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PubMed1993

The use of MDCK, MEK and LLC-MK2 cell lines with enzyme immunoassay for the isolation of influenza and parainfluenza viruses from clinical specimens.

Primary Monkey Kidney (PMK) epithelial cells or egg inoculation have been traditionally used for the culture of influenza and parainfluenza viruses. The high cost and variability of obtaining high quality PMK cells prompted us to investigate the use of other cell strains for the growth of these viruses. For this study we investigated three cell lines viz. MDCK, MEK and LLC-MK2 for the culture of influenza A and B and parainfluenza 1, 2 and 3 viruses. Clinical specimens were spun onto cell monolayers in microtitre wells. The growth of these viruses was then identified by specific antibodies in an enzyme immunoassay (EIA). The LLC-MK2 and MDCK cell lines were found to provide optimal growth of parainfluenza and influenza viruses respectively. During the period from November, 1990 to July, 1992, 6501 respiratory specimens were tested. There were 100 influenza A, 36 influenza B and 261 parainfluenza virus isolates. The influenza isolates were further subtyped by the WHO Influenza Reference Centre. The use of these cell lines and the EIA provided an effective method for the routine culture of these viruses.

Animals↗

PubMed1993

Polymerase chain reaction detection of human cytomegalovirus in over 2000 blood specimens correlated with virus isolation and related to urinary virus excretion.

The polymerase chain reaction (PCR) as applied to human cytomegalovirus (HCMV) detection should provide a valuable tool for rapid, reliable diagnosis of infection, thereby allowing prompt treatment. However, to date the high sensitivity of this technique and the lack of semi-quantitative interpretation have hindered establishing its validity for diagnosing systemic infection. We describe a rapid, simple, semi-quantitative PCR technique for HCMV detection. The validity of the technique was tested objectively by analyzing over 2000 leukocytes specimens by PCR and comparing the results with virus isolation from urine and blood in concomitant samples in the absence of any clinical data. It could thus be established that this technique had a sensitivity and specificity of 97%. When the PCR signal corresponded to > or = 8000 genome equivalents for 10(4) leukocytes, the predictive value for viremia was 86%. This semi-quantitative PCR technique should allow rapid diagnosis of systemic infection and provide a reliable means of monitoring clearance of CMV from blood during drug therapy.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Replication and physical parameters important for preparing purified Junin virus.

Junin virus (JV) is an Arenavirus and the causative agent of Argentine hemorrhagic fever (AHF), an often fatal human disease. The attenuated strain XJ-clone 3 (XJC13) of JV, after being tested in humans, has been considered a promising vaccine. We found that synthesis of JV XJC13 reaches a peak 2 days after infection and the kinetics of synthesis are little affected by the multiplicity of infection (MOI) in a range from 0.125 to 1.00. Virus synthesis is sensitive to actinomycin D, indicating that cellular biosynthesis is required for viral replication. Combined precipitation and ultracentrifugation of supernatant from virus-infected cells yielded large amounts of concentrated and purified virion that banded in sucrose as a single peak with average density 1.177 +/- 0.015 g/ml. Purified virions have an average diameter of 203 +/- 23 nm by electron microscopy and an average sedimentation coefficient of 454 +/- 27 S. The results from the present study should assist in the preparation of large amounts of attenuated Junin virus which are required for vaccination against and diagnosis of Argentine hemorrhagic fever.

Animals↗

PubMed1994

Physiological factors of growth and susceptibility to virus regulating Vero cells for optimum yield of vaccinia and cloned gene product (beta-hCG).

Replication of vaccinia virus carrying beta-hCG gene at the 'tk' locus was studied in microcarrier culture of Vero cell. The yields of recombinant vaccinia virus and its expressed product (beta-hCG) were influenced by the physiological state of the host cell, at which it was infected. Cells infected with the virus between the 3rd and 6th day gave maximum amount of virus and beta-hCG in a parallel manner, the yields of which were 2.3 x 10(9) pfu per 10(6) cells and 450 ng per 10(6) cells, respectively. The yields were reduced by 33-36% in the virus-infected stationary culture of the 8th day. The physiological state of the host cell has been quantified by the cellular activity parameter 'P' (mu(t)/mu max) in the scale of 1 to 0. The value of 'P' was highest in the exponentially growing culture, which reduced with decline in growth rate. The cellular energy generation has been related with the parameter 'P'. ATP generation was reduced to 55% in the stationary culture on the 8th day (P = 0). Also, the adsorption of virus was reduced by nearly 55% in extremely slow growing or stationary culture. Host cells at moderate growth rate (P > or = 0.15) were found to be optimum for maximizing the yield of viral products.

