用蚀斑法滴定痘苗病毒的准确性及其精确数学模型的探讨

用蚀斑法滴定病毒是确定感染病毒颗粒存在数量的一种较准确方法。本实验表明,痘苗病毒吸附4h后仍有大量病毒粒子未能吸附到细胞单层,进而测定出病毒接种量、维持液加量和所测病毒滴度间具有一种互为消长的非线性相关性。因而设计了几种检测方法,其准确性均优于常规痘苗病毒蚀斑测定法。利用装配有Mathematic软件包的计算机在痘苗病毒接种量、维持液加量和所测病毒滴度间建立了曲线拟合模型和曲面拟合模型。通过曲线拟合模型推断病毒感染滴度为常规法滴定值的近5倍。     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.     

Cellular autophagy machinery is not required for vaccinia virus replication and maturation

The origin of the primary membrane of the vaccinia virus, a double-membrane structure that surrounds the immature virions (IV), is not fully understood. Here we investigated whether the primary membrane originates from the autophagic membrane. Morphologic studies by electron microscopy (EM) showed no apparent difference in viral maturation in the autophagy-deficient cell lines, the atg5(-/-) mouse embryonic fibroblasts (MEFs) and the beclin1(-/-) embryonic stem (ES) cells, compared to their isogenic wild-type counterparts. Moreover, viral growth curves demonstrated that vaccinia viruses replicate and mature in the autophagy-deficient cell lines as efficiently as they do in their isogenic wild type counterpart cells. This study indicates that the cellular autophagy machinery is not required for the life-cycle of vaccinia virus, suggesting that the primary vaccinia viral membrane does not originate from the autophagic membrane.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo

An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L(-)) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L(-) mutant virus. IMV from the H3L(-) mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L(-) mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.     

Inhibition of the vaccinia virus associated ribonuclease by HeLa S3 cytosol

A ribonuclease associated with purified vaccinia virus is able to degrade into 3S fragments the viral 8–12S mRNAs synthesized $\text{in}\text{vitro}$. This RNase not detected on purified viral cores was shown to be located on the outer side of the viral envelope. When added to reaction mixture, a partially purified HeLa S₃ cytosol fully inhibits the vaccinia virus associated RNase. Other cytosols from very common mammalian cell lines also contain RNase inhibitor and are of interest in order to prepare large amounts of undegraded vaccinia virus mRNAs.     

Role of interferon in lymphocyte recruitment into the skin

Large numbers of lymphocytes are recruited from the blood into sites of cutaneous DTH reactions. Our goal was to investigate the factors controlling this recruitment. 111In-labeled peritoneal exudate lymphocytes were injected iv and the accumulation of these cells in skin sites injected with a variety of stimuli, was used to measure lymphocyte recruitment in rats. Large numbers of lymphocytes migrated into vaccinia- and KLH-injected sites in sensitized animals, but only into the viral and not the KLH lesions in non-immune animals. Lymphocytes also migrated efficiently into sites injected with the alpha-interferon (IFN) inducers, uv-inactivated vaccinia virus and poly I:C, as well as into sites injected with IFN. In each case there was a dose-response relationship. Analysis of the kinetics of lymphocyte recruitment demonstrated that the peak rate of migration occurred most rapidly after the injection of IFN, later after poly I:C, and was slowest to be reached after vaccinia virus. Rabbit anti-IFN blocked the recruitment of lymphocytes by uv-inactivated vaccinia and by IFN. Histologically, all of these sites demonstrated a dense mononuclear cell infiltrate in the dermis. It is suggested that IFN may be an important mediator in the recruitment of lymphocytes into inflammatory reactions.     

Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.     

