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1.
2.
A L Frank 《The Yale journal of biology and medicine》1982,55(3-4):201-205
The importance of virologically documented infections in influenza surveillance is well recognized and has been reaffirmed in recent reviews. The large number of specimens tested in surveillance make efficiency and low cost of virologic methods important. Based on observations made by others and our work with reisolation of stored specimens we have used the continuous line tissue cultures MDCK and LLC-MK2 for virus isolation in large-scale influenza surveillance studies for three years. Both cell lines were equally successful in detecting influenza A viruses in 77 fresh, virus-positive specimens. However, during the influenza B outbreak of 1979--80, of 473 specimens positive in either or both tissue cultures, 54 were positive only in MDCK and just six in LLC-MK2 only. For parainfluenza viruses, LLC-MK2 was much superior to MDCK. The most promising alternative to tissue culture at this time, based on a review of the literature, appears to be enzyme immunoassay. Sensitivity sufficient for direct detection of viral antigen in routine specimens currently requires fluorescent or radioactive substrates. Identification of early virus growth in continuous cell line cultures by enzyme immunoassay is practical now and can be considered. 相似文献
3.
Some biological properties of Murayama virus, a new paramyxovirus, were studied. The virus grew well in primary monkey kidney cells as well as embryonated eggs, while the virus yields in primary chick embryo and BHK-21 cells were much lower. The infected BHK-21 cells formed large syncytia and showed typical hemadsorption, but did not produce any detectable amount of hemagglutinin in the culture fluid. The virus yields were very low in Vero. LLC-MK2 and MDCK cells at first passages. The addition of trypsin to the medium enhanced virus growth in Vero and LLC-MK2 but not in MDCK cells. Cell fusion activity of the virus was observed in Molt-4 cells. Hemolytic activity was enhanced by freeze-thawing. Several species of mammals and birds were susceptible to experimental infections with the virus as evidenced by seroconversion and positive virus isolation without showing any clinical signs. 相似文献
4.
W. Adrian Chappell Charles H. Calisher Roberta F. Toole Kathryn C. Maness Donna R. Sasso Brian E. Henderson 《Applied microbiology》1971,22(6):1100-1103
During the 1969 dengue epidemic in Puerto Rico, human sera and Aedes aegypti mosquitoes were collected for virus isolation and identification. Three methods of isolation were used and compared. In the first method, we inoculated newborn mice by the intracranial route, noted any signs of illness, and serially passed specimens in mice until virus was isolated. In the second method, we inoculated tube cultures of LLC-MK(2) cells, noted any cytopathic effect (CPE), and assayed fluids for virus by plaque formation in LLC-MK(2) cell monolayers. The third method was different from the second only in that the original specimens were first inoculated into fluid cultures of Singh's A. albopictus cells. No significant CPE was seen in LLC-MK(2) cultures; however, distinct syncytial CPE was observed in A. albopictus cells. About the same number of virus isolates were made in each isolation system. Virus isolates from both sera and mosquitoes were identified as dengue type 2 by a plaque-reduction neutralization test in LLC-MK(2) cells. The utility of the three methods, individually or in combination, is discussed and related to diagnostic and epidemic situations. 相似文献
5.