Adenosine Triphosphate↗

PubMed1984

Measles virus gene expression in subacute sclerosing panencephalitis.

RNA was extracted from the diseased brain of a case of human subacute sclerosing panencephalitis (SSPE) and analysed for the expression of measles-specific RNA. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho- (P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. RNA obtained from the human brain was also translated in vitro and measles virus nucleocapsid and P protein was produced. However, in marked contrast to control reactions M protein was not detected in the products formed by translation in vitro. These results indicate an impaired measles virus M protein mRNA synthesis in infected brain tissue.

Adolescent↗

PubMed1988

In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus.

Infection with HBeAg and HBV DNA positive serum in primary cultures of cryopreserved human hepatocytes in the presence of human whole blood in the medium was performed in 8 consecutive experiments. HBsAg and HBV DNA release into the medium was increased in the second week after infection. Via immunostaining, HBcAg was first observed in the nucleus of hepatocytes approximately 3 days after infection. A maximal percentage of HBcAg positive cells in 0.1% of cultured hepatocytes was detected on the 7th day. HBsAg was also first demonstrated on the 3rd day, and predominantly localized in the cytoplasm. About 5% of hepatocytes were HBsAg positive on the 12th day after infection. The percentage of positive cells did not appear to increase after this time. Using in situ cytohybridization and agarose gel electrophoresis and Southern blot analysis, HBV DNA was first detected on the 4th day. In addition, electron microscopic studies revealed the presence of 42 nm virus-like particles in the cytoplasm of infected cells in the second week after infection. This in vitro system provides a model for studying the mechanism of HBV infection, viral replication and maturation. However, further improvement of culture systems is needed, to increase the number of infected cells and for active HBV replication.

Cell Nucleus↗

PubMed1992

Modifications of the 5' untranslated region of foot-and-mouth disease virus after prolonged persistence in cell culture.

The nucleotide sequence of the 5'-untranslated region (5'UTR) of the genome of foot-and-mouth disease virus (FMDV) R100, rescued after 100 passages of persistently infected BHK-21 cells, has been compared with that of the parental FMDV C-S8c1. The nucleotide sequence divergence between the two viruses in heteropolymeric regions is 1%. The few mutations located at the 5'-most terminal region (S fragment) and at the internal ribosome entry site (IRES) do not appear to affect significantly the tight secondary structure predicted for these RNA segments. Comparison of the 5'UTR of C-S8c1 or R100 RNA with that of other FMDV serotypes and subtypes indicates the presence of block deletions (or insertions) which do not correlate with the serological classification of FMDV. Remarkably, FMDV R100, a virus highly attenuated for mice and cattle, contains a polyribocytidylate (poly C) tract of about 420 nucleotides, 145 residues longer than its parental, virulent FMDV C-S8c1. This long poly C of R100 RNA includes a few uridine residues interspersed at fairly regular intervals. This is the longest highly homopolymeric tract described in a viral genome and, to our knowledge, in any informational biomolecule.

Animals↗

PubMed1994

Genetic variation of foot-and-mouth disease virus from field outbreaks to laboratory isolation.

Foot-and-mouth disease virus (FMDV), by nature of its RNA genome, possesses a high rate of mutation during replication. This results in extensive genetic polymorphism of virus populations in nature. The emergence of FMDV variants during replication has been reported. Genetic changes in the viral capsid protein (VP1) gene can result in amino acid changes affecting the immunodominant epitopes of FMDV. The genetic heterogeneity of FMDV in the field and the antigenic variants observed after cell culture isolation has been investigated by PCR sequencing and reactivity with monoclonal antibodies. These methods were applied to viruses causing two different outbreaks of FMD before and after replication in cell culture and in the animal host. The VP1 region of the genome was amplified by PCR and sequenced to reveal variant sequences identified after passage and to determine their presence in the original field tissue. In one case, reactivity with monoclonal antibodies was lost after passage as a result of an amino acid change in the subpopulation. These findings suggest that host cells can select specific virus genetic and antigenic subpopulations during virus isolation and propagation.

Amino Acid Sequence↗

PubMed1994

Fine mechanisms of ectromelia virus thymidine kinase-negative mutants avirulence.

