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1.
J Immunol ; 196(9): 3768-79, 2016 05 01.
Article in English | MEDLINE | ID: mdl-27036912

ABSTRACT

Microbial colonization of the infant gastrointestinal tract (GIT) begins at birth, is shaped by the maternal microbiota, and is profoundly altered by antibiotic treatment. Antibiotic treatment of mothers during pregnancy influences colonization of the GIT microbiota of their infants. The role of the GIT microbiota in regulating adaptive immune function against systemic viral infections during infancy remains undefined. We used a mouse model of perinatal antibiotic exposure to examine the effect of GIT microbial dysbiosis on infant CD8(+) T cell-mediated antiviral immunity. Maternal antibiotic treatment/treated (MAT) during pregnancy and lactation resulted in profound alterations in the composition of the GIT microbiota in mothers and infants. Streptococcus spp. dominated the GIT microbiota of MAT mothers, whereas Enterococcus faecalis predominated within the MAT infant GIT. MAT infant mice subsequently exhibited increased and accelerated mortality following vaccinia virus infection. Ag-specific IFN-γ-producing CD8(+) T cells were reduced in sublethally infected MAT infant mice. MAT CD8(+) T cells from uninfected infant mice also demonstrated a reduced capacity to sustain IFN-γ production following in vitro activation. We additionally determined that control infant mice became more susceptible to infection if they were born in an animal facility using stricter standards of hygiene. These data indicate that undisturbed colonization and progression of the GIT microbiota during infancy are necessary to promote robust adaptive antiviral immune responses.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Enterococcus faecalis/physiology , Gastrointestinal Microbiome , Streptococcus/physiology , Vaccinia virus/immunology , Vaccinia/microbiology , Adaptive Immunity , Animals , Animals, Newborn , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/adverse effects , Cells, Cultured , Female , Interferon-gamma/metabolism , Maternal Exposure/adverse effects , Mice , Mice, Inbred C57BL , Vaccinia/immunology
3.
J Immunol ; 162(8): 4527-35, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10201991

ABSTRACT

In animals, T cells often die rapidly after activation, unless activation occurs in the presence of inflammatory factors. To understand how such activated cells survive to participate in immune responses, we studied the effects of viral infection on T cells responding to an unrelated superantigen. Normal T cells activated by superantigen in uninfected mice died as a result of their activation, whereas T cells that were activated during vaccinia infection survived longer in vivo and in culture. This bystander effect of viral infection on activated T cells was independent of effects on the magnitude of the initial T cell response, on induction of Bcl-2 and Bcl-x, on T cell proliferation, and on Fas killing. The failure of such effects to predict the fate of activated T cells in vivo indicates that virus infections shape T cell responses via mechanisms that differ from those described previously. These mechanisms may contribute to the ability of viral infections to induce autoimmunity.


Subject(s)
Lymphocyte Activation , T-Lymphocyte Subsets/microbiology , T-Lymphocyte Subsets/virology , Vaccinia/immunology , Vaccinia/microbiology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/virology , Cell Death/immunology , Cell Survival/immunology , Cells, Cultured , Cytokines/genetics , Enterotoxins/immunology , Female , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/physiology , Receptors, Antigen, T-Cell, alpha-beta/physiology , Staphylococcus aureus/immunology , Superantigens/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Time Factors , Transcription, Genetic/immunology , Vaccinia/pathology , bcl-X Protein , fas Receptor/physiology
4.
Proc Natl Acad Sci U S A ; 93(5): 2037-42, 1996 Mar 05.
Article in English | MEDLINE | ID: mdl-8700881

ABSTRACT

Host protein synthesis is selectively inhibited in vaccinia virus-infected cells. This inhibition has been associated with the production of a group of small, nontranslated, polyadenylylated RNAs (POLADS) produced during the early part of virus infection. The inhibitory function of POLADS is associated with the poly(A) tail of these small RNAs. To determine the origin of the 5'-ends of POLADS, reverse transcription was performed with POLADS isolated from VV-infected cells at 1 hr and 3.5 hr post infection. The cDNAs of these POLADS were cloned into plasmids (pBS or pBluescript II KS +/-), and their nucleotide composition was determined by DNA sequencing. The results of this investigation show the following: There is no specific gene encoding for POLADS. The 5' ends of POLADS may be derived from either viral or cellular RNAs. Any RNA sequence including tRNAs, small nuclear RNAs and 5'ends of mRNAs can become POLADS if they acquire a poly(A) tail at their 3' ends during infection. This nonspecific polyadenylylation found in vaccinia virus-infected cells is probably conducted by vaccinia virus poly(A)+ polymerase. No consensus sequence is found on the 5' ends of POLADS for polyadenylylation. The 5' ends of POLADS have no direct role in their inhibitory activity of protein synthesis.


