Comparison of the 3′ Termini of Discrete Segments of the Double-Stranded Ribonucleic Acid Genomes of Cytoplasmic Polyhedrosis Virus, Wound Tumor Virus, and Reovirus

The 3' terminal nucleosides of the isolated components of double-stranded ribonucleic acids of reovirus, wound tumor virus, and cytoplasmic polyhedrosis virus were determined by labeling with tritiated sodium borohydride. All wound tumor virus and cytoplasmic polyhedrosis virus components appear to contain approximately equal amounts of U(OH) and C(OH) termini. Reovirus segments have essentially only C(OH) termini.     

Antigenic and Morphological Similarities of Progressive Pneumonia Virus, a Recently Isolated “Slow Virus” of Sheep, to Visna and Maedi Viruses

Progressive pneumonia virus, the causative agent of a slow, pulmonary disease of Montana sheep, was shown to be antigenically related to two other slow viruses of sheep, visna and maedi. Electron microscopic examination of infected cells revealed that the virus matures by a budding process and that the budding particles as well as the mature, extracellular virions bear striking resemblances to the oncogenic ribonucleic acid (RNA) viruses. Recent findings of an RNA-dependent deoxyribonucleic acid polymerase associated with the virions of this group of slow viruses lend further support to the notion that they may tentatively be classified with the oncogenic RNA tumor viruses.     

Transformation of Murine Cells by Two “Slow Viruses,” Visna Virus and Progressive Pneumonia Virus

Visna and progressive pneumonia virus (PPV), two antigenically related, non-oncogenic "slow viruses" which have ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, were examined for their ability to transform cells. Murine cells which had been exposed to either visna or PPV developed foci of altered, spindle-shaped cells 3 to 4 weeks after infection. Visna and PPV transformed lines were established from these cultures. There was no evidence that other oncogenic DNA or RNA viruses were involved in the observed transformation. Visna or PPV could be "rescued" from all transformed lines by co-cultivation with normal sheep testis cells. "Rescued" virus was identified as visna or PPV, and they retained the capacity to transform mouse cells. These experiments may have important implications in the understanding of both viral carcinogenesis and "slow" viral infections.     

Immunogenicity of Rabies Virus Inactivated by β-Propiolactone, Acetylethyleneimine, and Ionizing Irradiation

Ionizing radiation, beta-propiolactone, and acetylethyleneimine were compared for their ability as virus-inactivating agents for the preparation of rabies vaccine. Each agent reduced viral infectivity exponentially; ionizing radiation also destroyed viral hemagglutinin. The vaccine prepared by ionizing radiation was equal or superior to that prepared by beta-propiolactone in its ability to protect mice from rabies infection. The acetylethyleneimine-treated vaccine was a less potent immunogen.     

Specific Fragmentation of DNA of Adenovirus Serotypes 3, 5, 7, and 12, and Adeno-Simian Virus 40 Hybrid Virus Ad2+ND1 by Restriction Endonuclease R· EcoRI

The products of complete digestion of duplex DNA of each of seven human adenoviruses with restriction endonuclease R. EcoRI ranged from two fragments for adenovirus 7 DNA (Ad7) to six fragments for Ad12 and Ad2 DNA. Viral serotypes from the same subgroups appeared to have related cleavage sites; Ad3 DNA and Ad7 (cl E46-LL) DNA were each cleaved into three fragments, and Ad7 (cl 19) DNA lacked one of the cleavage sites present in Ad3 and Ad7 (cl E46-LL) DNA. One of the cleavage sites in Ad2 DNA was deleted in the DNA' of adeno-SV40 hybrid virus Ad2(+)ND1, and three of the cleavage sites in Ad2 DNA were missing in Ad5 DNA. Thus, Ad2(+)ND1 DNA was cleaved into five and Ad5 DNA into three fragments. Each fragment represented a unique segment of viral DNA since each fragment was obtained in equimolar amounts and since the sum of the molecular weights of the fragments equaled the molecular weight of the homologous intact adenovirus DNA.     

14. Studies on Poliomyelitis Virus—Concentration and Purification of the Virus

ABSTRACT

For the purpose of determining the immunogenic potency of polio virus, relatively large amounts of concentrated virus material were prepared which had titres of the order of 10¹⁰ T.C.I.D.jo per ml. These were obtained by pervaporating large quantities of tissue culture fluid containing approximately 10⁶⁵ T.C.I.D.JQ per ml.     

