Susceptibility of cell lines of primate and nonprimate origin to certain human viruses

A comparative study was made of the susceptibility of 11 cell lines of human and animal origin, the WI-38 cell strain and fresh cultures of human thyroid, monkey kidney and hamster embryo tissues to certain human viruses. The animal cell lines were derived from monkey, rabbit, mouse, pig and calf tissues. The viruses used were strains of influenza A₂ and B viruses, parainfluenza viruses types 1, 2 and 3, RS virus, adenoviruses types 3, 4 and 21, poliovirus type 1 and Coxsackie A type 21 and Coxsackie B type 3 viruses. Cell lines derived from nonprimate tissues were generally less susceptible than cell cultures of human and simian origin. The combined use of fresh cultures of human thyroid and monkey kidney tissues and of a human cell line seems to provide a satisfactory indicator system for the viruses employed in this study.     

Studies on Parainfluenza Virus Infections among Infants and Children in Sendai

A cell line sensitive enough for the recovery of all parainfluenza viruses and free of simian virus contamination frequently occurring in monkey kidney cells was sought. The VERO cell obtained from African monkey kidney was found suitable for the initial isolation of types 1, 2 and 3 parainfluenza viruses, although the cells did not always allow the successive transfer. Mixed cultures of VERO and HEp-2 cells were also useful in the recovery of various respiratory viruses including parainfluenza viruses. The characteristics of hemagglutinins of parainfluenza viruses were examined, and type 2 parainfluenza and SV5 viruses agglutinated both guinea pig and green monkey erythrocytes at 36 C, whereas types 1 and 3 parainfluenza viruses agglutinated only guinea pig erythrocytes. Thus parainfluenza viruses were divided into two groups by the presence or absence of hemagglutinins for green monkey erythrocytes. Identification of these parainfluenza isolates, employing HI microtechnique was simple and reliable, even with the first passage harvest, when guinea pig erythrocytes were used and the test read at 36 C. Specific standard antisera for these parainfluenza viruses were prepared by immunizing chickens intravenously and bleeding within a short period. These type-specific antisera were useful for the identification of parainfluenza isolates by HI test.     

Selected laboratory aspects of influenza surveillance

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.     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

Growth of Myxovirus parainfluenza type 3 in organ cultures of guinea pig tissue

Craighead, J. E. (Harvard Medical School, Boston, Mass.). Growth of Myxovirus parainfluenza type 3 in organ cultures of guinea pig tissue. J. Bacteriol. 92:751-761. 1966.-Organ cultures of adult guinea pig nasal mucosa, lung, and pleura were infected with Myxovirus parainfluenza type 3. Observations were made on the growth of virus at intervals after inoculation. An inoculum of 10(2.5) tissue culture infectious doses (tcid(50)) initiated infection in each of the tissues. Cultures of nasal mucosa yielded up to 10(6.0)tcid(50) per 6 hr for periods of as long as 2 weeks. Virus production was not affected by the "immune" status of the animal used as a source of tissue. Introduction of antiserum into the medium appeared to suppress virus release but failed to "cure" the infection. Interferon was not detected in fluids bathing the nasal mucosa. Cultured fragments of lung produced virus for 28 days after inoculation. As much as 10(5.0)tcid(50) per 6 hr was released by the tissue. Pleural mesothelial cells lining the diaphragm yielded up to 10(6.0)tcid(50) per 6 hr over a 14-day period. Histological sections showed that the tissues retained differentiated morphological features during maintenance in vitro. Cytological changes unequivocally associated with infection were not recognized. The techniques described give reproducible, quantitative results. Organ cultures are feasible for the study of virus growth and cytopathology in differentiated tissues.     

Rapid Identification of Viruses by Indirect Immunofluorescence: Standardization and Use of Antiserum Pool to Nine Respiratory Viruses

The indirect fluorescent antibody test employing treated and standardized antisera and conjugated antiglobulin has been used successfully, in conjunction with a technique for growing and staining virus cell systems in situ on microscope slides, in the identification of nine respiratory viruses. By using pooled antisera in a single test, the presence or absence of these viruses was determined in 18 to 45 hr after inoculation of slide microtissue culture.     

Observations on Clinical and Immunofluorescent Diagnosis of Parainfluenza Virus Infections

Immunofluorescent techniques have been applied to nasopharyngeal secretions for the rapid diagnosis of parainfluenza virus types 1, 2, and 3 infections. Seventy-five infections were found by isolation techniques; 55 of these had nasopharyngeal secretions taken and 53 were positive by direct examination. A comparison of the results of 60 neutralization tests with immunofluorescence applied to monkey kidney isolations showed complete agreement. Immunofluorescence appeared to be a satisfactory method for differentiating the various haemadsorption viruses. The importance of parainfluenza viruses and respiratory syncytial virus in croup was noted and the association of the parainfluenza viruses with acute respiratory virus infection was confirmed. The clinical relationship between respiratory syncytial virus and parainfluenza virus type 3 is discussed.     

