Amount Of Infectious Virus Research Articles

Biological functions of the NS1 protein of an influenza B virus mutant which has a long carboxyl terminal deletion.

To clarify the function of the NS gene of a highly cytolytic mutant of influenza virus B/Yamagata/1/73 which expresses an NS1 protein with a long carboxyl terminal deletion (clone 201), we prepared a single gene reassortant (201 L-77) and a control reassortant (YL-20) in which all the genes were of wild type influenza virus B/Lee/40 origin except NS gene which was derived from either clone 201 or wild type B/Yamagata. Comparative studies have revealed that 201 L-77 destructed infected cells more severely and much earlier after infection than did YL-20, although both produced comparable amount of infectious virus. The highly cytolytic reassortant 201 L-77 produced a small plaque, while the weakly cytolytic reassortant YL-20 produced a large plaque in MDCK cells. There was little difference between the two reassortants in the time course and the amount of synthesis of viral proteins within the infected cells. However, the mode of synthesis of viral RNA (vRNA) by 201 L-77 was greatly altered compared with YL-20.

Detection of foot-and-mouth disease antigen in bovine epithelial samples: comparison of sites of sample collection by an enzyme linked immunosorbent assay (elisa) and complement fixation test

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.

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.

Measles Virus Strain-dependent Variation in Outcome of Infection of Human Blood Mononuclear Cells

Eight measles virus strains including four subacute sclerosing panencephalitis (SSPE) isolates were compared on the basis of their growth characteristics in human peripheral blood mononuclear cells and effect on mitogen-stimulated lymphocyte proliferation. The Edmonston strain virus and some other strains of measles virus replicated in phytohaemagglutinin (PHA)-stimulated lymphocytes and released high titres of infectious virus into the culture medium. Inhibition of DNA synthesis was relatively low at the beginning of lymphocyte proliferation and could be detected only at high multiplicity of infection. Three to 4 days after initiation of proliferation a strong inhibition was seen also in cultures infected with low doses of virus, and was apparently related to the production of high titres of new infectious virus. Some virus strains originally isolated from SSPE patients produced only a small amount of infectious virus in lymphocytes at 37 degrees C but had a very strong inhibitory effect on lymphocyte proliferation leading to early cell death. The inhibitory effect was found over a wide range of virus concentrations, but was strictly dose-dependent and no increase in inhibition with low multiplicities of infection could be seen with longer culture times. The amount of interferon induced by different strains varied from 400 to 6400 international units/ml but the amount of interferon produced in the cultures did not correlate with the inhibitory effect on proliferation or with the amount of released new infectious virus. The Hallé measles strain, originally isolated from an SSPE patient, differed most from the Edmonston strain in its characteristics, and was studied in greater detail. Although only a small amount of infectious virus was produced, the Hallé strain had a very strong inhibiting effect on the proliferation of PHA-stimulated lymphocytes, and extensive syncytium formation was seen leading to cell death within 3 days.

In vitro infection of human monocytes with human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV).

We explored the possibility that normal human monocytes can be infected with the retrovirus human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). The T4 antigen, believed to be the receptor for HTLV-III/LAV binding to CD4 cells, is found on monocytes at low levels. Anti-T4A, which recognizes an epitope on the T4 molecule, inhibits viral binding to monocytes, and virus inhibits anti-T4A binding, although inhibition in both cases is not total. Virus particles were detected in HTLV-III/LAV-pulsed monocytes by electron microscopy as early as 10 min and for up to 3 days after inoculation, although budding virus was not observed. Monocytes were exposed to virus, were washed, and were cultured. Monocyte cultures were monitored by conventional assays for virus replication: immunofluorescence detection of cytoplasmic virus, supernatant reverse transcriptase activity, and supernatant virus antigen. These assays were either negative or at the lower limits of positivity. However, the amount of infectious virus was shown to increase over time in monocyte cultures by harvesting monocytes or their culture supernatants and titrating them into assay cultures containing stimulated T cells. Virus recovery from monocytes and virus recovery from T cells differed both quantitatively and qualitatively. Recovery from T cells and T cell supernatants peaked at 3 to 6 days and declined thereafter. Recovery from monocytes and monocyte supernatants increased over time in culture and never attained the levels of T cell cultures. Taken together, these studies indicate that HTLV-III/LAV binds to monocytes via the T4 molecule and enters the cells. Infectious virus is retained and increases with time in infected monocyte cultures. Both viral binding and infection are at low levels compared with levels in T cells. Unlike the usual infection of T cells characterized by high level virus replication with cell depletion, the infection appears to be persistent in monocytes.

