Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human sera.

The capacity of human sera genetically deficient in selective complement (C) components to enhance neutralization of enveloped viruses was examined by kinetic plaque reduction assays. Vaccinia virus, a DNA virus, and vesicular stomatitis virus (VSV), an RNA virus, were studied. Exogenous rabbit: or human antibody to vaccinia virus, and guinea pig or human antibody to VSV were provided in limiting, C-dependent concentrations. IgG antibodies predominated in most of the antisera employed. C5-deficient and C6-deficient human sera consistently supported normal rates of neutralization of either virus; this effect was heat-labile. C4-deficient human serum did hot exceed heat-inactivated serum in any neutralization assay. C1r-deficient serum displayed slight heat-labile neutralizing capacity against vaccinia but none against VSV. C2- and C3-deficient sera consistently exhibited measurable but clearly subnormal rates of neutralization. Two fresh agammaglobulinemic sera failed to inactivate either virus in the absence of added antibody. These results confirm and extend earlier evidence, based on neutralization of herpes simplex and Newcastle disease viruses in the presence of early (IgM) antibody and functionally pure guinea pig C components or C-deficient animal sera, that the late-acting components C5-C9 are not required for C-dependent neutralization. Data on four enveloped viruses now agree that this function is mediated by C1-C3, although C1 plus C4 appear to have some neutralizing capacity. This requirement for C1-C3 is overcome, however, in the presence of higher antibody cohcentrations, suggesting that the contribution of the C system to viral neutralization in vivo may be chiefly in the early phase of infection when antibody is limited.     

水泡性口炎病毒核蛋白基因的表达及初步应用

将水泡性口炎病毒编码群特异性抗原的N基因片段克隆至pMD18-T克隆载体质粒中,构建N基因克隆重组质粒,进行核苷酸序列分析。然后亚克隆插入pBAD/Thio TOPO表达载体,经PCR限制性内切酶分析、测序鉴定,筛选获得N基因正向插入、有正确读码框的阳性克隆,成功构建了水泡性口炎病毒N基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达N蛋白抗原。SDS-PAGE、Western blotting及间接ELISA试验结果表明,表达蛋白为融合蛋白,质量约63.5 kD,其表达产量约占菌体总蛋白的16％,相当于92mg/L。融合蛋白中含有水泡性口炎病毒群特异性的核蛋白抗原,应用表达的VSV核蛋白抗原建立了酶联免疫吸附试验,通过对186份山羊、豚鼠实验动物人工感染VSV的血清样品和参考血清样品的检测,并与微量血清中和试验进行了比较,结果表明：以表达的VSV核蛋白为包被抗原的酶联免疫吸附试验是一种特异性强、敏感性高、快速、简单、安全的检测方法,抗原制备成本低。     

狂犬病毒蚀斑方法在病毒研究和疫苗研制中的应用

本文采用狂犬病毒CTN-1和4aC株,经Vero细胞传代适应后,以Vero细胞为培养基质,建立了狂犬病毒蚀斑试验和蚀斑减少试验的方法。目前已将此方法应用于病毒鉴定、病毒克隆、病毒滴定以及抗狂犬血清的检测,并取得了较好的结果。     

Factors Affecting the Sensitivity of Different Viruses to Interferon

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV(pi), a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.     

Virion-Associated Complement Regulator CD55 Is More Potent than CD46 in Mediating Resistance of Mumps Virus and Vesicular Stomatitis Virus to Neutralization

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.     

Properties of DI particle resistant mutants of vesicular stomatitis virus isolated from persistent infections and from undiluted passages

We describe an assay procedure to quantitate relative DI resistance of a variety of DI particle resistant (Sdi^?) mutants of vesicular stomatitis virus (VSV). We show that numerous diverse Sdi^? mutants of VSV are selected continuously in a stepwise manner during persistent infections, and also during serial undiluted lytic passages initiated with cloned virus. Concurrently with the successive appearance and disappearance of different Sdi^? mutants of infectious VSV, new DI particle types with altered interference properites also appear and disappear, resulting in rapid “coevolution” of virus and DI particle populations. Complementation tests with Sdi^? mutants indicate that mutations in at least two different virus factors (presumably associated with replication-encapsidation) can give rise to Sdi^? mutants. Interference studies with chimeric DI particles indicate that DI particle template RNA rather than DI particle protein determines the interference properties of DI particles interacting with Sdi^? and Sdi⁺ mutants of helper virus.     

Removal of Viruses from Human Intravenous Immune Globulin by 35 nm Nanofiltration

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.     

Susceptibility of influenza viruses to interferon and to poly(I) . Poly(C) determined by the plaque reduction method

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) . poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) . poly(C) for influenza A and B viruses were as high as 3.0- to 4.5-fold and 6- to 18-fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0-6.2 and 7.3-15.2 units/ml, respectively. The ratio of PD50 of poly(I) . poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly(I) . poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) . poly(C).     