Subject(s)
Gene Expression Regulation, Viral , Polynucleotide Adenylyltransferase/metabolism , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Viral/chemistry , Vaccinia virus/genetics , Cell-Free System , HeLa Cells , Humans , RNA, Messenger/genetics , RNA, Viral/metabolism , Time Factors , Vaccinia/genetics , Vaccinia/microbiology
5.
Proc Natl Acad Sci U S A ; 91(23): 10987-91, 1994 Nov 08.
Article in English | MEDLINE | ID: mdl-7971996

ABSTRACT

The adenovirus (Ad) 14.7-kDa protein, which is called "14.7K," has been shown to function as a general inhibitor of tumor necrosis factor alpha (TNF) cytolysis in tissue culture assays, and the effect of this antagonism on viral pathogenesis in vivo has recently been explored. In infections of immunocompetent BALB/c mice, we have shown previously that Ad type 2 (Ad2) 14.7K, when cloned into a vaccinia virus (VV) vector in combination with the gene for murine TNF, is able to counteract much of the attenuating effect of TNF on VV virulence. In the present study we utilized VV constructs expressing various combinations of Ad 14.7K and TNF in infections of T- and B-cell-deficient C.B-17 severe combined immunodeficient (SCID) mice to determine whether these cells are directly necessary for 14.7K's reversal of TNF-mediated viral attenuation. The mice were infected by the intranasal route, and mortality, morbidity, histopathology, and virus replication in selected organs were evaluated at various times after infection. We found that, in the SCID murine pneumonia model, neither the attenuation by TNF nor its reversal by Ad 14.7K require the participation of T or B lymphocytes or their secreted products. SCID mice infected with VV expressing both 14.7K and TNF [VV 14.7(+)/TNF] were generally well clinically for the first 7-10 days after infection; however, they developed a subacute or chronic illness, succumbing to diseminated VV infection at least 3 weeks earlier than mice infected with VV expressing TNF alone [VV 14.7(-)/TNF]. Animals infected with VV 14.7(+)/TNF were shown to have higher initial titers of virus and delayed clearance from the lungs as well as more rapid spread of virus to internal organs than animals infected with VV 14.7(-)/TNF. SCID mice infected intranasally with VV without TNF showed a dramatic increase in acute disease and succumbed within the first 1-2 weeks after infection, independent of Ad 14.7K expression.


Subject(s)
Adenovirus E3 Proteins/toxicity , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Vaccinia virus/pathogenicity , Animals , Base Sequence , Chronic Disease , DNA Primers/chemistry , Gene Expression , Genes , Lymphocytes/immunology , Mice , Mice, SCID , Molecular Sequence Data , Pneumonia, Viral/microbiology , Tumor Necrosis Factor-alpha/genetics , Vaccinia/immunology , Vaccinia/microbiology
6.
Virology ; 202(1): 305-14, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8009842

