Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Antibody-dependent cellular cytotoxicity detects type- and strain-specific antigens among human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus SIVmac isolates.

Human cell lines were infected with different strains of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as with a simian immunodeficiency virus SIVmac isolate and used as targets in an antibody-dependent cellular cytotoxicity (ADCC) assay. Sera from HIV-1- or HIV-2-infected subjects provided the antibody, and lymphocytes from normal donors provided the effector cells. About 60% of HIV-1 antibody-positive sera mediated ADCC when tested against any given HIV-1 isolate-infected target cell (human T-cell lymphotropic virus type IIIB, B40, A2587), and about 75% of HIV-2 antibody-positive sera mediated ADCC when tested against target cells infected with HIV-2 isolates (lymphadenopathy-associated virus type 2 and SBL-6669) or simian immunodeficiency virus from macaques. Within each type, individual sera showed different reactivity patterns, and the probability that a serum was ADCC positive was higher when it was tested against several strains. When the ADCC reactivity of sera against different strains was compared, diversity as detected by ADCC appeared to be greater among HIV-1 strains than among HIV-2 strains. For HIV-1, 54 to 67% of the sera gave concordant ADCC reactions, whereas for HIV-2 and SIVmac, 91% of the sera gave concordant results. Almost no strain-specific differences were seen between SBL-6669 and lymphadenopathy-associated virus type 2. As we determined previously, HIV-1 and HIV-2 did not cross-react in ADCC. The results indicated that HIV-1 and HIV-2 antibody-positive sera mediate both strain- and type-specific ADCC. HIV-2 antibody-positive sera seem to mediate ADCC with broader reactivity and to a higher frequency compared with HIV-1 antibody-positive sera.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Induction of cytopathogenicity in mammalian cell lines challenged with culturable enteric viruses and its enhancement by 5-iododeoxyuridine

Cultures of 17 established cell lines were tested against 105 enteric virus types for capacity to support viral replication as indicated by cytopathogenic effect production. Enhancement of susceptibility by treatment of the cells with 5-iododeoxyuridine was evaluated in parallel with untreated cells. Cytopathogenic effect was produced in two or more cell lines by every virus tested except six strains of group A coxsackie virus. No cell line was found to be susceptible to these six virus types. In general, treatment with 5-iododeoxyuridine provided a more rapid onset of cytopathogenic effect in susceptible cells and in some instances resulted in refractory cells becoming permissive to viral replication. The use of 5-iododeoxyuridine allowed two human embryonic lines (HEL-299 and L-132), in combination, to be susceptible to all but the six group A coxsackie virus strains.     

Induction of cytopathogenicity in mammalian cell lines challenged with culturable enteric viruses and its enhancement by 5-iododeoxyuridine.

Cultures of 17 established cell lines were tested against 105 enteric virus types for capacity to support viral replication as indicated by cytopathogenic effect production. Enhancement of susceptibility by treatment of the cells with 5-iododeoxyuridine was evaluated in parallel with untreated cells. Cytopathogenic effect was produced in two or more cell lines by every virus tested except six strains of group A coxsackie virus. No cell line was found to be susceptible to these six virus types. In general, treatment with 5-iododeoxyuridine provided a more rapid onset of cytopathogenic effect in susceptible cells and in some instances resulted in refractory cells becoming permissive to viral replication. The use of 5-iododeoxyuridine allowed two human embryonic lines (HEL-299 and L-132), in combination, to be susceptible to all but the six group A coxsackie virus strains.     

