server will be useful for epidemiological surveillance and to monitor the circulation of MuV genotypes within and across geographic areas. This will also facilitate phylodynamics studies of mumps viruses.     

Simian virus 5 is a poor inducer of chemokine secretion from human lung epithelial cells: identification of viral mutants that activate interleukin-8 secretion by distinct mechanisms

We have compared chemokine secretion from human lung A549 cells infected with simian virus 5 (SV5) with other members of the Rubulavirus genus of paramyxoviruses. High levels of the chemokines interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were secreted from A549 cells infected with Human parainfluenza virus type 2 (HPIV-2) but not from cells infected with wild-type (WT) SV5. The lack of IL-8 secretion from SV5-infected cells was not due to a global block in all signal transduction pathways leading to IL-8 secretion, since SV5-infected A549 cells secreted IL-8 after stimulation with exogenously added tumor necrosis factor alpha or by coinfection with HPIV-2. A previously described, recombinant SV5 containing substitutions in the shared region of the P/V gene (rSV5-P/V-CPI-) induced IL-8 secretion by a mechanism that was dependent on viral gene expression. By contrast, an SV5 variant isolated from persistently infected cells (Wake Forest strain of Canine parainfluenza virus) induced IL-8 secretion by a mechanism that was largely not affected by inhibitors of viral gene expression. Together, these data demonstrate that SV5 is unusual compared to other closely related paramyxoviruses, since SV5 is a very poor inducer of the cytokines IL-8 and MCP-1. The isolation of two recombinant SV5 mutants that are defective in preventing chemokine induction will allow an identification of mechanisms utilized by WT SV5 to avoid activation of host cell innate immune responses to infection.     

Characterization of the F gene of contemporary mumps virus strains isolated in Japan

Mumps virus (MuV) strains isolated in Saitama Prefecture, Japan, from 1997 to 2001, were examined by analyzing the SH and the F gene nucleotide sequences. The results of the SH gene analysis showed that only genotype G was found in 2001 as well as in 2000, and that genotype J, which we proposed as a new genotype in a previous study, was from a different lineage than the genotype J described by Tecle et al. (J. Gen. Virol. 82, 2675-2680). We therefore, propose to rename the genotype as K to avoid confusion. Then, the F gene of genotypes G, H, and K strains were analyzed together with previously reported strains in this study. The results of phylogenetic analysis of the F gene nucleotide sequences showed that these strains formed a cluster as described by the SH gene analysis. Alignment of the F amino acid sequences showed that the F protein was well conserved among strains of different genotypes with a few amino acid differences. These results provide better information for the characterization of contemporary MuV strains in Japan.