Francis E. Payne

Isolation of measles virus from cell cultures of brain from a patient with subacute sclerosing panencephalitis.

Abstract Isolation of measles virus from the brain of a patient with subacute sclerosing panencephalitis was accomplished by in vitro propagation of the patients brain cells and cocultivation of the brain cells with a line of green-monkey-kidney cells. This observation offers direct evidence that measles virus has a a role in the etiology of the disease. Before the production of detectable, mature virus by the cell cultures, there was a prolonged period when they produced syncytia containing measles antigen, but they failed to hemadsorb.

Studies of the biosynthesis of protein and ribonucleic acid in HeLa cells infected with poliovirus

Abstract The amounts of protein, RNA (ribonucleic acid), virus, and the rate of incorporation of P 32 into RNA were determined in various subcellular fractions of HeLa cells at various times during a single sequence of infection with poliovirus. From these data interpretations are drawn concerning the biosynthesis at the cellular level of protein and RNA which are induced by virus infection. Within 1 hour after the initiation of infection, there is a detectable accumulation in the cellular cytoplasm of newly synthesized protein and RNA. The synthesis of protein continues at a constant rate until the seventh hour of infection. RNA in the cytoplasm increases at a constant rate until the fourth hour, at which time the rate is markedly enhanced, and the first virus, as infectious activity, appears in the cytoplasmic fraction. The synthesis of RNA stops by the sixth hour. Virus accumulates at an increasing rate in the cytoplasm between the fourth and seventh hours. At the seventh hour, 99% of the virus formed is present in the intracellular state. From the amounts of nucleic acid and protein produced, their distribution relative to virus among the various subcellular fractions, and from the nucleotide composition of the RNA, it was concluded that the major portion of the newly formed materials was not virus.

Initial stages of the interaction of HeLa cells with poliovirus.

As a c(~nsequence of the usual interaction of viruses and cells, one detects an initial loss of viral activity, a viral increase and destruction of cells. I n such cases the reaction may be followed in terms of any of the resultant~effects, ttowever, all interactions may not b e productive of virus; nor result in cellular ,destruction and viral activity m a y lend itself .to competing reactions. Td follow the reaction by a single effect may preclude the observation of unexpected phenomena. The present study is centered upon the cytopathogenic effect of the poliovirus and for this reason its reaction with the HeLa cell is followed primarily in terms of cellular destruction. For this purpose a method has been developed for quantitating cellular injury. By the application of the method, the kinetics of two early phases of the interaction of virus and cell have been investigated.

EFFECT OF 5-FLUORO-2'-DEOXYURIDINE ON THE SYNTHESIS OF VACCINIA VIRUS.

Abstract The effect of FUDR on vaccinia virus replication in HeLa cells was investigated. The incorporation of deoxyuridine-H 3 into viral DNA, as determined by autoradiography, was completely blocked by 10 −6 M FUDR. This concentration of the analog prevented production of infectious virus above levels present in the cultures after washing at the end of the viral adsorption period. The production of vaccinial HA was also inhibited. However, 20–70% of the inhibited cells produced NP and LS antigen detectable by the fluorescent antibody technique. These findings were in accord with the results of previous experiments using p -fluorophenylalanine which had suggested that viral antigen can be synthesized in the absence of synthesis of viral DNA. The proportion of cells that synthesized antigen was progressively reduced by increasing the concentration of FUDR or increasing the duration of pretreatment of cultures with the analog. The inhibition of synthesis of infectious virus and of viral antigen was not prevented by uridine added with the FUDR but was reversed by thymidylic acid added at the time of infection to cultures that had been pretreated for as long as 12 hours with FUDR. When FUDR was added to cultures at the time of infection and thymidylic acid was added 12 hours later, the proportion of cells that synthesized viral antigen was essentially the same as in cultures not treated with FUDR; however, the yield of infectious virus in the former cultures was only 15% of that from cultures not treated with the analog.

Effect of p-Fluorophenylalanine on the synthesis of vaccinia virus

Abstract The effect of the amino acid analog FPA on vaccinia virus replication in HeLa cells was investigated. By varying the dose of FPA or the time after infection at which the analog was added to cultures, it was possible to inhibit differentially the synthesis of viral components in vaccinia-infected cells. When sufficient FPA was added to cultures at the time of infection, the synthesis of viral DNA, NP antigen, infectious virus, and HA was inhibited. The inhibition of synthesis of LS antigen under all conditions studied was only partial. FPA added to cultures at 6 or more hours post-infection did not inhibit production of viral antigens or infectious virus. The inhibition was reversed by PA added with the FPA, but PA added at later times was progressively less effective in restoring the ability of inhibited infected cultures to produce virus. The proportion of nuclei incorporating thymidine-H 3 was decreased in cultures infected with vaccinia virus. This decrease was not seen in cultures receiving FPA at the time of infection.