Background
This study aims to investigate the role of microtubule dynamics in the initiation of NWS/33 human influenza A (NWS) virus infection in MDCK and LLC-MK2 mammalian kidney cells. We previously demonstrated a host-dependent role of the actin cytoskeleton in inducing restriction during the early phases of NWS infection. Furthermore, we showed the differential infectious entry of NWS virus in the above mentioned cell models.Methodology/Principal Findings
By first employing a panel of microtubule-modulators, we evidenced that microtubule-stabilization negatively interferes with NWS replication in LLC-MK2 but not in MDCK cells. Conversely, microtubule-depolymerization improves NWS growth in LLC-MK2 but not in the MDCK model. By using immunofluorescence labelling and Western blotting analyses upon NWS infection in mammalian kidney cells, it was observed that the occurrence of alpha-tubulin hyperacetylation - a post-translational modified form suggestive of stable microtubules - was significantly delayed in LLC-MK2 when compared to MDCK cells. Furthermore, mock-infected LLC-MK2 cells were shown to have higher levels of both acetylated alpha-tubulin and microtubule-associated protein 4 (MAP4), the latter being essential for the maintenance of normal microtubule polymer levels in interphase epithelial cells. Finally, to obtain highly dynamic microtubules in LLC-MK2 cells, we knocked down the expression of MAP4 by using a RNA-mediated RNA interference approach. The results evidenced that MAP4 silencing improves NWS growth in LLC-MK2 cells.Conclusion
By evidencing the cell type-dependent regulatory role of microtubule dynamics on NWS replication in mammalian kidney cells, we demonstrated that microtubule-stabilization represents a restriction factor for the initiation of NWS infection in LLC-MK2 but not in MDCK cells. 相似文献6.
A microplate method for isolation of viruses from infants and children with acute respiratory infections 总被引:7,自引:0,他引:7
Y Numazaki T Oshima A Ohmi A Tanaka Y Oizumi S Komatsu T Takagi M Karahashi N Ishida 《Microbiology and immunology》1987,31(11):1085-1095
Between December 1984 and December 1986, a microplate technique was adopted for isolation of viruses from infants and children with acute respiratory infections. By using two kinds of tissue culture microplates, i.e., the HHVM plate, containing human embryonic fibroblast (HEF), HEp-2, Vero and MDCK cells, and the MK plate which contains secondary monkey kidney cells, 1,080 field viruses were isolated from 1,061 (24.9%) out of 4,254 throat swabs. Of these 1,080 isolates, 1,003 (92.9%) were recovered in the HHVM plates and the remaining 77 (7.1%) in the MK plates. With the HHVM plate, influenza A and B viruses were cultivated in MDCK, RS virus in HEp-2, parainfluenza and mumps viruses in Vero, adenoviruses in both HEF and HEp-2, polioviruses in HEF, HEp-2 and Vero, coxsackie B viruses in both HEp-2 and Vero, rhino and echo viruses in HEF, herpes simplex virus in both HEF and HEp-2, and cytomegalovirus in HEF, although MK were more sensitive than Vero to parainfluenza and coxsackie B viruses. There was no difference in the rate of isolation of viruses between the microplate and ordinary tube methods. Cross contamination in the microplates was negligible for routine work. 相似文献
7.
I Orstavik 《Acta pathologica et microbiologica Scandinavica. Section B, Microbiology》1981,89(3):179-183
LLC-MK2, GMK AH-1, BSC-1, and Vero cells were compared in titrations of recent isolates and laboratory strains of influenza A and B and parainfluenza types 1, 2, and 3 viruses. About the same titres, as determined by haemadsorption in cell cultures, were obtained in LLC-MK2, GMK AH-1, and BSC-1 cells when trypsin had been added to the medium, whereas the Vero cells were less sensitive to the influenza virus strains tested. Virus titres were usually low in the absence of trypsin. A laboratory strain of parainfluenza 2 virus reached about the same titres in medium without as in medium with trypsin, possibly owing to prior adaptation by passages in Vero cells. Comparative titrations of influenza A, and parainfluenza 1 and 3 viruses suggested the same susceptibility of LLC-MK2 cells with trypsin as of primary monkey kidney cells. Re-isolation experiments from 38 clinical specimens showed LLC-MK2 cells to be as efficient as primary monkey kidney cells for isolation of influenza and parainfluenza viruses, whereas the susceptibility of the other cell lines to clinical material has not yet been tested on a larger scale. It is concluded that a continuous line of monkey kidney cell culture may be acceptable as an alternative to primary monkey kidney cells for the isolation of influenza and parainfluenza viruses from patients. 相似文献
8.
