CULTURE OF VIRUS OF TICK-BORNE ENCEPHALITIS IN VARIOUS TISSUE LINES.
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Correlation of cytopathic effect, fluorescent-antibody microneutralization, and plaque reduction test results for determining avian infectious bronchitis virus antibodies.
A microneutralization fluorescent-antibody (MFA) test was effective in determining the level of antibodies to avian infectious bronchitis virus. A comparison of the MFA test with the cytopathic effect microneutralization (MNT) test and 50% plaque reduction (PR) test resulted in positive correlations that were significant (P less than 0.001). The PR test was more sensitive than either the MFA or the MNT test, but there was no significant difference between the sensitivities of the MFA and MNT tests. The MFA test has advantages over the PR test in the capacity to test large numbers of sera in a shorter period of time. The MFA test also can be completed in one-half the time required for the MNT test.
Animals↗
Propagation of infectious bovine rhinotracheitis (bovine herpes-1) virus in murine primary cell cultures.
Virus synthesis in BALB/C mouse lung and kidney primary cultures infected with infectious bovine rhinotracheitis (IBR) virus started between 6 and 8 hours after virus inoculation and reached a maximum titer of 5.5 log10 plaque forming units (PFU) at 48 hours postinfection (PI). Cytopathic effect (CPE) in cell cultures occurred at 8-10 hours and over 90% of the cells had CPE by 48 to 72 hours PI. The bulk of the newly replicated virus (60-80%) was cell-associated as determined by plaque assay of extracellular and intracellular virus. Pulse-chase experiments demonstrated incorporation of radioactive precursors into viral DNA and protein macromolecules. Viral DNA synthesis was initiated between 2 and 4 hours PI and was maximum at 4 to 6 hours. Viral proteins were detected at 4 hours and peaked between 6 and 8 hours PI. Enzyme-linked immunosorbant assay (ELISA) confirmed synthesis of specific viral proteins, which gradually increased during the virus growth cycle. Abstract in French is given at the end of the article.
Animals↗
Selection of attenuated dengue 4 viruses by serial passage in primary kidney cells. IV. Characterization of a vaccine candidate in fetal rhesus lung cells.
A strain of primary dog kidney (PDK)-passaged dengue (DEN) 4 (H-241) virus cloned by terminal dilution (PDK 35-TD3) was propagated in large volumes in fetal rhesus lung (FRhL) cells to produce a candidate vaccine for evaluation in man. Production seed (FRhL p2) and candidate vaccine (FRhL p3) were subjected to rigorous safety tests to exclude contaminating microbial agents. There was no significant monkey neurovirulence of parental or PDK-passaged DEN-4 virus or of control fluid cultures. FRhL-passaged viruses retained the phenotypic characteristics: small (occasional medium) plaque; temperature sensitivity at 38.5 degrees C; and absence of plaque formation in African green monkey kidney cells, cytopathic effect in LLC-MK2 cells, and viral growth in human monocytes. FRhL p2 virus displayed low virulence for monkeys; only one of four animals was viremic and three of four developed low-titered antibody. FRhL p3 virus produced viremia in three monkeys and moderate to high hemagglutination-inhibition and neutralizing antibody titers in all animals. Virus at both passages in FRhL exhibited reduced neurovirulence in suckling mice as compared to parental DEN-4. Because of its safety and desirable monkey virulence attributes PDK 35-TD3 FRhL p3 is recommended for human phase I trial.
Animals↗
In vitro identification and characterization of a virus isolated from a dog with neurological dysfunction.
A virus, 78-238, isolated from the cerebrospinal fluid of a dog with neurological dysfunction, was characterized as a paramyxovirus. This conclusion was supported by viral cytopathic effects and morphological appearance of virions and nucleocapsids in infected cells. Nucleocapsids were found in the cytoplasm of all infected cells and in the nuclei of 0.001% of these cells. Growth curves revealed that a high percentage (>/=76%) of infectious progeny virus was cell released. Persistent infection of Vero cells with 78-238 showed a consistently high percentage of fluorescence-positive cells and a low proportion of hemadsorption-positive cells. Serological studies indicate that the virus was closely related to Simian virus 5 and reference canine parainfluenza virus.