Susceptibility of a Strain or Rat Virus to Statolon and Other Virus Inhibitors.

Summary Rat virus, strain XI4, a small (22 m/x) virus isolated from the mammary tissue of an X-irradiated rat, was found to be inhibited by statolon and by guanidine. Since statolon, a penicillium-derived antibiotic previously had been found to inhibit RNA viruses of the arbor- and entero-groups, it is noteworthy that rat virus was also inhibited by 5-fluoro-2-deoxyuridine and that the inhibition was prevented by thymidine, a conventional identification of a DNA virus.

Mycoplasma in the uterine cervix.

Abstract A study of Mycoplasma in the uterine cervix was made on 2 groups of 150 women each. One group consisted of women attending a venereal disease clinic; the other of participants of a family planning clinic. Ages ranged from 16 to 61 years in the venereal disease group and 15 to 45 years in the family planning women. Both groups were from lower income family units and of similar ethnic background. The prevalence of Mycoplasma in the cervix was 92 per cent in the venereal disease group, as compared to 38 per cent in the family planning group. Mycoplasma hominis, type 1 was the predominant species of mycoplasma isolated from both groups. Within either the venereal disease or family planning group, age, phase of the menstrual cycle, type of contraception, or non-Mycoplasma genitourinary tract infections did not significantly relate to the prevalence of cervical Mycoplasma. Mycoplasma isolations did not relate to cytologic evidence of premalignant or malignant alteration of cervical cells in either group of women. Coccoid bodies associated with the cytoplasm of squamous cells were present in cervical smears from about 50 per cent of women yielding M. hominis as opposed to about 10 per cent of women not yielding Mycoplasma.

Radioimmunoassay of Measles Virus Nucleocapsid Antigen

A double antibody competitive binding radioimmunoassay (RIA) was developed as a tool for investigating the involvement of measles virus in persistent virus infections. The assay employs 125I-labelled measles virus nucleocapsids as the labelled antigen. Nucleocapsids were purified from cytoplasmic extracts of virus-infected Vero cells, treated with trypsin to prevent clumping and iodinated to a specific activity of about 15 microCi/microgram. Electrophoretic analysis of iodinated trypsin-treated nucleocapsids revealed only labelled polypeptide with a relative mol. wt. (Mr) 40 000, indicating that trypsin had cleaved the 60 000 mol. wt. native polypeptide to a 40 000 mol. wt. subunit. The assay could detect as little as 0.1 ng of nucleocapsid protein. Either native (Mr 60 000) or cleaved Mr 40 000) nucleocapsid polypeptide was detected in the assay. As much as 100 micrograms of protein from uninfected Vero cells did not react in the RIA. Another measure of specificity was the fact that 400 ng of purified nucleocapsid protein from simian virus 5, Newcastle disease virus or canine distemper virus did not react in the assay. In the RIA, nucleocapsid antigen of virus isolated from subacute sclerosing panencephalitis (SSPE) was indistinguishable from that of Edmonston strain measles virus, indicating antigenic homology between the 40 000 mol. wt. nucleocapsid polypeptide of the SSPE virus and that of measles virus.

Evidence of precursors of defective measles virus

Cytoplasmic extracts of Vero cells infected with wild strain Edmonston measles virus were found to contain at least two distinct nucleocapsid species. The two most prominent species of nucleocapsids sedimented at 200S and 110S and contained RNA of molecular weight 6.0×106 and 0.6×106 daltons respectively. Both species of nucleocapsids had a density of 1.31 g/cm3 in CsCl. A third species sedimenting at 170S was not present in all experiments and was not characterized in detail. Infection of cells with undiluted-passage virus usually resulted in production of mostly 110S nucleocapsids while both 110S and 200S species were found when diluted-passage virus was used. These results suggest that measles virus may produce distinct classes of defective virus which contain segments of RNA representing as little as 10% of the complete viral genome.

Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains.

A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs 70-100% of the measles virus-specific RNA present these SSPE brain samples were recovered in this enriched fraction.