Role of heterogenized cellular antigens in cross immunity reactions in paramyxovirus infections]

Heterogenized antigens similar to the antigens of sheep and guinea pig red blood cells were revealed by indirect immunofluorescence in tissue cultures infected with parotitis virus. Participation of these antigens in cross immunofluorescence reactions observed in tissue cultures infected with various paramyxoviruses and in a suspension of erythrocytes loaded with these viruses was established. It was shown that immunization of children with parotitis virus was accompanied by a specific anamnestic reaction for heterogenized antigens. It is supposed that the corresponding antibodies can take part in cross serological tests with parainfluenza viruses.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V. Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.     

Replication of the Reticuloendotheliosis Virus (Strain T) in Chicken Embryo Cell Culture

Investigations were conducted on the in vitro replication of the reticuloendotheliosis (RE) virus (strain T) in specific-pathogen-free chicken embryo fibroblast (CEF) cultures. Active virus production was detected in the tissue culture fluid 24 hr after infection. When injected into chickens, samples taken 42 hr after infection of the cell cultures killed approximately 50% of the birds at a 1:100 dilution. The RE virus titer remained at this level for 5 days before declining. Cell-free virus preparations from tissue cultures rarely resulted in 100% mortality of the assay birds. The level of cell-associated virus was very low. Evidence that the reticuloendotheliosis was not induced by a mycoplasma was indicated by failure to isolate an organism on PPLO Agar (Difco) and failure of kanamycin or amphotericin B to inhibit multiplication of RE virus in vitro. RE virus appeared to be unrelated to members of the avian leukosis and sarcoma complex. It did not induce resistance in CEF cultures to sarcoma viruses of the A or B subgroup of this complex. Similarly, preinfection of cell cultures with leukosis viruses of the A or B subgroup did not inhibit or reduce the replication of RE virus.     

Susceptibility of continuous lines of monkey kidney cells to influenza and parainfluenza viruses in the presence of trypsin

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.     

Development of Four Arboviruses in Mice and Application to Rapid Test Procedures

Mice were inoculated with St. Louis encephalitis (SLE), Flanders (FLAN), California (CE), or Tensaw (TEN) viruses. At fixed intervals after inoculation, brains from these mice were collected and assayed for infective virus and complement-fixing, hemagglutinating, and precipitating antigens. Detectability of these antigens was correlated with the appearance of signs of illness in the mice. Infective virus appeared 64, 48, 48, and 40 hr before signs of illness and 90, 86, 64, and 56 hr before death in mice inoculated with SLE, FLAN, CE, and TEN viruses, respectively. Diagnostic antigens were also detected well before signs of illness appeared. These findings were applied to the isolation of viruses from field-collected specimens. It was shown that by harvesting tissues at appropriate intervals these viruses could be detected and identified more rapidly than by conventional techniques with mice.     

Immunofluorescence technique for the detection of salmonellae in various foods

Salmonella species have been detected in nine food varieties by use of fluorescent antibodies without false-positive or false-negative results. Test antisera were specially prepared, commercially available, conjugated polyvalent O globulin absorbed with cultures of Escherichia coli and Citrobacter freundii, and polyvalent phase II H globulin antibodies. Use of this technique permits a decrease of 24 hr in time normally required for Salmonella detection when compared with cultural Salmonella recovery methods.     

Procedure for the Evaluation of the Virucidal Effectiveness of an Ethylene Oxide Gas Sterilizer

A quantitative, reproducible method was developed for the evaluation of the virucidal activity of test gases. Using this method, we determined the virucidal effectiveness of a Steri-Vac ethylene oxide gas sterilizer. Wool gabardine material was exposed to high concentrations of herpes simplex, vaccinia, parainfluenza, or polio viruses and was processed through the sterilizer. Two time-temperature cycles of the machine, 29 C for 180 min and 60 C for 48 min, were used in separate experiments. The viruses were exposed to the gas when freshly pipetted onto the fabric or when pipetted on the material and allowed to dry 16 to 24 hr. In two experiments carried out under each condition, the virus titers were reduced by the sterilization process to less than detectable limits. These titer reductions were for the herpes virus >/= 2.7 to 5.0 log, for vaccinia virus >/= 4.0 to 6.1 log, for parainfluenza virus >/= 1.8 to 4.9 log, and for poliovirus >/= 4.9 to 7.7 log. The observed reductions in virus titers were the same whether the virus-contaminated fabrics were sealed in polyethylene packages or held in open petri dishes during exposure to ethylene oxide.     

Pneumonia in Ottoman vipers (Vipera xanthena xanthena) associated with a parainfluenza 2-like virus

A paramyxovirus related to parainfluenza 2 (PI2) virus was recovered from the lungs of two dead Ottoman vipers from a zoological collection. Snakes of other species in the collection were unaffected. Histologic examination of the vipers' lungs revealed interstitial pneumonia, and degeneration and hyperplasia of bronchial and atrial epithelia. Scattered vacuoles, some of which contain eosinophilic inclusion bodies, were seen in the cytoplasm of several cells of affected epithelial tissues. The virus recovered from pulmonary tissues of the snakes replicated optimally at 30 C in a variety of cell cultures and hemagglutinated chicken erythrocytes. Viral hemagglutination was inhibited by PI2 virus antiserum, but not by antisera to PI1, PI3, respiratory syncytial, and canine distemper viruses. Indirect immunofluorescence with PI2 antiserum specifically stained inclusions in the epithelial cells of respiratory tissues and infected cell cultures.     

