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  • Outbreak of human metapneumovirus detected by use of the Vero E6 cell line in isolates collected in Yamagata, Japan, in 2004 and 2005. 17460056

    A number of epidemiological studies have shown human metapneumovirus (hMPV) to be one of the most important viral agents associated with acute respiratory infections in humans. However, due to the difficulty in growing the virus, all epidemiological studies of hMPV infection have been performed on the basis of the molecular method. Thus, the development of a cell line suitable for the isolation of hMPV from clinical specimens is a crucial step for further research. Using the Vero E6 cell line, which could be stably maintained for 1 month without passage or medium change, we succeeded in isolating 79 strains from 4,112 specimens obtained in Yamagata, Japan, in 2004 and 2005. The total isolation rate was 1.9% (79/4,112). The monthly distribution revealed that hMPV infections occurred between February and April in 2004 and throughout most of the year in 2005. Phylogenetic analysis indicated that subgenogroup B2 was predominant in 2004, whereas three subgenogroups, A2, B1, and B2, had cocirculated in 2005. Although multiple subgenogroups cocirculated in 2005, each individual subgenogroup strain was found to predominate at specific sites. An infectivity assay of hMPV strains also indicated that the infection efficiency in Vero E6 cells was better than that in LLC-MK2 cells. Finally, we found that Vero E6 cells are useful for the isolation of hMPVs and that this utility might aid further research into hMPVs beyond the epidemiological data shown in this study.
    Document Type:
    Reference
    Product Catalog Number:
    AP108P
    Product Catalog Name:
    Goat Anti-Guinea Pig IgG Antibody, HRP conjugate

  • Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system. 21603631

    Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was greater than 10(6) TCID(50)/mL by 6 days post infection when a MOI of 10(-5) was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24-28% sucrose fractions had an icosahedral structure 30-31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35-38% sucrose were 33-35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211-225).These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.
    Document Type:
    Reference
    Product Catalog Number:
    MAB979
    Product Catalog Name:
    Anti-Enterovirus 71 Antibody, cross-reacts with Coxsackie A16, clone 422-8D-4C-4D

  • Isolation of measles virus from clinical specimens using B95a and Vero/hSLAM cell-lines. 19852319

    The clinical presentation of acute measles is normally quite typical, especially in the presence of Koplik's spots, that laboratory test is seldom required to confirm the diagnosis. However, with wide measles vaccination coverage and the extensive use of immunosuppressive chemotherapy, the diagnosis of atypical manifestations of acute measles may require laboratory confirmation. When compared with B95a cell-line, this study shows that the Vero/hSLAM cell-line is sensitive and is recommended for use in the primary isolation of wild-type measles virus from clinical specimens. Throat swab and urine specimens are the clinical specimens of choice and both are recommended for optimal isolation of measles virus from patients suspected of acute measles virus infection.
    Document Type:
    Reference
    Product Catalog Number:
    5030

  • Enterovirus 71 infection induces apoptosis in Vero cells. 16196375

    The effects of Enterovirus 71 (HEV71) infection on African green monkey kidney cells (Vero) were investigated. It was found that the infected cells showed progressive cellular morphological changes characteristic in apoptotic cells within 10 hours post-infection. The number of apoptotic cells correlated significantly with the number of HEV71 antigen positive cells when cells were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and stained for HEV71 antigen. Approximately 11, 26, 45 and 50% of the infected cells were apoptotic at 12, 24, 48 and 72 hours post-infection, respectively. Internucleosomal DNA fragmentation, characteristic in the late stage of apoptosis was noted beginning on day 2 post-infection. The DNA fragmentation, however, was absent in cells treated with the heat- and ultraviolet light-inactivated virus inocula. These results demonstrate the capacity of HEV71 to induce apoptosis in the infected cells. The induction, however, requires high level of HEV71 infectivity and the presence of live virus particles, suggesting the need for the presence of specific viral proteins for apoptosis to occur.
    Document Type:
    Reference
    Product Catalog Number:
    3324

  • The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. 2501082

    Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily. We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.
    Document Type:
    Reference
    Product Catalog Number:
    14-118

  • Characterization of a novel embryonic stem cell line from an ICSI-derived blastocyst in the African green monkey. 19955206

    Several cell types from the African green monkey (Cercopithecus aethiops), such as red blood cells, primary culture cells from kidney, and the Vero cell line, are valuable sources for biomedical research and testing. Embryonic stem (ES) cells that are established from blastocysts have pluripotency to differentiate into these and other types of cells. We examined an in vitro culture system of zygotes produced by ICSI in African green monkeys and attempted to establish ES cells. Culturing with and without a mouse embryonic fibroblast (MEF) cell monolayer resulted in the development of ICSI-derived zygotes to the blastocyst stage, while culturing with a buffalo rat liver cell monolayer yielded no development (3/14, 21.4% and 6/31, 19.4% vs 0/23, 0% respectively; P0.05). One of the nine blastocysts, which had been one of the zygotes co-cultured with MEF cells, formed flat colonies consisting of cells with large nuclei, similar to other primate ES cell lines. The African green monkey ES (AgMES) cells expressed pluripotency markers, formed teratomas consisting of three embryonic germ layer tissues, and had a normal chromosome number. Furthermore, expression of the germ cell markers CD9 and DPPA3 (STELLA) was detected in the embryoid bodies, suggesting that AgMES cells might have the potential ability to differentiate into germ cells. The results suggested that MEF cells greatly affected the quality of the inner cell mass of the blastocysts. In addition, AgMES cells would be a precious resource for biomedical research such as other primate ES cell lines.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Zaire Ebola virus entry into human dendritic cells is insensitive to cathepsin L inhibition. 19775255