Three independently selected spontaneous thymidine kinase-negative mutants (TK-phenotype) and a recombinant with Escherichia coli beta-galactosidase gene (LacZ+ phenotype) inserted in the viral thymidine kinase gene (tk) were derived from a plaque-cloned isolate of K-1 ectromelia virus strain (TK+ phenotype). Dramatically decreased virulence of TK- variants was observed for all routes of mouse inoculation. The kinetics of TK+ and TK- variants in various target organs indicated a significant decrease of production and dissemination of TK- mutants and recombinant in the organs of mice. In the spleen and liver of intranasally or intracerebrally infected mice TK- virus was not detected during the entire period of observation. Analysis of organs homogenates of mice intranasally infected by a mixture of recombinant with TK-LacZ+ phenotype and parental isolate with TK+LacZ- phenotype on the monolayers of TK- cells indicated that only white plaques (LacZ-) with the TK+ phenotype appeared from liver and spleen homogenates. Thus, the mouse acts as a live filter much more efficiently than any other selective systems. Ultrastructural studies showed that viral damage in animals infected by TK- variants was far less than that observed in mice, infected with wild type of ectromelia virus and pathological lessions were slight and reversible. Replication of ectromelia virus TK- variants was blocked at the viroplasma stage in cells with a high level of differentiation in contrast to TK+ variants. Most likely, such restriction of target cells assortment is the general reason of reduced virulence in the case of tk-gene inactivation.

Animals↗

PubMed1992

Molecular basis of virulence and growth of hepatitis A virus in cell culture.

The ability of engineering variants of hepatitis A virus (strain HM175) to replicate in cell culture or to cause disease in marmosets was evaluated. Virus variants were encoded by chimeric genomes constructed from infectious cDNA clones of two viruses (wild type and cell-culture-adapted) which differed in their ability to grow in vitro and to cause acute hepatitis in marmosets. Transfection and infectivity assays indicated that virus growth in vitro could be enhanced by subcloning the cell substrate prior to infection or by introducing multiple combinations of two or more mutations into the wild type genome. Various chimeric viruses induced liver enzyme elevations in marmosets, indicating that attenuation of virulence also required multiple mutations.

Animals↗

PubMed1983

Inducement of cytopathic changes and plaque formation by porcine haemagglutinating encephalomyelitis virus.

ESK cells were shown to be a good medium for propagating the 67N strain of porcine haemagglutinating encephalomyelitis virus, although no cytopathic effect was observed. The virus induced a readily recognizable cytopathic effect in ESK cells, when a noncytotoxic amount of diethylaminoethyl-dextran (DEAE-dextran) was incorporated in the culture medium. Based on this finding, a sensitive, practical assay method for the virus was developed. When DEAE-dextran was incorporated in the agar overlay medium, 67N virus formed plaques in ESK cell monolayers. The cytopathic effect as well as the plaque formation were specifically inhibited by antisera against the virus. Neutralization tests were developed on the basis of these findings. Neutralization and haemagglutination-inhibition tests on swine serum samples indicated a wide dissemination of haemagglutinating encephalomyelitis virus or antigenically-related viruses in Japanese pigs.

Animals↗

PubMed1983

Physical and biological properties of Nairobi sheep disease virus.

Thermal and pH stability of Nairobi sheep disease (NSD) virus were studied. The 180th mouse brain passage lost infectivity at a higher rate than "wild" virus at 4 degrees C. At 37 degrees C and neutral pH, "wild" virus again was more stable than cell culture and mouse brain attenuated strains with half-life periods of 104, 87 and 51 min, respectively. At 0 degrees C the cell culture attenuated virus was most stable at pH 7.4 with an estimated half-life of 164 h. The density of the virus in sucrose gradients came to 1.195 g cm -3. Metabolic growth inhibition studies using a halogenated nucleoside, and staining of RNase and DNase-treated infected cell cultures with acridine orange, indicated that NSD virus has a single stranded RNA genome. The growth of the cell culture adapted virus was assayed in monolayers of BHK21/13 cells at low multiplicity of infection. Cell-associated virus (CAV) was first detected at 6 h post-inoculation (PI). The titre increased rapidly until CPE appeared at 48 h and declined after 72 h PI. Cell-free virus (CFV) was first detected at 10 h PI. The titre of CFV increased up to 72 h, but on average was two log units less than the CAV titre.

Animals↗

PubMed1984

The isolation, propagation and characterization of tissue-cultured equine rotaviruses.

From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.

Animals↗

PubMed1989

A heteroploid permanent cell line originating from embryonic calf thyroid supporting the replication of all known bovine adenovirus serotypes.

An embryonic calf thyroid cell culture was established as a permanent heteroploid cell line, which is now in its 150th subculture. It allowed replication of all nine bovine adenovirus serotypes at its 15th as well as its 60-150th passages. All viruses induced typical cytopathic effects. Yields obtained on the permanent calf thyroid line were, on average, 0.8 log10 lower than those obtained on primary calf testicle cells.