ABSTRACT

Wild-type rabbitpox virus (RPV) and cowpox virus (CPV) produce red hemorrhagic lesions or pocks upon infection of the chicken chorioallantoic membrane (CAM) of 11-day-old embryonated chicken eggs. However, white, nonhemorrhagic pock variants arise spontaneously within wild-type (wt) populations of either virus at a frequency of about 1%, reflective of complex deletions/rearrangements in the termini of the viral DNA. A subpopulation of the RPV white-pock mutants fail to plaque on pig kidney (PK-15) cells and are referred to as host-range (hr) mutants. In the case of CPV, white-pock formation has been linked to mutations in the SPI-2 (crmA) gene. We show that five spontaneous RPV white-pock host-range mutants (RPV mu hr8sm, RPV mu hr23, RPV mu hr28, RPV mu hr30, and RPV mu hr31) each contain a SPI-2 (crmA) gene and express the crmA protein but lack instead a functional SPI-1 gene. Two other spontaneous RPV white-pock mutants, RPV mu 9 and RPV mu 12, which plaque on PK-15 cells (nonhost-range mutants) contain and express a SPI-1 gene but lack instead a functional SPI-2 gene. Targeted disruption of either the SPI-1 or SPI-2 genes of wtRPV, but only the SPI-2 gene of wtCPV, generates mutants which produce white pocks. The RPV delta SPI-1 mutant fails to plaque on PK-15 or human A549 cells, whereas the RPV delta SPI-2 mutant has a normal host range. No changes in host range compared to wtCPV for either the CPV delta SPI-1 or CPV delta SPI-2 mutants were noted. These differences in phenotypes observed between the two viruses may be reflective of either small sequence variations between the highly conserved SPI-1 or SPI-2 genes or the aggregate phenotypes provided by the other remaining genes.


Subject(s)
Peptides/genetics , Serine Proteinase Inhibitors/genetics , Vaccinia virus/genetics , Vaccinia/pathology , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chick Embryo , Cowpox virus/genetics , Cowpox virus/pathogenicity , DNA, Viral , Genes, Viral , Humans , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Mutation , Phenotype , Vaccinia/microbiology , Vaccinia virus/pathogenicity
7.
J Virol ; 68(6): 3642-9, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8189502

ABSTRACT

The vaccinia virus gene A18R is essential for virus infection. The loss of A18R protein function results in unregulated transcription late during virus infection from regions of the viral genome which are normally transcriptionally quiescent. We have characterized A18R protein expression in cells infected with wild-type virus and the A18R temperature-sensitive mutant Cts23. The A18R protein is expressed during early and late phases of infection. The A18R protein expressed by Cts23 virus at permissive and nonpermissive temperatures is significantly less stable than the wild-type A18R protein. The A18R protein was identified as a virion component and localized by detergent extraction to the virion core. Virions purified from cells infected with the A18R temperature-sensitive mutants Cts4, Cts22, and Cts23 are defective in early viral transcription in vitro. The mutant transcription defect is not attributable to gross defects in virion structure or virion DNA-dependent RNA polymerase activity. We conclude that the A18R protein plays a role in viral transcription during the early phase of infection as well as during the late phase.


Subject(s)
Vaccinia virus/genetics , Viral Proteins/genetics , Cell Line , Genes, Viral , Humans , Mutation , Temperature , Transcription, Genetic , Vaccinia/etiology , Vaccinia/genetics , Vaccinia/microbiology , Vaccinia virus/pathogenicity , Viral Proteins/metabolism
8.
Am J Ophthalmol ; 117(4): 480-7, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-8154530

ABSTRACT

A patient who used contact lenses and had a history of blunt trauma developed vaccinia keratouveitis after accidental ocular autoinoculation from a recent vaccination site. Corneal and conjunctival cultures were taken for bacteria, fungi, Acanthamoeba, and viruses. Viral-like cytopathic effects became evident in tissue culture within three days. Immunofluorescence studies were negative for varicella-zoster virus, herpes simplex virus, adenovirus, measles, mumps, parainfluenza, and influenza. Pox viral particles were identified in the infected tissue cultures by electron microscopy. The Hind III restriction endonuclease profile of the viral DNA isolate was similar to the Lister strain of vaccinia virus. Ocular vaccinia may manifest as a masquerade syndrome and may mimic signs of herpes simplex virus, varicella-zoster virus, and Acanthamoeba infection. Although vaccination with vaccinia is currently limited to a few populations throughout the world, vaccinia must still be considered in the differential diagnosis of infectious keratouveitis.