Effect of β-Propiolactone on Sendai Virus

The biological properties (infectivity, hemagglutination, hemolysis, cell fusion, neuraminidase) of Sendai virus were dissociated on the basis of sensitivity to beta-propiolactone, by freeze-thawing, by heating at different temperatures, and by adsorption-elution with formalinized chicken erythrocytes. Possible mechanisms whereby beta-propiolactone selectively destroys viral infectivity are discussed.     

Is the Gut the Major Source of Virus in Early Simian Immunodeficiency Virus Infection?

The acute phases of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection are characterized by rapid and profound depletion of CD4⁺ T cells from the guts of infected individuals. The large number of CD4⁺ T cells in the gut (a large fraction of which are activated and express the HIV/SIV coreceptor CCR5), the high level of infection of these cells, and the temporal coincidence of this CD4⁺ T-cell depletion with the peak of virus in plasma in acute infection suggest that the intestinal mucosa may be the major source of virus driving the peak viral load. Here, we used data on CD4⁺ T-cell proportions in the lamina propria of the rectums of SIV-infected rhesus macaques (which progress to AIDS) and sooty mangabeys (which do not progress) to show that in both species, the depletion of CD4⁺ T cells from this mucosal site and its maximum loss rate are often observed several days before the peak in viral load, with few CD4⁺ T cells remaining in the rectum by the time of peak viral load. In contrast, the maximum loss rate of CD4⁺ T cells from bronchoalveolar lavage specimens and lymph nodes coincides with the peak in virus. Analysis of the kinetics of depletion suggests that, in both rhesus macaques and sooty mangabeys, CD4⁺ T cells in the intestinal mucosa are a highly susceptible population for infection but not a major source of plasma virus in acute SIV infection.The acute phase of human immunodeficiency virus (HIV) infection is characterized by moderate CD4⁺ T-cell depletion in blood, followed by a transient partial restoration of CD4⁺ T-cell numbers and eventually by a slow long-term CD4⁺ T-cell decline in the chronic phase that lasts for several years. Studies of CD4⁺ T-cell depletion in mucosal sites, often conducted with simian immunodeficiency virus (SIV)-infected macaques, have demonstrated that mucosal CD4⁺ T-cell depletion is more rapid and profound (3, 10, 13, 19, 21). The severe depletion of cells in the gut in early infection is thought to be driven in part by the phenotype of the cells present, which are predominantly CCR5⁺ and in general more activated than their circulating counterparts. As such, these mucosal CD4⁺ T cells are highly susceptible to productive infection with the dominant CCR5-tropic strains of HIV and SIV present in early infection (20). The rapid depletion of CD4⁺ T cells at mucosal sites is accompanied by relatively high numbers of infected cells (10, 13) and is temporally associated with the peak viral load in plasma, suggesting that the infection of mucosal CD4⁺ T cells may be responsible for the majority of virus replication occurring during acute infection (10, 15, 21, 22).The size of the CD4⁺ T-cell pool in the gut is a matter of some controversy, with estimates ranging from ∼5 to 50% of the total body pool of these cells (reviewed in reference 5). Regardless of the precise numbers, the gut (and particularly the mucosal lamina propria) contains a significant proportion of the body CD4⁺ CCR5⁺ memory T cells, which are depleted very early in infection. However, whether CD4⁺ T cells in the gut are merely a target of early infection or whether they are a major driver of early viral growth and peak viral loads in acute infection is unclear. Here we use a combination of experimental data and modeling to demonstrate that the gut is unlikely to be a major source of virus production in acute SIV infection.     

Geochemistry of Virus–Prokaryote Interactions in Freshwater and Acid Mine Drainage Environments,Ontario, Canada

An extensive water sample survey was conducted in southern Ontario, Canada across a variety of freshwater systems in order to further understand the role of viruses in aquatic environments. Backwards stepwise multiple regression analysis found that VLP (virus-like particle) abundance, phosphate, pH, sulfate, and magnesium are predictors of prokaryotic abundance with the model describing 90% of the variability in the data (R² = 0.90). Statistically significant (P < 0.05) predictors of VLP abundance were mineral saturation indices (SI) of goethite (R² = 0.78) although moderate Pearson component analysis correlations (r) were noted with ferrihydrite, jarosite, and pyrolusite. These relationships indicate that viral inactivation through mineral attachment may be a contributor to the moderate relationship between VLP and prokaryotic abundance (r_s = 0.45). In addition, VLP abundance is shown to have a stronger correlation with minerals SI values than prokaryotes indicating a stronger mineral influence with viruses.     