Characteristics of serially propagated monkey kidney cell cultures with persistent rubella infection 总被引:6,自引:0,他引:6
Maassab, H. F. (University of Michigan, Ann Arbor), and J. A. Veronelli. Characteristics of serially propagated monkey kidney cell cultures with persistent rubella infection. J. Bacteriol. 91:436-441. 1966.-A persistent infection of LLC-MK(2) cells with rubella virus has been established and maintained for over 3 years. This "carrier culture," designated as LLC-MK(2)-RAL, possesses distinct morphological and biological characteristics when compared with the original uninfected LLC-MK(2) line. The mechanism of viral persistance has not been entirely elucidated, but available data suggest a regulated infection with transmission of the virus directly from cell to cell or through cell division. Interferon was isolated from RAL (rubella-associated line) culture, which explains partly the wide spectrum of resistance to superinfecting viruses. Amantadine, although inhibiting cultures of LLC-MK(2) cells infected with rubella virus, failed to cure the "carrier culture." 相似文献
9.
《Research in virology》1990,141(3):373-384
Samples of nasopharyngeal secretion (NPS) from 100 infants and small children admitted for acute respiratory disease during the period from January to March 1989 were examined for the presence of influenza A virus. All samples were tested by enzyme immunoassay (EIA), fluorescent antibody (FA) technique and by isolation in cell culture 3–6 h after they were obtained from the patients. Of 24 influenza strains found by isolation, 21 were detected by EIA and 19 were FA+. In comparison with virus isolation, EIA gave the following values: sensitivity 88 %, specificity 100 %, positive prognostic value (PPV) 100 %, and negative prognostic value (NPV) 96 %. A rabbit anti-influenza-A serum (A-13) was used as catching antibody and a monoclonal anti-influenza-A pool against NP protein was used as detector antibody in EIA. A-13 gave bands corresponding to influenza A core proteins (NP and M1) in Western blot (WB) studies when different H3N2 strains were employed as antigens. A-13 gave only a band corresponding to the NP protein when H1N1 strains were examined by WB. The detection level by EIA for both H3N2 and H1N1 strains precipitated by polyethylene glycol from tissue culture maintenance medium was 1–2 ng. 相似文献
10.
Electron Microscopy of Monkey Kidney Cell Cultures Infected with Rubella Virus 总被引:5,自引:1,他引:4
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Two rubella virus strains isolated in this laboratory were investigated in terms of their growth in LLC-MK(2) cell cultures and their effect on cell morphology. Rubella virus grew readily in LLC-MK(2) cells, but cytopathic effects of the virus were not observed in infected cultures. Such infected cultures can be subcultured indefinitely and continue to shed virus. Examination of rubella-infected cell cultures by electron microscopy showed the presence of annulate lamellae in the cytoplasm of 15% of the cells. No changes were evident in the nuclei. These membranous inclusions varied in complexity from parallel arrays of annulate lamellae to large lamellar structures of complex morphology. An occasional cell contained a crystal lattice structure in association with the lamellae. Larger inclusions, consisting of disorganized arrays of "unit" membranes, were also found. Uninfected cells were devoid of annulate lamellae, crystals, and complex membranous inclusions. No viruslike particles were observed in any part of the cells from infected cultures. The significance of the structures observed has not been determined. 相似文献
11.
The replication of type 2 dengue (D-2) virus in Aedes albopictus (Aal) mosquito cell cultures differed from that in vertebrate (LLC-MK2) rhesus monkey kidney cells. Virus readily replicated in Aal cells at either 30 or 37 C, but had no apparent effect on the host cell. Persistent infection was established with continual virus production for at least 6 months, although the virulence of progeny virus for both suckling mice and LLC-MK2 cells became attenuated. Density gradient analysis of infected Aal cell supernatant products indicated that only complete virus was released, in contrast to infected LLC-MK2 cells which also released incomplete virus. The surface antigens of the virus produced in Aal cells appeared to be considerably modified in that antiserum to vertebrate cell-produced D-2 virus did not block hemagglutination, whereas anti-Aal cell antiserum did. Virus infectivity could be neutralized by the antiserum to D-2 virus grown in vertebrate cells, however. Virus produced in LLC-MK2 cells did not demonstrate a similar host-cell modification. These results may reflect a difference in the mechanism by which D-2 virus matures in Aal cells. 相似文献
12.
