Lymphocytic Choriomeningitis virus. I. Concentration and purification of the infectious virus.

Two procedures for the purification of infectious lymphocytic choriomeningitis virus from cell culture fluid have been developed. If large quantities of very pure virus are to be prepared, infected L cells are maintained with a medium supplemented with calf serum, the proteins of which have been largely removed by pretreatment with polyethylene glycol. Two days after infection of the cultures, the media are collected and the virus is concentrated by treatment with polyethylene glycol 40,000. Purification with a 10,000-fold increase of specific infectivity is achieved with steric chromatography on controlled-pore glass beads with pore sizes of 42 to 44 nm and centrifugation in density gradients prepared with amido trizoate. An alternative method begins with precipitation of the virus from infected cell cuture medium with zinc acetate, followed by controlled-pore glass chromatography and density centrifugation in a discontinuous sucrose gradient. Purification thus obtained is 200-fold in terms of specific infectivity.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA.

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).     

Susceptibility of chickens to avian encephalomyelitis virus. III. Behavior of the virus in growing chicks.

A chick embryo-adapted strain of avian encephalomyelitis virus was inoculated subcutaneously and orally into 40-day-old (middle-aged) and 110-day-old (advanced-aged) chicks to examine the behavior of the virus in the chick body. In the middle-aged chicks, the virus appeared in the muscle at the site of inoculation, liver, spleen, pancreas, lumbar and cervical portions of the spinal cord, and brain 1 approximately 9 days after subcutaneous inoculation, and remained mostly in the central nervous system up to 17 days after the inoculation. The virus was found in large amounts in the muscle at the site of inoculation (10(3.1)), lumbar portion (10(2.5)) and cervical portion (10(2.1)) of the spinal cord, brain (10(1.9)), and in minute amounts in the other organs examined. It appeared in 11 of 21 organs examined. In the middle-aged chicks inoculated by the oral route, the virus was detected transiently in small amounts from esophagus, pancreas, and rectum 4 approximately 14 days after inoculation. In the advanced-aged chicks inoculated by the subcutaneous route, the virus was detected in titer of 10(2.1) approximately 10(3.0) from the muscle at the site of inoculation 2 approximately 7 days after inoculation. The virus was also found sporadically in several organs up to 17 days after inoculation. In the advanced-aged chicks inoculated by the oral route, no virus appeared in any organ, but these chicks turned to be weakly positive for neutralizing antibody in the 4th or later week after inoculation.     

Susceptibility of chickens to avian encephalomyelitis virus. IV. Behavior of the virus in laying hens.

Some laying hens 6 months of age were inoculated subcutaneously or orally with a chick embryo--adapted strain of avian encephalomyelitis virus and examined for propagation of the virus in the body. When inoculated subcutaneously, the virus appeared in liver, spleen, ovarian follicle, and muscle at the site of inoculation 1 day, in kidney and lumbar part of the spinal cord 3 days, in the pancreas 5 days, in heart, duodenum, and cervical part of the spinal cord 7 days, and in the brain 11 days after inoculation. After its appearance, it increased gradually in amount in liver, spleen, pancreas, muscle at the site of inoculation, and cervical and lumbar parts of the spinal cord, but remained at a low level in any other organ. When examined 14 days after inoculation and later, it was distributed mainly in the central nervous system. It was detected from 12 of 16 organs examined. The highest virus level in each organ was 10(2.6)/0.1 g in pancreas and lumbar part of the spinal cord, which were followed by muscle at the site of inoculation (10(2.0)/0.1 g), spleen (10(1.8)/0.1 g), cervical part of the spinal cord, heart, and liver in the order listed. When inoculated orally, the virus was found sporadically in spleen, pancreas, kidney, cecum, ovarian follicle, and lumbar part of the spinal cord. The virus level was low in these organs, of which pancreas, kidney, and lumbar part of the spinal cord showed the highest virus level, or 10(1.3)/0.1 g.     

Proteins of vesicular stomatitis virus. V. Identification of a precursor to the phosphoprotein of Piry virus.

A metabolic precursor to the major phosphoprotein of Piry virus (NSv) has been identified in extracts of Piry virus-infected L cells. The conversion of the precursor NSi to NSv occurs with a half-life of 20 min and is independent of continued protein synthesis. NSi has a greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than does the product NSv, suggesting an increase in molecular weight during maturation. The conversion is unaffected by cyclic AMP, cyclic GMP, or by theophilline and cordycepin. No decrease in isoelectric point of NSv relative to NSi was observed on isoelectric focusing acrylamide gels. These latter observations suggest that NSi and NSv do not differ in extent of phosphorylation. We also report, without further characterization, the identification of another phosphoprotein in Piry virus-infected cells having an electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis just slightly greater than the nucleocapsid N protein.     

Susceptibility of chickens to avian encephalomyelitis virus. II. Behavior of the virus in day-old chicks.