SJL/J Mice Are Highly Susceptible to Infection by Mouse Adenovirus Type 1

Mouse adenovirus type 1 (MAV-1) targets endothelial and monocyte/macrophage cells throughout the mouse. Depending on the strain of mouse and dose or strain of virus, infected mice may survive, become persistently infected, or die. We surveyed inbred mouse strains and found that for the majority tested the 50% lethal doses (LD(50)s) were >10(4.4) PFU. However, SJL/J mice were highly susceptible to MAV-1, with a mean LD(50) of 10(-0.32) PFU. Infected C3H/HeJ (resistant) and SJL/J (susceptible) mice showed only modest differences in histopathology. Susceptible mice had significantly higher viral loads in the brain and spleen at 8 days postinfection than resistant mice. Infection of primary macrophages or mouse embryo fibroblasts from SJL/J and C3H/HeJ mice gave equivalent yields of virus, suggesting that a receptor difference between strains is not responsible for the susceptibility difference. When C3H/HeJ mice were subjected to sublethal doses of gamma irradiation, they became susceptible to MAV-1, with an LD(50) like that of SJL/J mice. Antiviral immunoglobulin G (IgG) levels were measured in susceptible and resistant mice infected by an early region 1A null mutant virus that is less virulent that wild-type virus. The antiviral IgG levels were high and similar in the two strains of mice. Taken together, these results suggest that immune response differences may in part account for differences in susceptibility to MAV-1 infection.     

Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus.     

Susceptibility of varicella-zoster virus to thymidine analogues

Ten strains of varicella-zoster virus (VZV) were tested for susceptibility to 17 nucleoside analogues by a plaque reduction assay using human embryonic lung fibroblast cells. The compounds employed were 5-substituted arabinosyluracils and 2'-deoxyuridines, 2'-fluoro-arabinosylpyrimidines (F-araPyr) and acyclovir. In terms of the 50% plaque reduction dose (PD50), 4- to 40-fold difference were found between the 10 strains of VZV in susceptibilities to each compound. VZV was highly susceptible to 5-halogenovinyl-arabinosyluracils (XV-araUs); the PD50 values of these compounds were less than 0.001 micrograms/ml. VZV was much more susceptible than herpes simplex virus (HSV) type 1 to XV-araUs, but less susceptible than either HSV type 1 or type 2 to 5-ethyl-2'-deoxyuridine, 5-ethyl-arabinosyluracil and acyclovir.     

Interaction of herpes simplex virus type 2 with a rat glioma cell line

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.     

Modelling the relationship between antibody-dependent enhancement and immunological distance with application to dengue

When antibodies raised in response to a particular pathogen bind with immunologically similar pathogens it may facilitate infection through a phenomenon known as antibody-dependent enhancement (ADE). This process occurs between the four serotypes of dengue virus and, furthermore, secondary infection is a major risk factor in dengue hemorrhagic fever (DHF). Theory has suggested that ADE may be responsible for the large immunological distance between dengue serotypes. We investigate this hypothesis using an epidemic model for dengue in which immunological distance and the strength of immune cross-reaction are expressed separately. Cross-enhancement is considered in three alternative forms acting on susceptibility, transmission and mortality. Previous models have shown that transmission and mortality enhancement can lead to periodicity or chaos. We confirm this result for reasonable levels of susceptibility and transmission enhancement but not for mortality enhancement. We also show that when the two strains have identical basic reproductive numbers no form of enhancement leads to competitive exclusion. When the two strains have different basic reproductive numbers susceptibility or transmission enhancement allow strains with greater immunological similarity to stably coexist but mortality enhancement forces strains to be more distinct. All three forms of enhancement can be associated with DHF and we conclude that mortality enhancement must be dominant if ADE really is responsible for the immunological distance between dengue serotypes.     

Differential activities of the human immunodeficiency virus type 1-encoded Vpu protein are regulated by phosphorylation and occur in different cellular compartments.

The human immunodeficiency virus type 1 (HIV-1)-specific Vpu is an 81-amino-acid amphipathic integral membrane protein with at least two different biological functions: (i) enhancement of virus particle release from the plasma membrane of HIV-1-infected cells and (ii) degradation of the virus receptor CD4 in the endoplasmic reticulum (ER). We have previously found that Vpu is phosphorylated in infected cells at two seryl residues in positions 52 and 56 by the ubiquitous casein kinase 2. To study the role of Vpu phosphorylation on its biological activity, a mutant of the vpu gene lacking both phosphoacceptor sites was introduced into the infectious molecular clone of HIV-1, pNL4-3, as well as subgenomic Vpu expression vectors. This mutation did not affect the expression level or the stability of Vpu but had a significant effect on its biological activity in infected T cells as well as transfected HeLa cells. Despite the presence of comparable amounts of wild-type and nonphosphorylated Vpu, decay of CD4 was observed only in the presence of phosphorylated wild-type Vpu. Nonphosphorylated Vpu was unable to induce degradation of CD4 even if the proteins were artificially retained in the ER. In contrast, Vpu-mediated enhancement of virus secretion was only partially dependent on Vpu phosphorylation. Enhancement of particle release by wild-type Vpu was efficiently blocked when Vpu was artificially retained in the ER, suggesting that the two biological functions of Vpu are independent, occur at different sites within a cell, and exhibit different sensitivity to phosphorylation.     