Andre Coetzer Claude T. Sabeta Wanda Markotter Charles E. Rupprecht Louis H. Nel 《PLoS neglected tropical diseases》2014,8(9)
The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus. 相似文献
13.
Infection of a continuous cell line of dog kidney origin (MDCK) with herpes simplex virus type 2 (HSV-2) resulted in production of little to no new infectious virus. Serial subculture of MDCK cells inoculated with HSV-2 did not permit establishment of carrier cell cultures, as assessed by negative results of plaque assays for infectious virus and radioimmunoassay (RIA) for viral antigens. Group- and type-specific antigens were detected in lysates of non-permissive MDCK cells inoculated with HSV-2 and tested by RIA at 24 hours post-inoculation. Polypeptides produced in permissive (Vero) and non-permissive (MDCK) cell systems were labeled with [14C]-amino acids and analyzed by polyacrylamide slab gel electrophoresis and autoradiography. During non-permissive infection, two polypeptides of large molecular weight, not found in uninfected MDCK cells, one of which commigrated with a major HSV-2 structural polypeptide, were synthesized and reproducibly detected. 相似文献
14.
The growth of Rio Bravo virus (RBV) in eight cell culture systems was studied. Highest yields of virus were produced in BHK-21 (C13), L, and Vero cell lines, but L cells were resistant to low doses of virus. LLC-MK(2), HeLa, and human embryo skin cells produced moderate amounts of virus, but FL amnion and primary chick embryo fibroblasts supported little virus growth. Virus was rapidly inactivated by exposure to pH values below 7.0. Single-cycle growth in BHK-21, L, and LLC-MK(2) cell monolayers was characterized by a latent period of about 12 hr followed by rapid virus production that peaked at 36 to 48 hr. Vero cell cultures can remain chronically infected with RBV for more than 100 days. Such cultures show evidence of cell destruction, and their supernatant fluids contain virus at 10(4) to 10(5) log(10) per ml. 相似文献
15.
16.
Marius Ianconescu Geoffrey Y. Akita Bennie I. Osburn 《In vitro cellular & developmental biology. Animal》1996,32(4):249-254
Summary Recently, bluetongue virus (BLU) serotype 11 was detected in diseased dogs that had been inoculated with live attenuated vaccine
contaminated with this serotype of bluetongue virus (Akita et al., 1994). For various laboratory tests, BLU can be propagated
in different cell cultures. No information was found in the literature about the possibility of propagating this virus in
canine cells. To determine whether the BLU isolate from the contaminated canine vaccine (BLU-vac) is unique in its ability
to replicate in canine cells, this virus was studied in parallel with U.S. prototype strains of BLU (serotypes 2, 10, 11,
13, and 17), in hamster lung (HmLu-1) and canine kidney (MDCK) cell cultures. In HmLu-1 cell cultures, the BLU-vac produced
cytopathic effect (CPE) of the same type as the U.S. prototype BLU strains by 4 to 6 d postinoculation. In MDCK cell cultures,
all of the BLU strains tested were able to replicate but did not produce CPE. The BLU-inoculated MDCK cells became persistently
infected, and these cultures continued to produce infectious BLU even after six serial passages over 2 1/2 mo. In none of
these cultures was CPE observed. In mixed cultures containing both HmLu-1 and MDCK cells, CPE first affected the HmLu-1 islands;
subsequently, CPE spread also to the areas with MDCK cells. The silent persistent infection of the MDCK cells with BLU indicates
that more stringent screening of the cells used in the production of live vaccines for various contaminating viruses is necessary. 相似文献
17.