Latent infection of human ovarian teratocarcinoma cells with human cytomegalovirus. Brief report.

Persistent infection with human cytomegalovirus (HCMV) can be established in cultures of human ovarian teratocarcinoma (PA1) cells, and maintained for more than 200 days. Infected cultures maintained at 34 degrees C (PA1CMV34) and 37 degrees C (PA1CMV37) entered crisis and subsequently displayed massive cytopathic effects (CPE), whereas infected cultures maintained at 32 degrees C (PA1CMV32) and 39 degrees C (PA1CMV39) continued to release small amounts of infectious virus until 240 or 151 days post-infection (p.i.) respectively. PA1CMV32 cultures shifted to 37 degrees C at 258 days p.i. resumed synthesis of infectious virus which resulted in cell destruction, indicating that latent infection with HCMV was maintained in PA1 cells at 32 degrees C. In contrast, PA1CMV39 cells did not produce infectious virus even when cultured at 37 degrees C for more than 100 days after the temperature shift.

Preliminary Characterization of a Persistent Infection of HeLa Cells with Human Rhinovirus Type 2

We were able to initiate a persistent infection (PI) in HeLa cells with a temperature-sensitive (ts-) mutant of rhinovirus type 2 (TS-1), but not with the corresponding wildtype (wt) virus. The ability to initiate a PI may be related to the multiplicity of infection. Persistence was established at 37 degrees C but not at 32 degrees C and the virus isolated from the PI was no longer temperature-sensitive. Infectious virus was continually produced at low levels throughout the course of the PI and cell cultures underwent multiple episodes of partial destruction (crisis) and subsequent recovery. PI virus and the initiating virus were neutralized to the same extent by hyperimmune polyclonal TS-1 antiserum indicating that no significant change had occurred with respect to serological type. The presence of either interferon or virus-related interfering activity could not be demonstrated in the PI cultures. Superinfection experiments in cells that were 'cured' of PI virus indicated the selection of a cell population during persistence that could no longer support the growth of homologous-type virus. This effect became less pronounced upon further passage of the cured cells. When compared with the wt and ts viruses, the PI virus yielded comparable amounts of infectious virus in HeLa cells but with decreased synthesis of RNA.

Is progressive multifocal leukoencephalopathy a chronic disease because of defective interfering particles or temperature-sensitive mutants of JC virus?

JC virus was examined for temperature sensitivity and for evidence of defective interfering particles as a means of explaining the slow chronic nature of progressive multifocal leukoencephalopathy (PML). JC virus direct from the brain tissue of seven persons with PML was not temperature sensitive as indicated by in vitro assay at 37 and 39 degrees C. In fact, more cells contained viral antigen at 39 than at 37 degrees C. The amount of infectious virus also was increased at 39 degrees C. Virions isolated directly from diseased brain tissue had a higher buoyant density than did virus from the same PML patient passaged in culture and containing genomic deletions. In contrast to DNA from culture-passed virus, DNA extracted from virions direct from brain tissue was homogeneous in length. In 13 separate cases examined, the viral DNA direct from the brain was homogeneous although variations in length were noted among DNAs from different cases. Restriction enzyme cleavage patterns identified all as JC virus DNA. It was concluded that neither temperature sensitivity nor DI particles can be used to explain the slow, progressive nature of PML.