Genetic analysis of the susceptibility of mouse cytomegalovirus to acyclovir.

Eight independently derived mouse cytomegalovirus (MCMV) mutants resistant to acyclovir (ACV) were obtained by the sequential plating of wild-type virus in increasing concentrations of ACV. Results of complementation studies among these eight mutants suggest that all had mutations within the same or closely associated genes. A ninth MCMV mutant resistant to phosphonoacetate (PAA) derived by plating wild-type virus in the presence of 100 micrograms of PAA per ml displayed coresistance to ACV and was unable to complement any of the ACV-derived mutants. Recombination experiments among all combinations of the nine MCMV mutants were performed and supported the complementation data in that no recombination could be detected. Seven of the eight ACV-resistant mutants demonstrated cross-resistance to PAA and hypersensitivity to aphidicolin. The one mutant not coresistant to PAA was more susceptible to PAA than was the parent virus. Only a few mutants demonstrated coresistance when the mutants were tested against 9-beta-D-arabinofuranosyladenine (ara-A). The ACV mutant that demonstrated increased susceptibility to PAA was 30-fold more susceptible to ara-A but remained unchanged in susceptibility to aphidicolin. Two of the parent-mutant combinations were selected for DNA synthesis analysis in the presence of ACV (5 microM). A significant decrease in DNA synthesis was demonstrated for both parent viruses, and there was little effect on mutant virus DNA synthesis at the same drug concentration. These results suggest that susceptibility of MCMV to ACV is confined to a product of a single gene and that a mutation of this gene can lead to an altered phenotype when compared with parent virus in susceptibility of DNA synthesis to PAA, ara-A, and aphidicolin, drugs that are known to inhibit DNA polymerase activity.     

Prasinoviruses of the marine green alga Ostreococcus tauri are mainly species specific

Prasinoviruses infecting unicellular green algae in the order Mamiellales (class Mamiellophyceae) are commonly found in coastal marine waters where their host species frequently abound. We tested 40 Ostreococcus tauri viruses on 13 independently isolated wild-type O. tauri strains, 4 wild-type O. lucimarinus strains, 1 Ostreococcus sp. ("Ostreococcus mediterraneus") clade D strain, and 1 representative species of each of two other related species of Mamiellales, Bathycoccus prasinos and Micromonas pusilla. Thirty-four out of 40 viruses infected only O. tauri, 5 could infect one other species of the Ostreococcus genus, and 1 infected two other Ostreococcus spp., but none of them infected the other genera. We observed that the overall susceptibility pattern of Ostreococcus strains to viruses was related to the size of two host chromosomes known to show intraspecific size variations, that genetically related viruses tended to infect the same host strains, and that viruses carrying inteins were strictly strain specific. Comparison of two complete O. tauri virus proteomes revealed at least three predicted proteins to be candidate viral specificity determinants.     

白纹伊蚊和埃及伊蚊对基孔肯雅病毒的易感性和传播性的研究

用４株基孔肯雅病毒经口感染白纹伊蚊和埃及伊蚊,进行了易感性和传播性的研究。结果表明,这两种蚊虫对基孔肯雅病毒易感。无论白纹伊蚊或埃及伊蚊,感染后第５－６天即可通过吸血将病毒传播给乳鼠,至第８－１３天,传播率可高达５５．５５％－１００％。感染蚊亦可经叮咬将病毒传播给小鸡。埃及伊蚊的易感性和传播率高于白纹伊蚊。实验还发现,不同来源毒株之间存在一定差异,如分离自云南白纹伊蚊的Ｍ８１株的感染率和传播率均高于其它毒株。这些结果表明,白纹伊蚊和埃及伊蚊在基孔肯雅病毒的保存和传播中起重要作用。     

Cell-to-cell spread of wild-type herpes simplex virus type 1, but not of syncytial strains, is mediated by the immunoglobulin-like receptors that mediate virion entry, nectin1 (PRR1/HveC/HIgR) and nectin2 (PRR2/HveB)