    Cathepsins B and L contribute to Ebola virus (EBOV) entry into Vero cells and mouse embryonic fibroblasts. However, the role of cathepsins in EBOV-infection of human dendritic cells (DCs), important targets of infection in vivo, remains undefined. Here, EBOV-like particles containing a beta-lactamase-VP40 fusion reporter and Ebola virus were used to demonstrate the cathepsin dependence of EBOV entry into human monocyte-derived DCs. However, while DC infection is blocked by cathepsin B inhibitor, it is insensitive to cathepsin L inhibitor. Furthermore, DCs pre-treated for 48 h with TNFalpha were generally less susceptible to entry and infection by EBOV. This decrease in infection was associated with a decrease in cathepsin B activity. Thus, cathepsin L plays a minimal, if any, role in EBOV infection in human DCs. The inflammatory cytokine TNFalpha modulates cathepsin B activity and affects EBOV entry into and infection of human DCs.
    Document Type:
    Reference
    Product Catalog Number:
    AB3738

  • Comparison of different cell substrates on the measurement of human influenza virus neutralizing antibodies. 23284988

    Eight cell lines were systematically compared for their permissivity to primary infection, replication, and spread of seven human influenza viruses. Cell lines were of human origin (Caco-2, A549, HEp-2, and NCI-H292), monkey (Vero, LLC-MK2), mink (Mv1 Lu), and canine (MDCK). The influenza viruses included seasonal types and subtypes and a pandemic virus. The MDCK, Caco-2, and Mv1 Lu cells were subsequently compared for their capacity to report neutralization titers at day one, three and six post-infection. A gradient of sensitivity to primary infection across the eight cell lines was observed. Relative to MDCK cells, Mv1 Lu reported higher titers and the remaining six cell lines reported lower titers. The replication and spread of the seven influenza viruses in the eight cell substrates was determined using hemagglutinin expression, cytopathic effect, and neuraminidase activity. Virus growth was generally concordant with primary infection, with a gradient in virus replication and spread. However, Mv1 Lu cells poorly supported virus growth, despite a higher sensitivity to primary infection. Comparison of MDCK, Caco-2, and Mv1 Lu in neutralization assays using defined animal antiserum confirmed MDCK cells as the preferred cell substrate for influenza virus testing. The results observed for neutralization at one day post-infection showed MDCK cells were similar (less than 1 log(2) lower) or superior (greater than 1 log(2) higher) for all seven viruses. Relative to Caco-2 and Mv1 Lu cells, MDCK generally reported the highest titers at three and six days post-infection for the type A viruses and lower titers for the type B viruses and the pandemic H9N2 virus. The reduction in B virus titer was attributed to the complete growth of type B viruses in MDCK cells before day three post-infection, resulting in the systematic underestimation of neutralization titers. This phenomenon was also observed with Caco-2 cells.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8671
    Product Catalog Name:
    Anti-Influenza B Antibody, Core, clone 22D5-12-13

  • Full-length sequence analysis of subacute sclerosing panencephalitis (SSPE) virus, a mutant of measles virus, isolated from brain tissues of a patient shortly after onset ... 16858143

    Subacute sclerosing panencephalitis (SSPE) virus, a measles virus (MeV) mutant, was isolated from brain tissues of a patient shortly after the clinical onset, and the entire viral genome was sequenced. The virus, named SSPE-Kobe-1, formed syncytia on B95a and Vero/SLAM cells without producing cell-free infectious virus particles, which is characteristic of SSPE virus. Phylogenetic analysis classified SSPE-Kobe-1 into genotype D3. When compared with an MeV field isolate of the same genotype (Ich-B strain), SSPE-Kobe-1 exhibited mutation rates of 0.8-1.6% at the nucleotide level in each of the proteincoding regions of the viral genome. It is noteworthy that the mutation rate of the M gene (1.2%) of SSPE-Kobe-1 was considerably lower than for other SSPE virus strains reported so far, but that the majority of the mutations (75%) were the uridine-to-cytidine biased hypermutation characteristic of the SSPE virus M gene. At the amino acid level, the viral proteins, such as N, P, C, V, M, F, H and L proteins, had point-mutations on 3, 7, 1, 4, 3, 9, 8 and 14 residues, respectively, compared with the Ich-B strain. In addition, the F and H proteins had mutated C-termini due to single-point mutations near or at the stop codons. Two of the three mutations in the M protein were Leu-to-Pro mutations, which are likely to affect the conformation and, therefore, the function of the protein. Because of the relatively small number of mutations, SSPE-Kobe-1 would be a useful tool to study genetic evolution of SSPE virus.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8910
    Product Catalog Name:
    Anti-Measles Blend Antibody, matrix protein, clones CV8, CV9

  • Immunological and biochemical characterization of coxsackie virus A16 viral particles. 23226233

    Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be greater than 10(6) the tissue culture's infectious dose (TCID(50)) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10(-5) was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24-28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35-38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176-190.These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.
    Document Type:
    Reference
    Product Catalog Number:
    MAB979
    Product Catalog Name:
    Anti-Enterovirus 71 Antibody, cross-reacts with Coxsackie A16, clone 422-8D-4C-4D