Adenoviridae↗

PubMed1986

Characterization of virulent isolates of vesicular stomatitis virus in relation to interference by defective particles.

To investigate the role that defective interfering (DI) particles might conceivably play in the epizootiology of vesicular stomatitis, two virulent New Jersey (NJ) isolates from the 1982-1983 epizootic in the United States (US) were compared with three laboratory adapted strains of vesicular stomatitis virus (VSV): NJ Hazelhurst, NJ Ogden and Indiana San Juan. Successive undiluted passages showed that the virulent isolates did not readily exhibit 'autointerference' because they did not readily generate and amplify DI particles. Viral RNA synthesis of isolates that were exposed to homotypic or heterotypic DI particles generated from the laboratory strains showed that the isolates were totally resistant to the heterotypic DI particle and partially resistant to the homotypic DI particle. In contrast, Indiana San Juan and NJ Ogden were inhibited by hetero- or homotypic DI particles. NJ Hazelhurst more closely resembled the isolates. This demonstrates that virulence of VSV in its natural setting may be related to a number of factors, including the slower generation and amplification of endogenous DI particles, as well as the increased resistance of the virus to some pre-existing DI particles.

Defective Viruses↗

PubMed1990

Virological applications of the grid-cell-culture technique.

Whole mounts of intact virus-infected cells have been used for several decades to examine virus-cell relationships and virus structure. The general concept of studying virus structure in association with the host cell has recently been expanded to reveal interactions between viruses and the cytoskeleton. The procedure permits utilization of immuno-gold protocols using both the transmission and scanning electron microscopes. The grid-cell-culture technique is reviewed to explain how it can be exploited to provide valuable information about virus structure and replication in both diagnostic and research laboratories. The use of the technique at the research level is discussed using bluetongue virus as a model. The procedure can provide basic structural information about intact virions and additional data on the intracellular location of viruses and virus-specific structures and about the mode of virus release from infected cells. Application of immunoelectron microscopy reveals information on the protein composition of not only released virus particles but also cell surface and cytoskeletal-associated viruses and virus-specific structures. Collectively, this simple and physically gentle technique has provided information which would otherwise be difficult to obtain.

Bluetongue virus↗

PubMed1996

Isolation of an in vitro transmissible agent with reverse transcriptase activity from a blood donor with a borderline-positive HIV-1 serology for more than five years.

BACKGROUND: Reverse transcriptase (RT) is present in all infectious retrovirus particles. Sensitive RT tests should thus detect all such particles. A family of ultrasensitive RT tests, product-enhanced reverse transcriptase (PERT) assays, have been designed. Accumulated results show that (i) a first version of the PERT assay that uses microtiter/ELISA technology detects RT of only 3-11 retroviral particles, (ii) very different human and animal lenti- and oncoviruses are detected very sensitively, (iii) HIV-1 is detected as sensitively as with polymerase chain reaction (PCR) for viral RNA, and (iv) prevalence of elevated particle-associated RT in plasma of unselected Swiss blood donors was 1.9%. OBJECTIVE: To investigate whether the RT activity detected in one unselected donor with a chronically elevated level of the liver enzyme alanine aminotransferase and two selected donors with chronically indeterminate or borderline-positive HIV-1 serology was due to human immunodeficiency virus (HIV) or human T-cell leukemia virus (HTLV) infection. STUDY DESIGN: Blood samples were tested by PCR for viral DNA and/or RNA of HIV-1, HIV-2, HTLV-1, HTLV-2, hepatitis B and C virus. Serological tests and virus cultures were also employed. RESULTS: Infection with any of the above agents could not be demonstrated. Virus cultivation in one case of borderline-positive HIV-1 Western blot for more than 5 years yielded a peak of RT production that was repeatedly transmissible to fresh cells. CONCLUSIONS: The findings suggest the presence of an infectious RT-positive agent, probably different from HIV-1/2 or HTLV-1/2, in a healthy individual with chronically borderline-positive HIV-1 serology. The PERT assay may detect retroviruses currently undetectable by other tests. The use of more stringent Western blot interpretation guidelines other than those of CDC or WHO is strongly recommended.

Journal Article↗

PubMedDate not supplied

Mononuclear phagocytes and the human immunodeficiency virus.

Macrophages are an important in vivo reservoir for HIV. Conclusive evidence that CNS, pulmonary, lymph node and blood-derived mononuclear phagocytes harbor and support HIV replication is supported by numerous independent studies. HIV variants which preferentially replicate in macrophages have been recovered from infected individuals, suggesting that these cells and variant viruses are involved in the establishment and progression of HIV-related disease.

Biological Factors↗