Animals↗
Stability of neurotropic mouse hepatitis virus (JHM strain) during chronic infection of neuroblastoma cells.
A line of mouse neuroblastoma cells which was chronically infected with the neurotropic strain (JHM) of MHV, a member of the coronavirus group, was established. These cells, designated NJ, exhibited typical MHV cytopathic effects (CPE) at all passage levels along with the continual production of infectious virus. Most cells were positive for viral antigen by immunoflourescence. Viral particles consistent with the morphology of MHV were found by electron microscopy. The uninfected neuroblastoma cell line did not contain a detectable population of cells resistant to JHM, and persistence did not elicit the production of interferon. No plaque morphology or temperature sensitive mutants were selected for in the NJ culture, and we were unable to detect the presence of either a defective or defective interfering virus population. The addition of low concentration antiviral antibody modulated the infection to a carrier culture with viral antigen in the cytoplasm of the cells, but no infectious virus was produced, and the cells lacked both surface viral antigen and CPE. Possible mechanisms of viral persistence in vitro are discussed.
Animals↗
Detection of adenovirus by enzyme-linked immunosorbent assay.
A solid-phase direct enzyme-linked immunosorbent assay (ELISA) was developed for the detection of adenovirus antigen in extracts of infected cells by using antihexon serum. Results with simulated clinical specimens consisting of normal nasal wash specimens seeded with varying concentrations of adenovirus type 5 showed that antigen could be detected in extracts of HEp-2 cell cultures inoculated with 10(2.5) 50% tissue culture infective doses (TCID50) and 10(1.5) TCID50 after 2 and 4 days of incubation, respectively. Fifty-three clinical nasal wash specimens containing adenovirus type 5 (stored for 5 years at -70 degrees C) were used to evaluate antigen detection by ELISA in HEp-2 cell extracts and by manifestation of cytopathic effect in human embryonic kidney cells. After 2 days of incubation, 62% were positive by ELISA, whereas none was positive for cytopathic effect. After 4 days of incubation, 76% were ELISA positive and 47% were positive for cytopathic effect. The results according to infectivity titers indicated that clinical specimens containing 10(3.0) TCID50 or greater were all positive by ELISA after 2 days of incubation in HEp-2 cells, and by 4 days all but one specimen containing 10(2.0) TCID50 or greater were ELISA positive. ELISA and immunofluorescent methods for antigen detection were compared using 24 of the 53 clinical specimens containing adenovirus type 5. Nearly equivalent sensitivities were demonstrated. These results suggest that ELISA may provide an alternative method of detecting and identifying adenoviral infections in humans.
Adenoviridae Infections↗
Mouse hepatitis virus (MHV-2). Plaque assay and propagation in mouse cell line DBT cells.
Various factor sinfluencing the plaque formation of mouse hepatitis virus (MHV-2) in DBT cell monolayers were studied and a practical method for plaque assay was developed. Infected DBT cells yielded high-titered virus and were a satisfactory source of complement-fixing viral antigen. The predominant cytopathic effect of MHV-2 in DBT cells was cell rounding and detachment, but no syncytial formation was observed. Fluorescent antibody staining revealed specific fluorescence only in the cytoplasm of infected DBT cells. In one-step growth experiment, newly formed virus was first recognized within 4-hr postinfection and showed subsequently a rapid exponential increase. Release of newly formed virus from the cell was rapid, and a continuous release lasted for a certain period of time. The average per-cell yield of active virus was estimated to be about 6-7 X 10(2) plaque-forming units.
Animals↗
Vaccinia keratouveitis manifesting as a masquerade syndrome.