Palak Galhotra Pankaj Prabhakar Himanshu Meghwani Soheb A. Mohammed Sanjay Kumar Banerjee Sandeep Seth Milind P. Hote K. H. Reeta Ruma Ray Subir Kumar Maulik 《Molecular and cellular biochemistry》2018,437(1-2):185-201
Viruses depend on cellular machinery to efficiently replicate. The host cytoskeleton is one of the first cellular systems hijacked by viruses in order to ensure their intracellular transport and promote the development of infection. Our previous results demonstrated that stable microfilaments and microtubules interfered with human influenza A/NWS/33 virus (H1N1) infection in semi-permissive LLC-MK2 cells. Although formins play a key role in cytoskeletal remodelling, few studies addressed a possible role of these proteins in development of viral infection. Here, we have demonstrated that mammalian Diaphanous-related formin-1 (mDia1) is involved in the control of cytoskeleton dynamics during human influenza A virus infection. First, by employing cytoskeleton-perturbing drugs, we evidenced a cross-talk occurring between microtubules and microfilaments that also has implications on the intracellular localization of mDia1. In influenza A/NWS/33 virus-infected LLC-MK2 cells, mDia1 showed a highly dynamic intracellular localization and partially co-localized with actin and tubulin. A depletion of mDia1 by RNA-mediated RNA interference was found to improve the outcome of influenza A/NWS/33 virus infection and to increase the dynamics of microfilament and microtubule networks in LLC-MK2 cells. Consistent with these findings, observations made in epithelial respiratory cells from paediatric patients with acute respiratory disease assessed that the expression of mDia1 is stimulated by influenza A virus but not by respiratory syncytial virus. Taken together, the obtained results suggest that mDia1 restricts the initiation of influenza A/NWS/33 virus infection in LLC-MK2 cells by counteracting cytoskeletal dynamics. 相似文献
18.
Monoclonal antibodies (MAbs) raised against porcine adipocyte plasma membranes were used to demonstrate complement-mediated cytotoxicity of adipocytes and preadipocytes in primary stromal-vascular (SV) cultures. Five of the six MAbs tested significantly reduced the number of fat cell clusters in cultures maintained in medium supplemented with pig serum and dexamethasone (PS/DEX) but not in cultures supplemented with insulin, transferrin, and selenium (ITS). Neither MAb nor complement alone affected fat cell cluster number. Treatment of both ITS and PS/DEX cultures with pools of 2 or more MAbs, in combination with complement, eliminated fat cell clusters in all instances. Treatment of cultures prior to appearance of cells containing lipid demonstrated that preadipocytes, or adipose lineage cells, could also be eliminated by MAb/complement treatment. Finally, injection of young rats with a pool of three of the MAbs produced a 30% reduction in inguinal fat pad weight without affecting other tissues. Adipocyte/ preadipocyte-depleted cultures can now be used as a model system to examine progression of cells through the adipose cell lineage at a time not previously possible with primary cells. 相似文献
19.
Weiguo Zhai Dan Ning Zhang Cecilia Mai Justin Choy Gary Jian Kuldip Sra Mark S Galinski 《PloS one》2012,7(12)
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 (<1 log2 lower) or superior (>1 log2 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. 相似文献
20.
Class I major histocompatibility proteins as cell surface receptors for simian virus 40 总被引:9,自引:2,他引:7
Class I major histocompatibility complex proteins appear to be the major cell surface receptors for simian virus 40 (SV40), as implied by the following observations. Adsorption of SV40 to LLC-MK2 rhesus monkey kidney cells specifically inhibited binding of a monoclonal antibody (MAb) against class I human lymphocyte antigen (HLA) proteins. Conversely, pretreatment of LLC-MK2 cells with anti-HLA MAbs inhibited infection by SV40. The ability of anti-HLA to inhibit infection was greatly reduced when the order of addition of the anti-HLA and the virus was reversed. Infection was also inhibited by preincubating SV40 with purified soluble class I protein. Finally, human lymphoblastoid cells of the Daudi line, which do not express class I major histocompatibility complex proteins, were infected at relatively low levels with SV40 virions. In a control experiment, we found that pretreatment of cells with a MAb specific for the leukocytic-function-associated antigen LFA-3 actually enhanced infection. This finding may also support the premise that class I major histocompatibility complex proteins are receptors for SV40. 相似文献