Subject(s)
Corneal Diseases/diagnosis , Uveitis, Anterior/diagnosis , Vaccinia/diagnosis , Adult , Antibodies, Viral/analysis , Corneal Diseases/microbiology , Corneal Diseases/pathology , Cytopathogenic Effect, Viral , DNA, Viral/analysis , Humans , Male , Smallpox Vaccine/adverse effects , Syndrome , Uveitis, Anterior/microbiology , Uveitis, Anterior/pathology , Vaccinia/microbiology , Vaccinia/pathology , Vaccinia virus/genetics , Vaccinia virus/immunology , Vaccinia virus/isolation & purification , Vaccinia virus/ultrastructure
9.
Arzneimittelforschung ; 44(3): 357-61, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8192703

ABSTRACT

The effects of propagermanium (3-oxygermylpropionic acid polymer) on various virus-infected mice were investigated. Propagermanium did not inhibit the multiplication of various DNA or RNA viruses in vitro. Oral administration of propagermanium in mice infected with herpes simplex virus type I (HSV-1) significantly prolonged the mean survival days. The efficacy of propagermanium at doses of 1 and 10 mg/kg daily was 13.4 +/- 2.3 and 14.2 +/- 2.3 mean survival days in comparison with 7.7 +/- 0.5 mean survival days at control group. In vaccinia virus-infected mice, oral doses of propagermanium ranging from 0.2 to 10 mg/kg suppressed the number of pocks on the tail which induced by the virus. Propagermanium (0.5-10 mg/kg) orally given to HSV-1-infected mice induced cytotoxic T lymphocytes (CTL) against HSV-1 antigen. In addition, propagermanium (1-10 mg/kg) enhanced interferon-gamma (IFN-gamma) induction in mice treated with Mycobacterium bovis (BCG). In mice spleen cells cultured with Concanavalin A, 0.1 to 10 micrograms/ml of propagermanium stimulated interleukin 2 (IL-2) production. It seems likely that the antiviral activity of propagermanium was exerted via enhancement of host immune resistance against viral infection.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antiviral Agents/pharmacology , Organometallic Compounds/pharmacology , Adjuvants, Immunologic/adverse effects , Animals , Antiviral Agents/adverse effects , Behavior, Animal/drug effects , Female , Germanium , Herpes Simplex/drug therapy , Herpes Simplex/microbiology , Herpesvirus 1, Human , In Vitro Techniques , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , Lymphocytes/drug effects , Lymphocytes/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mycobacterium bovis/immunology , Organometallic Compounds/antagonists & inhibitors , Propionates , Spleen/cytology , T-Lymphocytes, Cytotoxic/drug effects , Vaccinia/drug therapy , Vaccinia/microbiology , Vaccinia virus
10.
J Virol ; 68(1): 453-62, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8254756

ABSTRACT

The 14.7-kilodalton protein (14.7K protein) encoded by the adenovirus (Ad) E3 region inhibits tumor necrosis factor alpha (TNF-alpha)-mediated lysis of cells in tissue culture experiments, but the relevance of this effect in vivo is incompletely understood. To examine the effect of the ability of the Ad 14.7K protein to block TNF lysis upon viral pathogenesis in a murine model, we cloned the 14.7K protein-encoding gene into vaccinia virus (VV), permitting its study in isolation from other Ad E3 immunomodulatory proteins. The gene for murine TNF-alpha was inserted into the same VV containing the 14.7K gene to ensure that each cell infected with the VV recombinant would express both the agonist (TNF) and its antagonist (14.7K). VV was utilized as the vector because it accommodates large and multiple inserts of foreign DNA with faithful, high-level expression of the protein products. In addition, infection of mice with VV induces disease with quantifiable morbidity, mortality, and virus replication. The results of intranasal infections of BALB/c mice with these VV recombinants indicate that the Ad 14.7K protein increases the virulence of VV carrying the TNF-alpha gene by reversing the attenuating effect of TNF-alpha on VV pathogenicity. This was demonstrated by increased mortality, pulmonary pathology, and viral titers in lung tissue following infection with VV coexpressing the 14.7K protein and TNF-alpha, compared with the control virus expressing TNF-alpha alone. These results suggest that the 14.7K protein, which is nonessential for Ad replication in tissue culture, is an immunoregulatory protein which functions in vivo to help counteract the antiviral effects of TNF-alpha.