A patient who used contact lenses and had a history of blunt trauma developed vaccinia keratouveitis after accidental ocular autoinoculation from a recent vaccination site. Corneal and conjunctival cultures were taken for bacteria, fungi, Acanthamoeba, and viruses. Viral-like cytopathic effects became evident in tissue culture within three days. Immunofluorescence studies were negative for varicella-zoster virus, herpes simplex virus, adenovirus, measles, mumps, parainfluenza, and influenza. Pox viral particles were identified in the infected tissue cultures by electron microscopy. The Hind III restriction endonuclease profile of the viral DNA isolate was similar to the Lister strain of vaccinia virus. Ocular vaccinia may manifest as a masquerade syndrome and may mimic signs of herpes simplex virus, varicella-zoster virus, and Acanthamoeba infection. Although vaccination with vaccinia is currently limited to a few populations throughout the world, vaccinia must still be considered in the differential diagnosis of infectious keratouveitis.
Adult↗
Secondary heterotypic versus homotypic infection by Coxsackie B group viruses: impact on early and late histopathological lesions and virus genome prominence.
The impact of prior exposure to a different or identical strain of Coxsackievirus B (CVB) on murine CVB myocarditis was studied using a susceptible murine host (A/J[H-2a]) and myocarditic CVB3 or avirulent CVB2 as primary or secondary infectants. The effects of secondary heterotypic infection (CVB2 followed by CVB3) and homotypic infection (CVB3 followed by CVB3) 28 days after primary inoculation, versus CVB2 or CVB3 alone, on injury and viral genomic replication, both early (day 7) and late (days 28 and 56), were evaluated. After the primary infection by CVB2, trivial viral RNA was present in the heart and other organs, and a substantial positivity was observed with CVB3 infection. Seven days after secondary heterotypic (CVB2-CVB3) infection, the quantity of CVB genome in heart, pancreas, liver, and spleen was increased compared with the virus genome in the CVB3-CVB3 group and in the group with primary CVB3 infection alone. This phenomenon was seen in the heart and spleen up to day 28 postsecondary infection. Tissue inflammation and necrosis in heart and pancreas were prominent 7 days postsecondary infection with CVB2-CVB3 and correlated well with an increased quantity of CVB genome. Virus genome was present in heart and spleen 28 days after CVB3 infection alone. Serum CVB3 neutralization titer was increased to 1:128 in CVB2-CVB3 group at days 7 and 28 postsecondary infection, and serum completely neutralized cytopathological effects of CVB3 in the CVB3-CVB3 group at day 7 and 28 postsecondary infection. Our results indicate that secondary heterotypic infection by CVB causes increased injury, inflammation, and CVB replication in target organs such as the heart and pancreas, as well as in immune compartments like the spleen. Compared with CVB3 alone, the intense inflammatory infiltriate in the CVB2-CVB3 group is as not due solely to postviral sensitization of the immune system, but rather to the inability of the host to eradicate the virus.
Animals↗
[The isolation of measles virus strains and the study of their hemagglutinating activity].
Three measles strains Gag, Il, and Buk, were isolated from a suspension of mononuclear cells derived from measles patients in the active stage of the disease. Continuous Vero cell cultures were used for virus isolation. In the infected cell culture, all the isolates produced symplasts of different sizes and star-shaped or spindle-shaped multinuclear cells. The specificity of the cytopathic effect was proved by the adsorption of monkey erythrocytes on the surface of virus-affected cells. The isolates were identified in neutralization and HI tests with different immune preparations: measles gamma-globulin (national standard), hyperimmune rabbit sera to measles (Edmonston strain), rubella, and mumps viruses, S. sonnei and S. flexneri, as well as with sera from measles patients and subjects vaccinated with live measles vaccine L-16. The results of identification attest to isolation of 3 measles virus strains, one of which (Buk) possesses particularly high hemagglutinating activity.
Animals↗
Interferon-directed inhibition of chronic murine leukemia virus production in cell cultures: lack of effect on intracellular viral markers.
