Serial Undiluted Passage Research Articles

Reassortment of simian virus 40 DNA during serial undiluted passage.

Alterations occur in the supercoiled form of viral DNA after the serial undiluted passaging of simian virus (SV) 40. We have identified a portion of the viral genome which is amplified during this process. These SV40 DNA sequences represent about 30% of the viral genetic information and are present in a reiterated form in twisted circular molecules prepared from purified virions. In addition, reiterated and unique green monkey DNA sequences are incorporated into supercoiled viral DNA. The cellular DNA appears to be inserted at numerous locations in the DNA I molecules.

Acquisition of sequences homologous to host DNA by closed circular simian virus 40 DNA. II. Further studies on the serial passage of virus clones.

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.

The evolution of new species of viral DNA during serial passage of simian virus 40 at high multiplicity

Viral DNA isolated during serial undiluted passage of SV40 was analysed by electrophoresis of fragments produced by H. influenzae restriction endonuclease and by DNA-DNA hybridization. Compared to parental SV40 DNA, early passage DNA showed alterations at many sites in the population of DNA molecules, although certain sites were more frequently altered than others as evidenced by the reduced yield of some original DNA fragments and the appearance of new fragments. With continued passage, new dominant species of viral DNA evolved which yielded simple restriction endonuclease digest patterns. DNA molecules from all passages retained SV40 DNA sequences; however, the percentage of total DNA sequences which corresponded to SV40 sequences diminished to about 30% or less in late passage DNA. Repetitive host cell DNA sequences were detected in a high percentage of late passage DNA molecules, but accounted for 20% or less of total sequences present. Therefore the new species of viral DNA which have evolved probably contain predominantly nonrepetitive host DNA sequences.

Defective Virions of Herpes Simplex Viruses

Serial undiluted passage of clonally purified herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in 4 different types of cells resulted in (i) partial loss (84–98%) of infectivity and (ii) the app

Electron microscopic study of development of vesicular stomatitis virus using ferritin-labelled antibodies.

SUMMARY Significant virus autointerference was not observed in serial undiluted passages of vesicular stomatitis virus in Madin–Darby canine kidney cells. The ultra-structural features of vesicular stomatitis virus development into Madin–Darby canine kidney cells were studied by staining frozen-thawed cells with ferritin-conjugated antibody. The granular and fibrillar antigen could be readily identified in the cytoplasm within 4 hr after infection. Its appearance was followed closely by the differentiation of virus filaments at the cell surface. The virus antigen could be recognized in close proximity to virus particles; this material may represent virus nucleoprotein incorporated into budding virus particles.

The RNAs of infective and incomplete influenza virions grown in MDBK and HeLa cells

The MDBK line of bovine kidney cells inoculated with the WSN strain of influenza virus at high or low multiplicity produce a high yield of infective virus containing at least five distinct pieces of RNA. There is a significant decrease in infectivity and PFU HA ratio only after three or four serial undiluted passages of virus in MDBK cells, but when this decrease occurs, it is accompanied by a decrease in the largest piece of viral RNA and an increase in the amount of the small, heterogeneous RNA. HeLa cells inoculated with the WSN strain of influenza virus grown in MDBK cells produce infective virus containing at least 5 pieces of RNA similar to those in virus obtained from chick embryo and MDBK cells; however, HeLa cells inoculated with virus grown in the chick embryo produce noninfective virus with a deficiency of the largest piece of viral RNA and an increase in the amount of small, heterogeneous RNA. These results confirm the partial host cell dependence of incomplete virus formation, the loss of the largest piece of viral RNA, and the increase in the amount of small RNA in incomplete virus. They also indicate that HeLa cells can produce infective influenza virus if the inoculum is relatively free of incomplete virus, and suggest that the mechanism involved in the abortive infection of HeLa cells is similar to that involved in the von Magnus type of incomplete virus formation in other cells.

The lipids of incomplete influenza virus

Standard influenza virus (A 0/PR8/34) and incomplete virus of the von Magnus type were analyzed and compared. Virus propagated under conditions of serial undiluted passage had a lower molar ratio of cholesterol-phospholipid (0.6) than did standard virus. A comparison of phospholipids revealed decreased amounts of sphingomyelin and phosphatidylcholine together with increased amounts of phosphatidylethanolamine. Fatty acid profiles of the polar lipid fractions revealed a shift toward short-chain acids and long-chain polyunsaturated fatty acids. Neutral lipids, separated by a unidimensional, two-solvent system, revealed that incomplete virus had an increased amount of glycerides with a shift toward mono- and diglycerides. Such changes would tend to alter the packing of the lipids of influenza virus and lead to a more expanded and fluid envelope—accounting for the pleomorphism and defects in the envelope of the von Magnus type virus.

Antigen-forming defective viruses of simian virus 40

The presence of two kinds of defective virus particles was demonstrated in SV40 preparations obtained by serial undiluted passage in African green monkey kidney (GMK) cell cultures. They were designated T and V particles, respectively. The former produces only T antigen and the latter V antigen in the nuclei of GMK cells, without producing infectious progeny. A tentative method of the titration of each particle is presented. The purified virion preparation obtained by the third undiluted passage was shown to consist mainly of physically heterogeneous defective particles. The average buoyant density of defective virions was significantly lower than that of plaque-forming particles. T particles were present in the light virion population.