Subject(s)
Adenovirus E3 Proteins/pharmacology , Pneumonia, Viral/microbiology , Tumor Necrosis Factor-alpha/pharmacology , Vaccinia virus/drug effects , Vaccinia virus/pathogenicity , Adenovirus E3 Proteins/antagonists & inhibitors , Adenovirus E3 Proteins/biosynthesis , Administration, Intranasal , Animals , Disease Models, Animal , Female , Humans , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred BALB C , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , Survival Analysis , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Vaccinia/microbiology , Virulence/drug effects , Virus Replication/drug effects
11.
J Med Virol ; 41(3): 242-6, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8263505

ABSTRACT

Severe combined immune deficient (SCID) mice inoculated intravenously with vaccinia virus (VV) became sick within 6-8 days and died 10-12 days after infection. Tail lesions developed and the number depended on the virus inoculum. Age-matched immunocompetent NMRI mice similarly infected also developed tail lesions but did not become sick. When the infected SCID mice were treated with the acyclic nucleoside phosphonate HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine], either for 5 consecutive days starting on the day of infection or for 5 consecutive days starting on day 2, 4, or 6 post infection, or as a single dose at 7 days or 1 day before infection, VV-associated death was significantly delayed. VV-infected SCID mice that received two doses of 20 mg/kg of HPMPC every week survived the infection for about 130 days. The period during which the mice remained disease-free following HPMPC treatment correlated with the absence of detectable virus in their organs. The VV/SCID mouse model employed here may be useful for determining whether (attenuated) recombinant VV (carrying HIV genes) may have detrimental effects in the immunodeficient host. HPMPC may be considered as a drug candidate for the treatment and prophylaxis of such complications.


Subject(s)
Antiviral Agents/therapeutic use , Cytosine/analogs & derivatives , Organophosphonates , Organophosphorus Compounds/therapeutic use , Vaccinia virus/drug effects , Vaccinia/drug therapy , Animals , Antiviral Agents/pharmacology , Brain/microbiology , Cidofovir , Cytosine/pharmacology , Cytosine/therapeutic use , Disease Models, Animal , Dose-Response Relationship, Drug , Kidney/microbiology , Liver/microbiology , Lung/microbiology , Mice , Mice, SCID , Organophosphorus Compounds/pharmacology , Vaccinia/complications , Vaccinia/microbiology , Vaccinia virus/physiology , Virus Replication/drug effects
12.
Microb Pathog ; 15(5): 347-57, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8015416

ABSTRACT

To evaluate individual genes responsible for virulence of vaccinia virus, a shuttle vector containing a viral fusion fragment from a deletion mutant (Z-19) was constructed. The vector also included a beta-galactosidase cassette which contained the LacZ under the control of the vaccinia late promoter p11 to screen recombinants. The gene in question was reinserted into the attenuated mutant by homologous recombination. The vaccinia growth factor and the vaccinia complement-binding protein genes were inserted into this shuttle vector and transferred by recombination into Z-19. The presence of the inserted gene was examined by Southern hybridization and its expression by Northern blot hybridization. The virulence of the recombinants was investigated by inoculation into mice. Results indicate that the recombinants contained and expressed the inserted gene. Although virulence was recovered, it did not reach the level of the wild-type, either when the recombinant viruses were inoculated alone or in combination. Construction of the shuttle vector allows reintroduction of genes into Z-19 which is completely attenuated in vivo and could be useful as a vaccine vector.


Subject(s)
Carrier Proteins/genetics , Complement Inactivator Proteins , Gene Deletion , Genes, Viral , Genetic Vectors/genetics , Glycoproteins , Peptides/genetics , Vaccinia virus/genetics , Viral Structural Proteins/genetics , Animals , Carrier Proteins/biosynthesis , Female , Intercellular Signaling Peptides and Proteins , Mice , Mice, Inbred BALB C , Peptide Biosynthesis , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Smallpox Vaccine , Vaccines, Attenuated , Vaccinia/microbiology , Vaccinia virus/pathogenicity , Virulence/genetics
14.
J Virol ; 66(1): 386-98, 1992 Jan.
Article in English | MEDLINE | ID: mdl-1727494