Extracellular murine leukemia virus (MLV) reverse transcriptase activity was decreased by interferon treatment in four interferon-sensitive mouse cell lines which were chronic MLV producers. In three cell lines which were relatively insensitive to interferon, extracellular enzyme activity remained unchanged by interferon treatment. The concentrations of interferon used had no effect on DNA synthesis or cell replication of AKR,C+ cells which were chronic producers of AKR-MLV. In AKR,C+ cultures interferon treatment also had no effect on the level of intracellular viral reverse transcriptase activity in spite of an inhibition of extracellular enzyme activity. Treatment of AKRC+ cultures with interferon for 9 days inhibited extracellular viral reverse transcriptase levels throughout the period of treatment; however, the intracellular enzyme activity remained unchanged, and concentrations of viral p30 (gs) antigen were increased in the interferon-treated cells. When the cells were washed to remove interferon, however, virus production rapidly rose and intracellular p30 antigen fell to the levels of untreated AKR,C+ cells. These and previously reported results suggested that in interferon-treated AKR,C+ cells virus production is inhibited at a late step in the MLV replication cycle, either directly or through the inhibition of the production of a protein required for virus assembly.
Animals↗
Cell culture propagation of porcine rotavirus (reovirus-like agent).
Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.
Animals↗
Infection of human peripheral blood mononuclear cells by varicella-zoster virus.
The infection of human peripheral blood leukocytes by varicella-zoster virus (VZV) was studied using an infectious center assay, indirect immunofluorescence and electron microscopy. Subsets of freshly isolated leukocytes were prepared, including granulocytes, mononuclear cells from Ficoll-Hypaque gradients, lymphocytes, and glass-adherent monocytes. When each of these populations was inoculated with VZV (MOI = 0.1), there was no evidence of effective infection. However, when monocytes were cultured in vitro for 7 days, they differentiated into macrophages that were productively infected with VZV. Peak percentages of infectious macrophages were detected 8-24 h after inoculation (mean 17.5%; range 10.2-30.4%). Using indirect immunofluorescence, viral antigens were detected in the cytoplasm and at the nuclear membranes of infected macrophages between 24 and 72 h after infection. Electron microscopy demonstrated the appearance of viral particles in the nucleus by 24 h. Large numbers of virions, often collected in tubules or vacuoles, were present in the cytoplasm at 48 h. The difference between the infection of fresh monocytes and cultured macrophages by VZV might reflect differences in their metabolic or differentiation state. The possible significance of these observations to VZV infection of immunocompromised hosts is discussed.
Antigens, Viral↗
Serological differences between human T-lymphotropic virus type-I (HTLV-I) and HTLV-III/LAV.
Sera from each of five preselected groups of patients with acquired immune deficiency syndrome (AIDS), AIDS-related complex (ARC), hemophilia, adult T-cell leukemia (ATL), and healthy controls were examined for antibodies to human T-cell leukemia (T-lymphotropic) virus type-I (HTLV-I) and HTLV-III by indirect immunofluorescence (IF) and radioimmunoprecipitation (RIP) methods. All sera from five patients with AIDS, ARC, and hemophilia reacted at titers from 1 : 512 to 1 : 5,120 with fixed H9/HTLV-III cells by IF but not with fixed MT-1 cells carrying HTLV-I. Similarly, sera from patients with AIDS, ARC, and hemophilia precipitated HTLV-III-specific polypeptides of 120K, 46K, and 24K. In contrast, sera from five patients with ATL did not react with fixed H9/HTLV-III cells, but reacted with fixed MT-1 cells. Moreover, HTLV-I-specific polypeptides of 68K, 28K, and 24K were precipitated with sera from ATL-patients but not with anti-HTLV-III-positive sera. Recently, we infected HTLV-I-carrying MT-4 cells with HTLV-III and provoked strong cytopathic effects. This system enabled testing for neutralizing antibodies to HTLV-III. Neutralizing titers to HTLV-III of five anti-HTLV-III-positive sera ranged from 1 : 720 to 1 : 9,000. In contrast, all five seronegative controls showed no or only low reactivity to HTLV-III envelope (1 : 80 and 100). However, three out of five anti-HTLV-I-positive sera exhibited weak cross-reactivities with HTLV-III. The reactivities were expressed as less than 1 : 160, except for one case (1 : 720). They were considered to be nonspecific since they were negative for HTLV-III antibodies in the radioimmunoprecipitation studies.