Monoclonal antibodies against phloem P-protein from plant tissue cultures. I. Microscopy and biochemical analysis

Most research involving phloem proteins is done with phloem exudates, which are not easily obtained from many plants. We report here on the use of tissue cultures to study phloem proteins. Monoclonal antibodies against the filamentous phloem protein, P-protein, were made by injecting mice with a phloem-enriched fraction isolated from Streptanthus tortuosus callus grown on a medium that stimulates the differentiation of xylem and phloem (phloem[+] cultures). Monoclonal antibodies specific for P-protein were identified by incubating free-hand stem sections of S. tortuosus in hybridoma supernatants, then in a goat anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC), and observing the FITC under an epifluorescence microscope. Antibodies specific for P-protein in stem sections were used to probe nitrocellulose blots of polyacrylamide gels separating proteins isolated from both phloem(+) and phloem(-) tissue cultures. Immunoblots were incubated overnight in hybridoma supernatants followed by a secondary antibody conjugated to alkaline phosphatase. Three monoclonal antibodies—RS21, RS22, and RS23—bound to an 89-kD band in the phloem(+) lanes but failed to bind to any proteins in the phloem(—) lanes. In leaf sections of Arabidopsis thaliana processed by freeze-substitution, a mixture of RS21 and RS22 bound to the P-protein filaments in sieve elements, but not to any proteins in adjacent cells. A control antibody specific for tubulin did not bind to the P-protein filaments.     

Application of a multiple surface tissue culture propagator for the production of cell monolayers,virus, and biochemicals

A new concept of tissue culture equipment and procedures was developed for the mass-scale growth of several types of animal tissue cells in monolayers on multiple glass surfaces. Continuous, cell lines, primary and diploid cell strains were grown in this equipment. Cells studied include primary bovine kidney, human diploid WI-38, human foreskin, and mouse CCL1 cells. Photomicrographic comparisons of cells grown by these techniques indicate they are morphologically identical to tissue culture cells grown in glass bottles or tubes. The growth of the tissue culture cells in the propagator was monitored by carbohydrate Utilization and acid production. Large-scale production of viruses and biochemicals on cells grown in the multiple-plate tissue culture propagator was accomplished. Virus titers were equal to those obtained from conventional bottle or tube cultures for several strains of influenza, parainfluenza, and respiratory syneytial viruses. High-titred mouse interferon was also produced in this system. In addition to tissue culture cell production, Eaton agent, Mycoplasma pneumoniae was grown on the multiple glass surfaces on a mass scale.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

Developmental Sequence and Intracellular Sites of Synthesis of Three Structural Protein Antigens of Influenza A2 Virus

Specific antisera for hemagglutinin (HA) and neuraminidase antigens of influenza A(2) virus (A(2)E) were produced through the segregation of the two proteins in reciprocal viral recombinants of A(2)E and A(0)e viruses. Gamma globulin fractions of these specific antisera and of antiserum specific for the nucleoprotein (NP) antigen of A(0)e virus were conjugated with fluorescein isothiocyanate and employed to follow the synthesis of the three structural proteins in clone 1-5C-4 human aneuploid cells, with parallel measurement of serological and biological activity of the antigens by other techniques. In this system, NP antigen appeared first (at 3 hr) in the cell nucleus, whereas HA and neuraminidase appeared coincidentally, at 4 hr after infection, in the cytoplasm. The initial detectability of biological or complement-fixing activity of the proteins coincided with their demonstrability as stainable antigens. Late in infection, all three antigens were detected at the cell surface. Antibody specific for HA partially blocked the intracellular staining of neuraminidase and inhibited the enzymatic activity of both extracted and intact extracellular virus. These observations suggest the close intracytoplasmic proximity of the two envelope antigens and perhaps their initial association in a larger protein.     

Recombinant vaccinia viruses carrying the N gene of human respiratory syncytial virus: studies of gene expression in cell culture and immune response in mice.

The construction and characterization of vaccinia virus recombinants carrying the nucleocapsid (N) protein gene of human respiratory syncytial (RS) virus are described. Recombinant viruses were constructed that contained the N gene oriented either positively or negatively with respect to the 7.5-kilodalton vaccinia virus promoter. In addition, a positively oriented recombinant was constructed that lacked an out-of-frame AUG codon in the 5'-terminal noncoding region. In HEp-2 cells, both positive-orientation recombinants induced the synthesis of a protein which comigrated with N protein and was precipitated by antisera to RS virus. Sera from mice immunized with these recombinants specifically precipitated the RS virus N protein. Analysis of mRNA and protein expressed from the recombinant N genes showed that deletion of the upstream AUG codon markedly improved the efficiency of protein synthesis. Mice were vaccinated with the high-expressing recombinant and subsequently challenged with live RS virus. The results of these experiments demonstrated that the immune response to N protein afforded a significant degree of protection against RS virus disease.