The mechanisms of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: clearance of local infection

We have shown that immunization of mice with a vaccinia virus recombinant expressing glycoprotein D of Herpes simplex virus (HSV)-1 will induce a variety of L3T4+ T cell responses. These included a HSV-specific delayed-type hypersensitivity response, T cell help for the induction of antiviral antibodies, and the ability to eliminate a challenge dose of HSV from the pinna. This protection against a subcutaneous virus challenge was not mediated by the delayed-type hypersensitivity response because intravenous inoculation of the vaccinia virus recombinant expressing HSV-1-gD induced a state of split tolerance. Thus, mice could still clear a HSV challenge inoculum from the pinna yet were unable to mount a HSV-specific delayed-type hypersensitivity response. Evidence is presented that suggests the protective response was, at least, in part mediated by a T cell-dependent induction of virus-neutralizing antibodies. Evidence is also presented that may suggest the failure of a vaccinia virus recombinant expressing HSV-1-gD to induce HSV-specific cytotoxic T cell responses appears to minimize the protective response to only efficiently clearing low 10(4) 50% tissue culture infective dose) challenge populations of virus. These findings are discussed with relevance to the immune control of HSV infections and to the future development of anti-HSV vaccines.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Immunogenicity and safety of defective vaccinia virus lister: comparison with modified vaccinia virus Ankara

Potent and safe vaccinia virus vectors inducing cell-mediated immunity are needed for clinical use. Replicating vaccinia viruses generally induce strong cell-mediated immunity; however, they may have severe adverse effects. As a vector for clinical use, we assessed the defective vaccinia virus system, in which deletion of an essential gene blocks viral replication, resulting in an infectious virus that does not multiply in the host. The vaccinia virus Lister/Elstree strain, used during worldwide smallpox eradication, was chosen as the parental virus. The immunogenicity and safety of the defective vaccinia virus Lister were evaluated without and with the inserted human p53 gene as a model and compared to parallel constructs based on modified vaccinia virus Ankara (MVA), the present "gold standard" of recombinant vaccinia viruses in clinical development. The defective viruses induced an efficient Th1-type immune response. Antibody and cytotoxic-T-cell responses were comparable to those induced by MVA. Safety of the defective Lister constructs could be demonstrated in vitro in cell culture as well as in vivo in immunodeficient SCID mice. Similar to MVA, the defective viruses were tolerated at doses four orders of magnitude higher than those of the wild-type Lister strain. While current nonreplicating vectors are produced mainly in primary chicken cells, defective vaccinia virus is produced in a permanent safety-tested cell line. Vaccines based on this system have the additional advantage of enhanced product safety. Therefore, a vector system was made which promises to be a valuable tool not only for immunotherapy for diseases such as cancer, human immunodeficiency virus infection, or malaria but also as a basis for a safer smallpox vaccine.     

In vitro proliferation of rabbit bone marrow-derived and thymus-derived lymphocytes in response to vaccinia virus

Peripheral blood leukocytes from rabbits immunized with vaccinia virus were incubated in vitro with vaccinia antigen, and resultant lymphocyte proliferation was measured by incorporation of tritiated thymidine into acid-insoluble material. Significant lymphocyte stimulation was observed at a time when antiviral antibody was being synthesized in vivo. The extent of proliferation by bone marrow-derived lymphocytes after culture with viral antigen was determined by simultaneous detection of complement receptor lymphocytes (CRLs have been shown to be B cells) and uptake of tritiated thymidine in these CRLs by radioautography. The results indicate that both bone marrow-derived and thymus-derived lymphocytes participate in the in vitro proliferative response of rabbit peripheral blood lymphocytes to vaccinia antigen.     

Preexisting vaccinia virus immunity decreases SIV-specific cellular immunity but does not diminish humoral immunity and efficacy of a DNA/MVA vaccine

The influence of preexisting immunity to viral vectors is a major issue for the development of viral-vectored vaccines. In this study, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/modified vaccinia Ankara (MVA) SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of an SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively, in preimmune animals compared with those of control animals. The peak and set point viremia correlated directly with colorectal virus at 2 wk postchallenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included 1) the presence of anti-SIV IgA in rectal secretions, 2) high-avidity binding Ab for the native form of Env, and 3) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral coreceptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 wk postchallenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.