Acquired Immunodeficiency Syndrome↗
Latent infection and reactivation of human herpesvirus 6 in two novel myeloid cell lines.
It has been reported that reactivation of human herpesvirus-6 (HHV-6) causes a failure of hematopoiesis. To clarify the mechanisms of bone marrow suppression induced by HHV-6 infection, it is necessary to establish an in vitro model of HHV-6 infection in hematopoietic progenitor cells. We have established two novel Philadelphia chromosome-positive myeloid cell lines, SAS413 and SAS527, which possess different hematologic characteristics and show distinct susceptibility to infection by HHV-6, from a patient with blast crisis of chronic myelogenous leukemia (CML). HHV-6 subgroup A (HHV-6A) showed marked replication in SAS413, forming syncytia and inducing cell lysis in short-term culture. On the other hand, HHV-6A-inoculated SAS527 continued to proliferate without cell lysis and only a few cells showed HHV-6 antigen expression. In contrast to HHV-6A infection, inoculation with HHV-6 subgroup B (HHV-6B) did not induce any cytopathic effect (CPE) or viral antigen expression in either of the cell lines. Although HHV-6B replication was undetectable, the presence of the HHV-6 genome in both cell lines was shown by polymerase chain reaction (PCR) during culture for more than 10 months, suggesting that HHV-6B latently infected SAS413 and SAS527. Phorbol ester treatment of SAS527 latently infected with HHV-6B resulted in reactivation of HHV-6, as shown by the appearance of a CPE, positive reactivity for the HHV-6 antigen, and isolation of infectious HHV-6. These novel cell lines should be useful for studying the mechanisms of HHV-6-induced hematopoietic failure and HHV-6 latency and reactivation, as well as differentiation, of the myeloid cell lineage.
Adult↗
Infection of a human leukemia K-562 cell line with Semliki Forest virus.
Infection of the human leukemia hematopoietic stem cell line, K-562, with Semliki Forest virus (SFV) can be characterized by three stages: (1) an early virus-proliferating stage lasting 1 to 4 days post-infection (pi) in which infectious virus is produced in high titers (10(3)pfu/cell) but in which there is minimal cytopathic effect. All cells appear viable by trypan blue dye exclusion, although they do not proliferate, and DNA and cell protein synthesis decrease to less than 3% of uninfected controls within 24 hours; (2) an intermediate stage extending from day 5 to about day 24-30 pi in which the amount of infectious virus declines to low levels. During this stage, viral protein synthesis decreases to undetectable levels, although viral gylcoproteins are readily demonstrated by immunofluorescence and by immunoblot; however, capsid protein appears to degrade within 21 days pi. Cell numbers remain constant but the viability of the non-proliferating cells determined by trypan blue exclusion could not be determined with confidence; (3) a final long-term stage in which viral glycoproteins, E1 and E2, are detectable by immunoblots and immunofluorescence for many months but the cells are metabolically inactive and do not synthesize viral proteins. These non-viable cells do not lyse for as long as 2 years.
Antigens, Viral↗
Isolation and antibody prevalence of a parapoxvirus in wild Japanese serows (Capricornis crispus).
An epizootic suspected to be caused by parapoxviruses occurred in winter, 1984-85, among wild Japanese serows (Capricornis crispus) with nodular or papular lesions in Gifu Prefecture, Japan. Virus isolations were attempted on 30 animals using bovine fetal testicle cell cultures. Viral agents growing with cytopathic effects were isolated from six animals. The agents were identified as parapoxvirus from electron microscopic findings and physico-chemical characteristics. Antibody prevalence to the isolated agent, named S-1, was examined by enzyme-linked immunosorbent assay. No antibodies were detected among 153 sera obtained during two winters 1981-82 and 1982-83, but one of 189 sera collected in the winter of 1983-84 was positive. In contrast, 75 (32%) of 237 sera had antibodies to S-1 in 1984-85, when the disease was first detected. Antibody prevalences of 1984-85 were highest (39%) in December 1984, the first month of animal capture, and declined gradually to 20% in March 1985.
Animals↗