ABSTRACT

Vaccinia virus (VV) is a potent immunogen, but the nature of VV proteins involved in the activation of the immune response of the host is not yet known. By screening a lambda gt11 expression library of rabbitpox virus DNA with serum from humans vaccinated against smallpox or with serum from VV-immunized animals, we identified several VV genes that encode highly antigenic viral proteins with molecular masses of 62, 39, 32, 25, 21, and 14 kDa. It was found that VV proteins of 62, 39, 25, and 21 kDa are part of the virus core, while proteins of 32 and 14 kDa are part of the virus envelope. All of these proteins were synthesized at late times postinfection. Proteins of 62 and 25 kDa were produced by cleavage of larger precursors of 95 kDa (p4a) and 28 kDa, respectively. The 21-kDa protein was the result of a cleavage of p4a, presumably at amino acid Gly-697. DNA sequence analysis, in comparison with the known nucleotide sequence of VV, provided identification of the corresponding open reading frames. Expression of the viral genes in Escherichia coli was used to monitor which of the viral antigens elicit immunodominant responses and the location of antigenic domains. Three viral antigens of 62, 39, and 32 kDa exhibited immunodominant characteristics. The most antigenic sites of 62 and 39 kDa were identified at the N terminus (amino acids 132 to 295) and C terminus (last 103 amino acids), respectively. Immunization of mice with the 62-, 39-, or 14-kDa antigenic proteins conferred different degrees of protection from VV challenge. Proteins of 32 and 14 kDa induced cellular proliferative responses in VV-infected mice. Our findings demonstrate the nature of VV proteins involved in the activation of host immune responses after vaccination, provide identification of the viral gene locus, and define structural and immunological properties of these antigenic VV proteins.


Subject(s)
Immunodominant Epitopes/genetics , Vaccinia virus/genetics , Viral Proteins/genetics , Adult , Amino Acid Sequence , Animals , Antibodies, Viral/biosynthesis , Antibodies, Viral/immunology , Base Sequence , Blotting, Western , Cloning, Molecular , DNA, Viral , Electrophoresis, Polyacrylamide Gel , Female , Genes, Viral , Humans , Immunity, Cellular , Male , Mice , Mice, Inbred BALB C , Middle Aged , Molecular Sequence Data , Neutralization Tests , Vaccinia/immunology , Vaccinia/microbiology , Vaccinia/prevention & control , Vaccinia virus/immunology , Viral Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology
15.
Virology ; 185(2): 929-33, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1720593

ABSTRACT

We have analyzed the role of exogenous administration of mouse interferon (IFN alpha + beta) on the replication of vaccinia virus in peritoneal cells and in the spleen of Balb/c mice. Mice were pretreated for 16 hr with IFN and then infected with a vaccinia virus recombinant expressing luciferase under an early or late virus promoter, and the enzyme activity was measured in the course of virus infection. A dose of IFN as low as 10(3) units/mouse abolished the appearance of luciferase activity in cells of the peritoneal cavity and in spleen cells. The IFN-mediated inhibition of luciferase activity was observed even when mice were infected 4 days after the administration of IFN. The IFN-treated animals were considered free of virus since neither luciferase nor viral proteins were detected in target cells several days after virus infection. Despite a severe IFN-mediated inhibition of luciferase activity, the appearance of luciferase on mRNA levels was not inhibited 6 hr after virus infection. Our finding revealed that replication of vaccinia virus in Balb/c mice is exquisitively sensitive to inhibition by IFN and that this effect occurs at early times postinfection, most likely as a result of a translational block.


Subject(s)
Gene Expression , Interferons/pharmacology , Vaccinia virus/genetics , Vaccinia/microbiology , Virus Replication , Animals , Kinetics , Luciferases/genetics , Luciferases/metabolism , Mice , Mice, Inbred BALB C , Peritoneum/microbiology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spleen/microbiology , Vaccinia virus/physiology
16.
J Immunol ; 147(12): 4327-32, 1991 Dec 15.
Article in English | MEDLINE | ID: mdl-1684375