Cells inhibited by n-butyrate support adenovirus replication

The ability of adenoviruses to replicate viral DNA and produce infectious virus particles in tissue culture cells blocked with n-butyrate has been examined. Cells are treated with millimolar amounts of n-butyrate, gradually stopping cell division and DNA synthesis; the block is reversible when n-butyrate is removed. When HeLa cells which have been thus inhibited are infected with human adenovirus type 5 (Ad5) in the continued presence of inhibitor, the virus adsorbed and penetrated the cells normally and viral DNA synthesis ensued. A normal rate and time course of DNA synthesis was observed, and butyrate-treated cells yielded the same amount of infectious virus as uninfected cells. Adenoviral DNA synthesis occurred without detectable stimulation of cellular DNA replication or of histone synthesis. The mechanism of butyrate inhibition of cellular DNA replication and unique features of adenovirus replication are discussed.

Viruses in sewage: effect of phosphate removal with calcium hydroxide (lime)

During calcium hydroxide (lime) treatment (pH 9.6 to 10.5) of wastewaters for phosphate removal there was also a two-log removal of added poliovirus (type I, Sabin) from effluents. A similar virus reduction was seen in the sludge generated in these experiments. However, in view of the limitations of techniques for virus recovery from sludge, only a small portion of the infectious virus present in lime sludge may have been detected. Storage of lime sludge at 28 degrees C for up to 48 h produced no appreciable reduction in the virus titre. Five sets of field samples of sewage, effluents, and sludge from a sewage treatment plant (Kemptville, Ont.) which utilizes lime for phosphate removal were also examined for indigenous viruses being BS-C-1 cells. All of the sample of lime sludge and 80% of the samples of both sewage and lime-treated effluent revealed virus; after chlorination only 20% of the lime-treated effluent samples were positive for virus. In contrast, in an earlier study with essentially the same experimental set up, 76% of the sample of chlorinated primary effluent were found to contain virus. Because of the easily detectable quantities of infectious virus in lime sludge and due to the lack of virus inactivation during storage of such sludge, caution must be exercised in its handling and disposal.

Immunopathogenicity and oncogenicity of murine leukemia viruses. I. Induction of immunologic disease and lymphoma in (BALB-c times NZB)F1 mice by Scripps leukemia virus.

This report clearly demonstrates that a systemic lupus erythematosus (SLE)-like syndrome and lymphoma can be induced in immunologically normal (BALB/c x NZB)F(1) mice by infection of neonates with a murine leukemia virus (MuLV) (Scripps leukemia virus [SLV] 60A) isolated from NZB lymphoblasts. SLV 60A was titered in vitro (XC test) and administered to newborn and adult (BALB/c x NZB)F(1) mice over six log(10) dilutions. Propagation of MuLV in the newborn recipients was indicated by greatly elevated serum Mu gs-1 levels which were proportional to the dose of virus given. The SLE-like syndrome was characterized by antinuclear antibodies (ANA) and immune complex-type glomerulonephritis. ANA production was related to the dose of virus and reached the highest levels at 8-16 wk. The incidence of glomerulonephritis was also correlated with the dose of virus and reached nearly 50% in the animals given the highest virus dose. Both titers of ANA and incidence of glomerulonephritis were greater in females than in males, although the amounts of Mu gs-1 in sera of both sexes were equal. The incidence of direct Coombs' positivity was not significantly affected by inoculation of this virus. The incidence and time of onset of thymocytic lymphoma were linearly related to the amount of virus inoculated. High serum Mu gs-1 levels predicted lymphoma development and reflected increases in the amount of infectious virus in the spleen. No induction of tumors, autoimmunity, or high serum Mu gs-1 levels followed administration of SLV 60A to 6-wk old (BALB/c x NZB)F(1) mice or inactivated 60A or active AKR virus to newborns.

Development of an equine herpesvirus in two cell culture systems: light and electron microscopy.