Comparative Effects of β-Propiolactone on Mice, Mouse-derived Cell Cultures, and Venezuelan Equine Encephalomyelitis Virus

Studies were made comparing the toxicity of β-propiolactone (BPL) for mammalian (mouse) cells in vitro and for mice and for Venezuelan equine encephalomyelitis (VEE) virus which is highly cytopathogenic for each. The mammalian cells grown in tissue culture were found to be adversely affected by BPL in concentrations ranging from 0.001 to 0.1 mg/ml of supernatant fluid. The difference in response was influenced by the menstruum in which the BPL was suspended and the difference in cell types tested. Tenfold less BPL appeared to be required to destroy the cells when it was suspended in a balanced salt solution than when it was suspended in protein-containing solutions such as beef heart infusion broth or medium 199 plus 20% horse serum. Secondary embryonic mouse lung cells seemed slightly more adversely affected by BPL than the established embryonic lung or L cells. BPL given to mice by intranasal instillation and by intracerebral injection was lethal to half of the animals within 2 days at doses of 0.31 and 0.39 mg, respectively. Higher concentrations of BPL were required to rapidly inactivate the virus in vitro than were required to kill mice or to cause a toxic effect on cells in culture. It required 10 mg/ml of BPL to completely inactivate a high-titered VEE virus preparation in 5 min and 1 mg/ml to inactivate most, but not all, of the virus in 15 min. A concentration of 0.1 mg/ml of BPL had only a slight effect on the virus after a period as long as 60 min. Evidence is presented indicating that simultaneous inactivation of all of the properties of the VEE virus particles by BPL aerosols did not occur at the same time but that, after treatment, the virus possessed a limited ability to immunize mice despite a loss in infectivity.     

An ITAM in a Nonenveloped Virus Regulates Activation of NF-κB,Induction of Beta Interferon,and Viral Spread

Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: μ2, σ2, and λ2. We demonstrate for the first time that μ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, μ2 and μNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the μ2 ITAM. Moreover, both the μ2 ITAM and Syk are required for maximal μ2 activation of NF-κB. A mutant virus lacking the μ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-β) than does wild-type virus despite similar replication. Notably, the consequences of these μ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the μ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the μ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-β. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.     

Analysis of Minimal Functions of Simian Virus 40 III. Evidence for “Host Cell Repair” of Oncogenicity and Infectivity of UV-Irradiated Simian Virus 40

The in vitro transforming capacity of simian virus 40 (SV40) for Syrian hamster cells is highly resistant to inactivation by UV light in comparison to infectivity. In the same cell system, we demonstrated a "host cell repair mechanism" sensitive to caffeine which is, to a large extent, responsible for the high resistance to UV inactivation of the transforming capacity of SV40. The survival of infectivity of UV-irradiated SV40 in CV-1 cells was also sensitive to caffeine, again indicating host cell repair. On the other hand, depression of normal cell DNA synthesis by hydroxyurea during the first 24 h postinfection only modestly reduced, and to a similar extent, the transforming capacity of UV-irradiated and nonirradiated SV40.     

Analysis of Influence of Baicalin Joint Resveratrol Retention Enema on the TNF-α, SIgA,IL-2, IFN-γ of Rats with Respiratory Syncytial Virus Infection

Explore the influence of baicalin joint resveratrol retention enema on TNF-α, SIgA, IL-2, and IFN-γ of rats with respiratory syncytial virus (RSV) infection. The 60 SD rats were randomly divided into normal group, model group, baicalin group, resveratrol group, joint group, and ribavirin group. For model group, baicalin group, resveratrol group, joint group, and ribavirin group, rats were given RSV virus suspension intranasally for 3 days, and model group was not given administration. Baicalin group, resveratrol group, joint group, and ribavirin group were, respectively, given baicalin 100 mg/kg/day, resveratrol 30 mg/kg/day, baicalin joint resveratrol, and ribavirin 1 g/kg/day retention enema. After continuously given administration 7 days, rats were measured in serum TNF-α, IL-2, IFN-γ levels and SIgA levels in bronchoalveolar lavage fluid. Model group, TNF-α, IL-2, IFN-γ, and SIgA were significantly higher than the normal group (P < 0.05); Baicalin group, resveratrol group, ribavirin group, TNF-α, IL-2, IFN-γ, and SIgA were significantly higher than the model group (P < 0.05); TNF-α, IL-2 between baicalin group, resveratrol group, ribavirin group, have no significant difference (P > 0.05); Baicalin group, resveratrol group, joint group, IFN-γ, and SIgA were significantly higher than the ribavirin group (P < 0.05); Joint group TNF-α, IL-2, IFN-γ, and SIgA were significantly higher than baicalin group, resveratrol group, and ribavirin group (P < 0.05). Baicalin joint resveratrol retention enema can increase RSV infection model in rats serum TNF-α, IL-2, IFN-γ levels and SIgA levels in bronchoalveolar lavage fluid, which may anti-virus through this mechanism.     