Defective T particles of vesicular stomatitis virus: I. Preparation, morphology, and some biologic properties

Infection of chick embryo cells with vesicular stomatitis virus under conditions of high multiplicity and serial undiluted passage (VSV up) results in two kinds of progeny: a low yield of infectious bullet-shaped (B) particles and a high yield of noninfectious (T) particles. The smaller T particles can be partially purified by rate zonal centrifugation in sucrose gradients and readily identified by electron microscopy. T particles appear almost spherical with a diameter of ~65 mμ compared with the typically cylindrical B particles with dimensions of ~65 × 180 mμ. Despite this difference in size, T is strikingly similar to B in its ultrastructure, buoyant density, antigenicity, incorporation of uridine- 3H during growth, and capacity to inhibit cellular RNA and interferon synthesis. The data strongly suggest that T is a distinct, truncated form of B which contains only a portion of the VSV genome. The accompanying paper provides proof that T is the physical equivalent of the transmissible interfering component of Cooper and Bellett (1959).

Stepwise inactivation of an influenza virus by serial undiluted passages

Stepwise inactivation of an influenza virus by serial undiluted passages

Incomplete growth of simian virus 40 in African green monkey kidney culture induced by serial undiluted passages

A phenomenon similar to that described for influenza viruses by von Magnus was found in the growth of SV40 in African green monkey kidney cultures. In serial undiluted passages, the yield of infectious virus decreased markedly, but the yield of virus-associated CF antigen was only slightly reduced. It was also shown that a factor, probably an incomplete form of the virus, in undiluted passage stocks, interferes with the replication of virus.

The in ovo production of incomplete virus by B-Lee and A-PR8 influenza viruses.

The formation of incomplete virus by the influenza B/Lee strain on serial undiluted passages in the allantoic cavity has been studied in growth curve experiments with both the allantoic membranes and fluids. It is shown that large amounts of incomplete virus are produced by the B/Lee strain after 5 to 7 undiluted transfers. Comparative studies on the A/PR8 and B/Lee strains show that maximal incompleteness is consistently attained in UP-2 to UP-4 with the PR8 strain and in UP-5 to UP-7 with the Lee strain indicating that the pattern of behavior is an inherent characteristic of the strains. The Lee strain is found to be capable of eliciting at least the same amounts of incomplete virus as is the PR8 strain and this vitiates the sequence of the strains in the “incomplete virus gradient” suggested byFazekas de St. Groth andGraham, and makes its existence questionable.

Production and action of interferon in HeLa cells.

In preliminary experiments it was found that UP mumps virus (obtained by serial undiluted passages in chick allantois) interfered in HeLa cells with polio types 1, 2, 3 and vaccinia viruses, but poorly if at all with adeno virus type 7. These findings led to studies of interference and interferon production by egg adapted mumps virus in HeLa cells. Polio virus type 1, was chosen as the challenge virus.

The Structure of Fowl Plague Virus and Virus N

Fowl plague (KP) virus and virus N have been examined in the electron microscope as both metal shadowed and negatively stained preparations. The particles of KP virus are very similar to those of influenza A. The products of ether splitting are (1) the G antigen, a ribonucleoprotein whose helically arranged protein subunits can be resolved and (2) the haemagglutinin, a star-shaped structure about 350 A diameter made up of the spikes which project from the surface of the intact particle. Incomplete forms have been prepared by serial undiluted passage, and these show great pleomorphism, but the same outer coat as complete virus. Ether splitting of incomplete forms yields a haemagglutinin like that from complete virus, but only traces of G antigen. Virus N is more pleomorphic than KP virus, but the products of ether splitting are very similar.

Studies on the structure of influenza virus: II. Ultrathin sections of infectious and noninfectious particles

The morphology of infectious and heat-inactivated influenza virus, as well as of incomplete particles obtained under various conditions, was studied in the electron microscope. Purified preparations of elementary bodies were examined in ultrathin sections and in sprays from a volatile suspending medium. The composition of the materials under study was determined by relating infectious titers to hemagglutinin content and by assaying for the amount of internally bound soluble antigen released on treatment with ether. Standard virus in spray drop preparations consisted of spherical particles of uniform size and measuring on the average 118 mμ in diameter. Sections revealed spheres with a mean diameter of 70 mμ which contained an electrondense core limited by an inner membrane, an external coat of lower density, and a less distinct external membrane. Sprays of incomplete virus derived from serial undiluted passages (UP) and from the inoculation of heated standard seed (ΔSTP), contained particles of variable sizes and flatter than infectious virus, as well as large baglike structures. In addition to these, completely flattened and apparently empty ghosts were encountered with virus grown in HeLa cells and with noninfectious elementary bodies extracted from chorioallantoic membranes of standard eggs (CAM). In sectioned particles of UP, ΔSTP, and HeLa-grown virus, electron-dense centers were usually lacking and no internal details could be discerned. Their outer structures did not differ essentially from those of the infectious agent. Sections of preparations extracted from chorioallantoic membranes containing considerable quantities of noninfectious hemagglutinins revealed mainly masses of amorphous material. Those viral structures which could be recognized, however, resembled the other incomplete viruses described. Heat-inactivated standard virus, although somewhat changed due to prolonged exposure to 37°C, possessed all the internal structural characteristics of the infectious agent. It is thought, therefore to represent an entity distinct from other noninfectious forms.