Development of equine herpesvirus strain 82A was studied in cells from primary horse kidney (HOK) cultures and an equine dermis (ED) cell strain. HOK and ED cells are equally susceptible to the 82A virus infection and yield about the same amount of infectious virus. Intranuclear inclusions were present in both cell systems, but a ring-shaped syncytial formation was observed only in infected ED cells. Ultrastructural studies revealed the presence of dense granules 30 nm in diameter and characteristic star-like clusters of granules in the infected HOK cells, but these granules were rarely seen in the infected ED cells. Viral nucleocapsids were associated with homogenous nuclear matrices, with moderate electron density in both cell systems. Viral nucleocapsids acquired envelopes by budding into the nuclear vacuoles in both HOK and ED cells. Budding from inner nuclear membranes into perinuclear cisterna or into cytoplasmic vacuoles also was observed frequently in HOK cells but was not seen often in infected ED cells. Multiple, membrane-bound intranuclear inclusions of fibrillar material which may be associated with virus envelopes were seen only in infected ED cells. Enveloped virus particles seen in nuclear vacuoles or perinuclear cisterna were more regular in shape and had a 130-nm diameter, whereas the enveloped virus particles seen in the cytoplasm and extracellular space were more irregular in shape and had a 130- to 160-nm diameter.

In vitro sensitivity of measles virus to 6-azauridine.

SummaryThe effect of 6-azauridine, an inhibitor of RNA synthesis, has been shown by 2 experimental methods to inhibit the replication of measles virus within mammalian cells in vitro. In a direct virus titration in which the drug was incorporated in the agar overlay, 6-AZ not only reduced the infectivity of measles virus but also delayed plaque development. Experiments designed to test the effectiveness of 6-azauridine when added to cultures at intervals after infection, showed that this uridine analog was capable of reducing the amount of infectious virus produced by each infected cell, and that the drug is more effective when added early in the infection cycle.

Inheritance of Susceptibility to Friend Mouse Leukemia Virus

Two newly established mouse strains which are congenic with standard inbred strains were used for the study of the locus Fv which controls the susceptibility to Friend leukemia virus in mice. A strain in each congenic pair shares the major histocompatibility gene with the corresponding partner strain but differs from the latter in the Fv locus. Mice with Fv(r)/Fv(r) genotype (DDD-Fv(r), C57BL/6) do not develop marked spleen enlargement upon virus challenge, whereas spleens of mice with Fv(s)/Fv(s) genotype (DDD, C57BL/6-Fv(s)) become large even with a virus inoculum 1/10(3) to 1/10(5) times that used for the resistant strains. Mice of each strain were heavily irradiated, inoculated with bone marrow cells taken from either syngenic or corresponding congenic mice, and challenged later with the leukemia virus. When Fv(s)/Fv(s) mice had been restored with bone marrow cells taken from Fv(r)/Fv(r) mice, the spleens remained small after the virus inoculation. In contrast, Fv(r)/Fv(r) mice receiving Fv(s)/Fv(s) cells responded to the virus with marked spleen enlargement. In the enlarged spleens of the C57BL/6 mice which do not otherwise allow the virus multiplication, a considerable amount of infectious virus was found. The altered response seems to be due to repopulation of destroyed tissues by the transplanted bone marrow cells. It is concluded that the locus Fv is expressed on hemopoietic cells, and cells derived from bone marrow play a predominant role in the development of splenomegaly by Friend leukemia virus.

Mechanism of resistance to enteroviruses of some primate cells in tissue culture.

Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.

Characterization of Defectiveness of Human Adenoviruses in Green Monkey Kidney Cells.

SummaryIn the absence of foreign helper viruses, human adenoviruses are defective in simian cells, but the degree of effectiveness varies with the adenovirus. Adenovirus 2 initiates an infection in the simian cells but there is no increase in virus titer. When simian cells are infected with adenovirus 7, a small amount of infectious virus is produced but the amount never exceeds the input level. Each cell in the population produces a small amount of virus. The increase in infectious virus is dependent upon DNA synthesis. The simian cells therefore appear to be “leaky” for some human adenovirus serotypes.

Effect of interferon on early enzyme and viral DNA synthesis in vaccinia virus infections.