ABSTRACT

CD8+ cytotoxic T (Tc) lymphocytes mediate recovery from vaccinia virus (VV) infection. In mice, anti-VV Tc cells are detectable on or after day 3 after infection, and cytolytic activity peaks between days 5 and 6. A rVV encoding murine IL-2, VV-hemagglutinin (HA)-IL-2, was cleared more rapidly, compared with a control rVV, VV-HA-thymidine kinase (TK), from tissues of infected euthymic normal mice. The mechanism of VV-HA-IL-2 clearance was operative early in infection and correlated with an elevated NK cell response, before the induction of anti-VV Tc cell response. We have investigated the roles of NK cells, T cells, and IFN-gamma in the rapid clearance of VV-HA-IL-2, by using specific mAb. Depletion of NK cells with mAb significantly enhanced VV-HA-IL-2 but not VV-HA-TK titers 3 days after infection. NK cells alone could not account for rapid viral clearance, because VV-HA-IL-2 titers in NK cell-depleted mice were not comparable to VV-HA-TK titers. Treatment with a mAb to IFN-gamma completely abrogated the IL-2-induced mechanism(s) of VV-HA-IL-2 clearance, and titers of the IL-2-encoding virus were comparable to control virus titers. In addition, the elimination of CD4+ but not CD8+ T cells resulted in significant increases in VV-HA-IL-2 titers.


Subject(s)
Interleukin-2/physiology , Vaccinia/immunology , Animals , CD4-Positive T-Lymphocytes/physiology , Cytotoxicity, Immunologic , Female , Interferon-gamma/physiology , Interleukin-2/genetics , Killer Cells, Natural/physiology , Lymphocyte Depletion , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Recombination, Genetic , T-Lymphocytes/physiology , Vaccinia/microbiology , Vaccinia virus/isolation & purification , Virus Replication
17.
J Gen Virol ; 72 ( Pt 1): 125-30, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1990060

ABSTRACT

A recombinant vaccinia virus vector was constructed which expressed the major surface glycoprotein G of human respiratory syncytial virus (RSV) and the thymidine kinase (tk) gene of vaccinia virus. The virulence of this tk+ recombinant virus was compared with that of a tk- recombinant and the wild-type (wt) virus after intranasal inoculation of mice. Respiratory infection with wt virus resulted in a lethal infection with widespread dissemination of virus. In contrast, infection with the tk- recombinant was not lethal and the virus had a reduced ability to disseminate to extrapulmonary tissue compared with wt virus. Insertion of the tk gene restored the virulence of the recombinant virus to the level of that of the wt virus. Despite a dramatic reduction in virulence of the tk- recombinant, virus could occasionally be recovered from the brains of mice. The expression of the attachment glycoprotein of RSV appeared to enhance the ability of the tk- recombinant virus to replicate in the lungs when compared with recombinants expressing fusion or nucleoprotein genes. The results confirm that inactivation of the tk gene results in a dramatic reduction of virulence for mice but suggest that there is still a potential danger of infection of the brain following intranasal administration of virus.


Subject(s)
HN Protein , Lung/microbiology , Thymidine Kinase/genetics , Vaccinia virus/pathogenicity , Vaccinia/microbiology , Viral Proteins , Animals , Antigens, Viral/genetics , Genes, Viral , Mice , Mice, Inbred BALB C , Phenotype , Recombination, Genetic , Respiratory Syncytial Viruses/genetics , Vaccinia virus/genetics , Vaccinia virus/physiology , Viral Envelope Proteins , Virulence/genetics , Virus Replication
18.
Biochem Pharmacol ; 39(10): 1573-80, 1990 May 15.
Article in English | MEDLINE | ID: mdl-2337414