The immunoglobulin-like receptors that mediate entry of herpes simplex virus type 1 (HSV-1) into human cells were found to mediate the direct cell-to-cell spread of wild-type virus. The receptors here designated Nectin1alpha and -delta and Nectin2alpha were originally designated HIgR, PRR1/HveC, and PRR2alpha/HveB, respectively. We report the following. (i) Wild-type HSV-1 spreads from cell to cell in J cells expressing nectin1alpha or nectin1delta but not in parental J cells that are devoid of entry receptors. A monoclonal antibody to nectin1, which blocks entry, also blocked cell-to-cell spread in nectin1-expressing J cells. Moreover, wild-type virus did not spread from a receptor-positive to a receptor-negative cell. (ii) The antibody to nectin1 blocked transmission of wild-type virus in a number of human cell lines, with varying efficiencies, suggesting that nectin1 is the principal mediator of wild-type virus spread in a variety of human cell lines. (iii) Nectin1 did not mediate cell fusion induced by the syncytial strains HSV-1(MP) and HFEM-syn. (iv) Nectin2alpha could serve as a receptor for spread of a mutant virus carrying the L25P substitution in glycoprotein D, but not of wild-type virus, in agreement with its ability to mediate entry of the mutant but not of wild-type virus.     

Neuraminidase reversal of resistance to lysis of herpes simplex virus-infected cells by antibody and complement.

The susceptibility to lysis by antibody and complement was examined in four human cell lines. The cells were infected with herpes simplex virus type 1 and lysis was assessed by the 51Cr release test by using antibodies to herpes simplex virus and guinea pig serum as a source of complement. The four cell lines were found to differ in their susceptibility to lysis, although virus replication was readily demonstrated in the different cell lines. By indirect immunofluorescence, no differences in the expression of virus antigens at the surface of the cells could be found between the different cell lines. Treatment of cells with neuraminidase markedly enhanced the sensitivity of the cells which were relatively insensitive to lysis. The enhancement of susceptibiltiy to lysis by neuraminidase occurred if cells were treated before reaction of the cells with antibody and if the cells were reacted with antibody before treatment with the enzyme. No enhancement was observed when cells were reacted with antibody and complement before neuraminidase treatment. Neuraminidase treatment did not seem to enhance appreciably the quantity of antibody which reacted at the cell surface. The observations suggest that surface properties of certain cells render the cells resistant to lysis by antibody and complement and that the resistance to lysis can be abrogated by treating the cells with neuraminidase.     

Glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing CD4, CXCR4, and/or CCR5

Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca(2+) mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.     

Extent of measles virus spread and immune suppression differentiates between wild-type and vaccine strains in the cotton rat model (Sigmodon hispidus)

Infection of humans with wild-type measles virus leads to strong immune suppression and secondary infections, whereas immunization with an attenuated vaccine strain does not. Using the cotton rat model (Sigmodon hispidus), we investigated whether vaccine and wild-type viruses differ in viral spread and whether this is correlated with inhibition of of proliferation of spleen cells ex vivo after mitogen stimulation. After intranasal infection of cotton rats with wild-type and vaccine strains, it was found that wild-type virus replicates better in lung tissue, spreads to the mediastinal lymph nodes, and induces a more pronounced and longer-lasting inhibition of proliferation of spleen cells ex vivo after mitogen stimulation than does vaccine virus. To induce the same degree of proliferation inhibition, 1,000-fold less wild-type virus was required than vaccine virus. With this system, the virulence of various measles virus isolates and recombinant viruses was tested. Four (in humans and/or monkeys) highly pathogenic virus strains were immunosuppressive, whereas viruses of vaccine virus genotype A were not. Using virus pairs which, due to passage on fibroblasts versus lymphoid cells or due to a point mutation in the hemagglutinin (N481 --> Y), differed in their usage of the two receptor molecules CD46 and CD150 on human cells, it was found that viruses using exclusively CD150 in vitro spread to mediastinal lymph nodes and induced strong immune suppression. These data demonstrate that important parameters of virulence seen in humans, such as viral spread and immune suppression, are reflected in the cotton rat model.