The quantities of infectious virus and viral DNA-polymerase produced and the rate of viral DNA synthesis were all inversely related to the interferon concentration in vaccinia virus-infected monolayers of whole chicken-embryo fibroblasts. However, virus production was a least 10 times more sensitive to interferon inhibition than were the other parameters studied. Increasing concentrations of interferon depressed the initial rate of synthesis of the DNA polymerase as well as its final yields. The response of the rate of DNA synthesis to interferon concentration was not affected by varying the virus inoculum from 0.1 to 4.0 plaque-forming units per cell. Radioautographic studies of viral DNA synthesis demonstrated that inhibition by interferon is not “all or none” even at the level of the vaccinia inclusion. Instead, increasing concentrations of interferon reduced the quantity of DNA synthesized per site in addition to the proportion of cells initiating DNA synthesis and the number of DNA synthesizing sites per cell. A large proportion of the cells initiated viral DNA synthesis even after treatment with an interferon concentration that reduced virus yield 98%.

Differentiation of plants from tobacco mosaic virus inclusion-bearing and inclusion-free single tobacco cells.

Abstract Tobacco callus cells from normal and tobacco mosaic virus (TMV) infected Nicotiana tabacum Havana 38 plants were studied by phase contrast microscopy. The cells infected with TMV frequently had hexagonal crystals, gray plates, paracrystalline needles of various shapes, and spherical bodies occasionally bearing hexagonal plates. Early stages of formation of hexagonal plates and paracrystalline fibers were observed. The spherical bodies started as blebs in cytoplasmic strands which enlarged and eventually separated. The number of inclusion-bearing cells gradually declined during successive passages, but two isolates showed inclusions even after 16 passages. Inclusion-bearing cells had a higher virus concentration than inclusion-free cells. Only 10 and 70 cells, respectively, divided out of 100 inclusion-bearing cells and 150 inclusion-free cells. Eventually 5 and 65 formed colonies when cultured singly in microchambers. The amount of infective virus in these clones varied considerably. Three clones were free of virus. The virus-containing and virus-free single cell clones readily differentiated when grown on Murashige and Skoog medium with 4 mg/liter kinetin and 1.5 mg/liter indoleacetic acid. Plantlets were transferred to White's medium, rooted, and subsequently grown in pots. Healthy and virus-infected plants were thus obtained from infected single cells. Single cells infected with TMV were capable of division and colony formation, and supported virus multiplication. The morphogenetic potential of the cell to develop into a plant appeared unaltered by TMV; infected cells were still totipotent.

In vivo- undin vitro-verhalten temperatursensitiver mutanten des Tabakmosaikvirus

It has been found that certain mutants of TMV with known amino acid exchanges produce abnormally low quantities of infectious virus particlesin vivo at 32°C, although the particles themselves are stable at even higher temperatures. A correlation between temperature sensitivity of the mutantsin vivo and the inability of their A-Proteins to reaggregate to rodsin vitro at 30°C is demonstrated. It is concluded that the amino acid exchanges in coat protein lead to temperature sensitivity of the mutants described.

Antibody response in rabbits to adenovirus type 12.

The report by Trentin and associates (1) on the oncogenic properties of Adenovirus Type 12 has been confirmed by Huebner et al.(2) who have further demonstrated the production of tumors in hamsters with Adenovirus Type 18. These studies suggest a need for an in vitro test to differentiate strains of a particular Adenovirus type. The following pilot study on antibody production to Adenovirus in rabbits was initiated with the ultimate goal of demonstrating immunological differences among Adenovirus Type 12 strains. Previous reports(3–7) involved hyperimmunization with multiple doses of large amounts of infectious virus. As here shown, satisfactory antibody titers have been obtained following a single injection of relatively small amounts of viral antigen.Methods. New Zealand rabbits, 3.5 to 4.5 kg, were bled immediately prior to inoculation, and at 1, 7, 15, 21, 28, 30, 35 and 42 days thereafter. Sera were clarified by light centrifugation and stored at −20°C until tested.Adenovirus Type 12 antigen was prep...