ABSTRACT

Alkyl esters of (RS)-3-(adenin-9-yl)-2-hydroxypropanoic acid (AHPA) were shown recently to be broad spectrum antiviral agents (De Clercq E and Holy A, J Med Chem 28: 282-287, 1985). It was postulated that these alkyl esters function as prodrugs by undergoing hydrolysis catalyzed by cellular esters to AHPA, a known inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. In this study, we describe the metabolic fate of the 2-methylpropyl ester of AHPA (AHPA-iBu) in murine L929 cells. When AHPA-iBu was included in the culture medium, it was taken up rapidly by murine L929 cells. The uptake was time- and concentration-dependent, resulting in the intracellular accumulation of the free acid, AHPA. Treatment with AHPA-iBu caused inhibition of cellular AdoHcy hydrolase in both a time- and a dose-dependent manner. Complete inhibition of the enzyme was achieved after a 1-hr incubation in culture medium containing 50 microM AHPA-iBu. The inhibition of the enzyme caused cellular accumulation of AdoHcy and a significant increase in the ratio of AdoHcy/S-adenosyl-L-methionine (AdoMet). Partial recovery of the AdoHcy hydrolase activity in L929 cells treated with 50 microM AHPA-iBu was observed after 24 hr. This recovery of enzyme activity was paralleled by a significant decrease in the cellular levels of AdoHcy and the ratio of AdoHcy/AdoMet. AHPA-iBu also exerted an inhibition (IC50 = 0.17 microM) of vaccinia virus plaque formation in monolayers of L929 cells. A 1 microM concentration of AHPA-iBu, which caused 80% inhibition of plaque formation, produced a 17-fold increase in AdoHcy content in drug-treated, virus-infected cells versus non-drug-treated, virus-infected cells and a 15% undermethylation of the poly(A)+ RNA. These data show that AHPA-iBu is a prodrug for AHPA which inhibits cellular AdoHcy hydrolase. The inhibition of this enzyme elevates cellular levels of AdoHcy, creating an unfavorable environment which suppresses replication of vaccinia virus.


Subject(s)
Adenine/analogs & derivatives , Antiviral Agents/pharmacology , L Cells/drug effects , Vaccinia virus/drug effects , Adenine/analysis , Adenine/pharmacology , Adenosylhomocysteinase , Animals , Cell Line , Chromatography, High Pressure Liquid , Hydrolases/antagonists & inhibitors , Mice , S-Adenosylhomocysteine/analysis , S-Adenosylmethionine/analysis , Vaccinia/microbiology , Virus Replication/drug effects
19.
Microb Pathog ; 6(3): 219-26, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2739561

ABSTRACT

Understanding the molecular basis of virulence in poxvirus is of great importance for the development of recombinant vaccines using vaccinia virus as a vector. We have previously described mutants of vaccinia virus with deletions ranging from 20 to 21 kb at the left terminus and with attenuated phenotype. The virulence of these mutants was studied, using different routes of inoculation, for protection from wild-type challenge in mice and for replication in vivo. Regardless of the route of inoculation, the LD50 of the deletion mutants is at least 1000-fold higher than that of the wild-type. Results from protection experiments using viable and ultraviolet-inactivated viruses, and from determination of infectivity in different organs, indicated that the mutants were unable to replicate in vivo. Southern blot hybridization of viral DNA with pSC16, a plasmid containing the vaccinia growth factor (VGF) gene, revealed that in the IHD-W strain of vaccinia virus this gene is localized at the left terminus exclusively and that the mutants lack this gene. The results suggest that absence of the VGF gene is correlated with inability to replicate in vivo and decreased virulence.


Subject(s)
Growth Substances/genetics , Peptides/genetics , Vaccinia virus/genetics , Animals , Blotting, Southern , DNA, Viral/analysis , Female , Intercellular Signaling Peptides and Proteins , Liver/microbiology , Mice , Mutation , Nucleic Acid Hybridization , Phenotype , Spleen/microbiology , Vaccinia/microbiology , Vaccinia virus/pathogenicity , Vaccinia virus/physiology , Virulence , Virus Replication
20.
Article in Russian | MEDLINE | ID: mdl-3176779

ABSTRACT

The present work describes a case of human disease resulting from the bite of a white rat and caused by a biological variant of cowpox virus. The isolates obtained from the sick man and the white rats which had been the source of this infection proved to be identical and did not differ from the biological variants of cowpox virus, isolated earlier from white rats and carnivorous animals of the family Felidae. Thus, the possibility of ratpox (cowpox) transmission from sick rodents to man was established.


Subject(s)
Occupational Diseases/diagnosis , Vaccinia/diagnosis , Adult , Animals , Bites and Stings/complications , Bites and Stings/microbiology , Disease Vectors , Female , Humans , Occupational Diseases/microbiology , Occupational Diseases/transmission , Rats , Rodent Diseases/microbiology , Rodent Diseases/transmission , Time Factors , Vaccinia/microbiology , Vaccinia/transmission , Vaccinia/veterinary , Vaccinia virus/isolation & purification
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