High Prevalence of Hepatitis E Virus in Swedish Moose – A Phylogenetic Characterization and Comparison of the Virus from Different Regions

Background

Hepatitis E virus (HEV) infects a range of species, including humans, pigs, wild boars and deer. Zoonotic transmission may contribute to the high HEV seroprevalence in the human population of many countries. A novel divergent HEV from moose (Alces alces) in Sweden was recently identified by partial genome sequencing. Since only one strain was found, its classification within the HEV family, prevalence in moose and zoonotic potential was unclear. We therefore investigated samples from 231 moose in seven Swedish counties for HEV, and sequenced a near complete moose HEV genome. Phylogenetic analysis to classify this virus within the family Hepeviridae and to explore potential host specific determinants was performed.

Methods and Findings

The HEV prevalence of moose was determined by PCR (marker for active infection) and serological assays (marker of past infection) of sera and 51 fecal samples from 231 Swedish moose. Markers of active and past infection were found in 67 (29%) animals, while 34 (15%) were positive for HEV RNA, 43 (19%) were seropositive for anti-HEV antibodies, and 10 (4%) had both markers. The number of young individuals positive for HEV RNA was larger than for older individuals, and the number of anti-HEV antibody positive individuals increased with age. The high throughput sequenced moose HEV genome was 35-60% identical to existing HEVs. Partial ORF1 sequences from 13 moose strains showed high similarity among them, forming a distinct monophyletic clade with a common ancestor to HEV genotype 1-6 group, which includes members known for zoonotic transmission.

Conclusions

This study demonstrates a high frequency of HEV in moose in Sweden, with markers of current and past infection demonstrated in 30% of the animals. Moose is thus an important animal reservoir of HEV. The phylogenetic relationship demonstrated that the moose HEV belonged to the genotype 1-6 group, which includes strains that also infect humans, and therefore may signify a potential for zoonotic transmission of this HEV.     

Effect of Formalin, β-Propiolactone, Merthiolate, and Ultraviolet Light Upon Influenza Virus Infectivity, Chicken Cell Agglutination, Hemagglutination, and Antigenicity

Four strains of influenza virus were treated with Formalin, Merthiolate, Merthiolate and Formalin, ultraviolet light, and beta-propiolactone (BPL) for 18, 48, and 72 hr. Infectivity, chicken cell agglutination (CCA), hemagglutination (HA), and antigenicity determinations were made. Except for Merthiolate, each method of inactivation was equally effective in reducing infectivity. Loss of infectivity was related to length of treatment. CCA determinations were higher for all treated groups except for BPL-treated samples; these had lower determinations. BPL treatment also lowered the HA titer. Antigenicity was lessened by BPL treatment and by Merthiolate and Formalin treatment. Generally, the length of inactivation up to 72 hr did not affect CCA, HA, or antigenicity determinations. For the most part, there was no significant differences in the reactivity of the four strains.     

Isolation of Temperature-Sensitive Conditional Lethal Mutants of “Fixed” Rabies Virus

In an attempt to induce temperature-sensitive (ts) conditional lethal mutants of rabies virus, stocks of a plaque-purified substrain of strain CVS fixed rabies virus were subjected to mutagenesis by HNO₂, 5-fluorouracil, or 5-azacytidine. It was necessary to prepare virus stocks from clones of mutagenized virus selected at random and to test subsequently each stock for possible ts characteristics by measuring its relative capacity for growth at permissive (33 C) and nonpermissive (40.5 C) temperatures. Five ts mutants were detected in tests of 161 clones of mutagenized virus. Each of the mutants exhibited a remarkably low incidence of reversion and little demonstrable “leakiness.” One of the five ts mutants (ts2), which formed formed very small plaques, and another (ts1), which formed plaques of only slightly reduced size, were further characterized. Virus ts1 was more thermostable at 40.5 C than the parental virus, but the ts2 mutant was unchanged in this respect. The ts1 virus exhibited normal pathogenicity for mice, but ts2 virus caused a very irregular death pattern. Both deaths and survivors immune to rabies virus challenge were noted in all groups of mice inoculated with ts2 virus, regardless of the virus dose.