Dissecting Virus Entry: Replication-Independent Analysis of Virus Binding,Internalization, and Penetration Using Minimal Complementation of β-Galactosidase

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

Construction of Recombinant HVT Expressing PmpD,and Immunological Evaluation against Chlamydia psittaci and Marek’s Disease Virus

Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek’s Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek’s disease was developed and examined. The 5''-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek’s Disease virus (MDV) RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.     

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.     

Hepatitis B Virus Infection—Current Concepts of Chronicity and Immunity

Among the three types of viral hepatitis agents—A, B and non-A, non-B—the hepatitis B virus (HBV) has been best characterized by immunologic and recombinant DNA technologies. The indefinite persistence of hepatitis B virus infection in 85% to 90% of perinatally infected infants and in about 10% of those infected later in life accounts for a worldwide epidemiologic reservoir of more than 200 million carriers who are at a high risk for the development of δ-infection, chronic liver disease and hepatocellular carcinoma. Active immunization with a safe and effective vaccine, derived from the plasma of carriers of hepatitis B surface antigen (HBsAg), is envisaged to avoid viral hepatitis type B and its chronic sequelae. In addition to serologic and immunohistochemical markers of hepatitis B virus infection, hybridization assays using cloned HBV DNA have provided new insight into the biology of this virus, its persistence and its oncogenic potential in humans and in animal models. Genetic similarities have been recognized between HBV and the antigenically distinct non-A, non-B agents implicated in some cases of transfusion-associated chronic hepatitis. Structurally this unique group of HBV and HBV-like agents are DNA viruses with functional attributes of integration and replication analogous to the retroviruses.     

Virus or not? Phylogenetics of polydnaviruses and their wasp carriers

Our current, still limited, understanding of the comparative biology and evolution of polydnaviruses (PDVs) is reviewed, especially in the context of the possible origins of these parasitoid viruses and of their coevolution with carrier wasps. A hypothetical scenario of evolution of PDVs from ascovirus (or ascovirus-like) ancestors is presented, with examples of apparent extant transitional forms. PDVs appear, in the case of bracoviruses, to show phylogenetic relationships that mirror those of their wasp carriers: with ichnoviruses, the picture is less clear. Ongoing sequencing studies of entire PDV genomes from diverse wasp species are likely to greatly contribute to our understanding of PDV evolution.     

Plate Tectonics of Virus Shell Assembly and Reorganization in Phage Φ8, a Distant Relative of Mammalian Reoviruses

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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.     

Expansion,Reexpansion, and Recall-Like Expansion of Vγ2Vδ2 T Cells in Smallpox Vaccination and Monkeypox Virus Infection

Little is known about the in vivo kinetics of T-cell responses in smallpox/monkeypox. We showed that macaque Vγ2Vδ2 T cells underwent 3-week-long expansion after smallpox vaccine immunization and displayed simple reexpansion in association with sterile anti-monkeypox virus (anti-MPV) immunity after MPV challenge. Virus-activated Vγ2Vδ2 T cells exhibited gamma interferon-producing effector function after phosphoantigen stimulation. Surprisingly, like αβ T cells, suboptimally primed Vγ2Vδ2 T cells in vaccinia virus/cidofovir-covaccinated macaques mounted major recall-like expansion after MPV challenge. Finally, Vγ2Vδ2 T cells localized in inflamed lung tissues for potential regulation. Our studies provide the first in vivo evidence that viruses, despite their inability to produce exogenous phosphoantigen, can induce expansion, reexpansion, and recall-like expansion of Vγ2Vδ2 T cells and stimulate their antimicrobial cytokine response.Human γδ T cells appear to contribute to both innate and adaptive immune responses (4, 6, 10, 19). Vγ2Vδ2 T cells exist only in primates, and in humans, they constitute 60 to 95% of total blood γδ T cells. The capacity of Vγ2Vδ2 T cells to undergo major clonal expansion in primary infection and to mount rapid recall expansion upon reinfection has been proposed as an adaptive immune response (6), which is consistent with memory phenotypes of Vγ2Vδ2 T cells (7), long-term expansion of memory-like Vδ2 T cells, and in vitro recall expansion of blood γδ T cells in vaccinated or infected humans (1, 15a, 16, 17, 25). It is important to note that the microbial antigen recognized by Vγ2Vδ2 T cells is temporally limited to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), commonly referred to as phosphoantigen, produced in the newly discovered 2-C-methyl-d-erythritol-4-phosphate pathway of isoprenoid biosynthesis in bacteria, but not viruses (8). Our recent study has demonstrated that HMBPP is presented by a putative molecule on antigen-presenting cell membranes and recognized by Vγ2Vδ2 T-cell receptor (TCR) (24). Since viruses do not produce exogenous HMBPP recognized by Vγ2Vδ2 T cells, in vivo Vγ2Vδ2 T-cell expansion usually occurs only in HMBPP-producing bacterial or protozoal infections.Monkeypox virus (MPV) (an orthopoxvirus) has biological features similar to those of smallpox virus, and MPV infection is clinically similar to smallpox in humans (9, 12, 22). Immune responses of Vγ2Vδ2 T cells during lethal MPV infection have not been studied, although some laboratories have undertaken in vitro studies of γδ T-cell immune responses to vaccinia virus (1, 2, 15). We presume that initial vaccinia virus immunization and subsequent MPV challenge of macaques would provide an ideal in vivo setting in which to determine whether Vγ2Vδ2 T cells can mount innate-like or recall-like responses to orthopoxvirus infections. We made a novel observation indicating expansion, reexpansion, and recall-like expansion of Vγ2Vδ2 T cells with effector function in response to smallpox vaccination and MPV infection in macaques.