The VR strain of avian encephalomyelitis virus, which had been adapted to embryonated hen's eggs, was inoculated into 2-day-old chicks by the subcutaneous route (10(2.5) approximately 10(3.0) EID50) or by the oral route (10(4.8) EID50). The chicks were examined chronologically for the distribution of the virus in the body. As a result, minute amounts of the virus were detected from the liver, spleen, pancreas, and muscle at the site of inoculation one day after inoculation and various amounts from almost all the organs 3 days and more after inoculation. The virus titer could nearly reach a maximum 7 to 9 days after inoculation. Above all, such high virus titers as ranging from 10(4.3) to 10(5.8) EID50/0.1 g were demonstrated in the brain, heart, liver, spleen, and pancreas. After that, there was a tendency for virus titer to decrease in most organs and for virus to multiply persistently in the pancreas, brain, and eyeball. Virus titer was maintained at a level of 10(2.3) approximately 10(2.8) EID50/0.1 g in these three organs even 21 days after inoculation. In the group of subcutaneous inoculation, all the chicks manifested clinical signs of infection 5 to 10 days after inoculation. On the other hand, no chicks were involved in clinical infection in the group of oral inoculation. Multiplication of the virus was delayed in the body of these chicks. Small amounts of the virus were detected from the spleen and pancreas 11 days after inoculation. Low titers (10(2.7) EID50/0.1 g at the highest) of the virus were only detected from the brain, spinal cord, spleen, pancreas, esophagus, and other organs 14 and 21 days after inoculation.     

Deletion of the vaccinia virus growth factor gene reduces virus virulence.

The vaccinia virus growth factor (VGF) gene encodes a polypeptide with amino acid sequence homology to epidermal growth factor (EGF) and transforming growth factor alpha and is present twice, once at each end of the virus genome within the inverted terminal repetition. Recombination procedures were used to replace more than half of both VGF genes with a beta-galactosidase cassette which served as a color indicator for isolating an unconditionally viable VGF- mutant. The VGF- mutant genotype and phenotype were confirmed by Southern blot analysis and assays for functional growth factor. The plaque-forming efficiencies of VGF- and wild-type (WT) viruses were similar in a variety of cell types containing low or high densities of EGF receptors, suggesting a lack of a specific requirement for either VGF or the EGF receptor in the initiation of virus infection. The yield of VGF- virus was similar to that of WT virus in growing BS-C-1 and Swiss 3T3 cells, but lower in resting Swiss 3T3 cells. The greatest differences between VGF- and WT virus occurred in vivo: higher doses of VGF- virus than WT virus were required for intracranial lethality in mice and for production of skin lesions in rabbits. Thus, expression of the VGF gene is important to the virulence of vaccinia virus.     

Varicella-zoster virus vaccine DNA differs from the parental virus DNA.

The DNAs of a varicella-zoster virus vaccine and its parental virus were compared by CsCl buoyant density centrifugation and restriction enzyme cleavage analysis. The varicella-zoster virus vaccine DNA showed a heterogeneous buoyant profile and altered restriction enzyme cleavage patterns. These changed properties are probably the result of the accumulation of virus containing defective varicella-zoster virus DNA during extensive cell culture passage of the vaccine virus.     

Epstein-Barr virus DNA. X. Direct repeat within the internal direct repeat of Epstein-Barr virus DNA.

The 3,360-base-pair internal direct repeat (IR) in Epstein-Barr virus DNA separates the short and long unique DNA domains. IR has a single BamHI site. The juncture between the short unique domain and IR has been mapped by restriction endonucleases and is less than 2,600 nucleotides before the BamHI site in IR. The junction between IR and the long unique domain has been sequenced and is approximately 650 nucleotides after the BamHI site in IR. Thus, relative to the start of IR at the juncture with the short unique domain, the last repeat is at least 90 base pairs short of being complete. There is homology between the 250-nucleotide fragments to the left and the right of the unique BamHI site in IR. A 35-base-pair sequence of the left fragment is directly repeated within the right fragment, once fully and once partially. The implications of these findings are discussed.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Measurements of the genome sizes of simian virus 40 and polyoma virus.

We have measured the genome sizes of simian virus 40 and polyoma virus and found them to be 5,010 +/- 125 base pairs and 5,080 +/- 125 base pairs, respectively.     

Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus.

The recently described protein kinase activity in hepatitis B virus core antigen particles (Albin and Robinson, J. Virol. 34:297-302, 1980) has been demonstrated here in the liver-derived core particles of ground squirrel hepatitis virus. Both protein kinase activities were initially associated with DNA polymerase-positive heavy core particles in CsCl density equilibrium gradients and shifted to polymerase-negative cores during the course of purification. The major core-associated polypeptide of each virus was the dominant species labeled. A variable number of other polypeptide species were also labeled by this reaction. Tryptic peptide mapping of both major and minor phosphorylated polypeptides of each virus resulted in similar patterns, suggesting that many of the sites of phosphorylation were the same in the components of each core particle. Hydrolysis of these phosphorylated core particles revealed a major phosphoamino acid as serine and a minor phosphoamino acid as threonine. The products of the protein kinase reaction in both human hepatitis B and ground squirrel hepatitis virus core particles, then, share many characteristics. The possible function(s) of this protein kinase activity is discussed in the light of similarly characterized activities in other animal viruses.