Identification of a cellular receptor for mouse mammary tumor virus and mapping of its gene to chromosome 16

Pseudotypes of vesicular stomatitis virus (VSV) containing envelope glycoproteins provided by C3H mammary tumor virus (MTV) instead of the normal VSV G-proteins were prepared and used to assay the presence of an MTV receptor on cells. The assay was specific as demonstrated by competition studies with excess MTV particles and neutralization of the pseudotypes with anti-MTV serum or monoclonal antibodies directed against MTV gp52. The MTV receptor was abundantly present on mouse cells but hardly detectable on nonmurine cells, including the Chinese hamster cell line E36. Somatic cell hybrids between E36 cells and GRS/A spontaneous leukemia cells (GRSL cells) and between E36 and GRS/A primary mammary tumor cells were made. The hybrids retained all Chinese hamster chromosomes but segregated mouse chromosomes. From the analysis of the isoenzymes and chromosomes of the hybrid cell lines we conclude that the gene for the receptor (MTVR-1) is located on mouse chromosome 16.     

VSV Pseudotype Particles with the Coat of Avian Myeloblastosis Virus

PSEUDOTYPES of vesicular stomatitis virus (VSV) with the coat of avian myeloblastosis (AMV) or murine leukaemia viruses—VSV(AMV) and VSV(MLV)—can be produced by growing VSV in chick cells preinfected with AMV or in mouse cells preinfected with MLV¹. The VSV particles carrying their own neutralization antigen and double-neutralizable particles may be inactivated with antiserum against VSV. The surviving pseudotypes possess neutralization, host-range and interference specificities corresponding to the tumour virus donating their coat. It has also been shown that a conditional lethal mutant of VSV in which a structural protein is affected is complemented under restrictive conditions with AMV. This mutant, ts-45, when complemented with AMV again predominantly produces the pseudotype VSV(AMV).     

速成单层法半微量病毒空斑技术的研究

本文报道一种不需预先制备细胞单层的速成单层法半微量病毒空斑技术(简称速成法)及其在空斑中和试验与空斑纯化试验中的应用。作者以脊髓灰质炎病毒为代表,对速成法的实验条件、可靠性、敏感性、重复性等作了系统研究,并以此法对单纯疱疹病毒、呼吸道合胞病毒和水泡性口炎病毒进行了空斑滴定。结果提示速成法简单快速、经济方便、敏感稳定、可靠易行,可能具有较大的实用和推广价值。     

检测流行性出血热病毒滴度和中和抗体效价的半微量空斑法

建立了检测流行性出血热病毒滴度和中和抗体效价的半微量空斑法。小牛血清与胎牛血清的培养效果无差异。7株不同来源的出血热病毒均能在E6细胞上形成空斑。接种的病毒浓度与形成的空斑数呈直线关系。用空斑法测得的病毒滴度稍低于荧光TCIE50滴定法。空斑减少中和试验的敏感性较荧光中和试验高30倍左右。同时还初步表明了本方法可用于流行性出血热病毒的抗原性分析。     

PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice

We are developing oncolytic vesicular stomatitis viruses (VSVs) for systemic treatment of multiple myeloma, an incurable malignancy of antibody-secreting plasma cells that are specifically localized in the bone marrow. One of the presumed advantages for using VSV as an oncolytic virus is that human infections are rare and preexisting anti-VSV immunity is typically lacking in cancer patients, which is very important for clinical success. However, our studies show that nonimmune human and mouse serum can neutralize clinical-grade VSV, reducing the titer by up to 4 log units in 60 min. In addition, we show that neutralizing anti-VSV antibodies negate the antitumor efficacy of VSV, a concern for repeat VSV administration. We have investigated the potential use of covalent modification of VSV with polyethylene glycol (PEG) or a function-spacer-lipid (FSL)–PEG construct to inhibit serum neutralization and to limit hepatosplenic sequestration of systemically delivered VSV. We report that in mice passively immunized with neutralizing anti-VSV antibodies, PEGylation of VSV improved the persistence of VSV in the blood circulation, maintaining a more than 1-log-unit increase in VSV genome copies for up to 1 h compared to the genome copy numbers for the non-PEGylated virus, which was mostly cleared within 10 min after intravenous injection. We are currently investigating if this increase in PEGylated VSV circulating half-life can translate to increased virus delivery and better efficacy in mouse models of multiple myeloma.     

Use of microplate cell culture and enzyme immunoassay in titration of serum neutralizing antibody against Hochi strain of serotype 4 human rotavirus

The tube neutralization test read by enzyme immunoassay developed by Wyatt et al. (1983) for serotype determination of human rotavirus was modified so as to use stationary cultures of MA104 cells in a microtiter plate instead of roller tube cultures. Sera obtained from different age groups were titrated for neutralizing antibody against serotype 4 human rotavirus Hochi strain by this test and the results were compared with those obtained by the plaque neutralization test. There was a good correlation between the titers obtained by the two tests and the age distribution pattern of serotype 4 neutralizing antibody was similar to those of serotype 1 and 3 antibodies previously reported.     

Antibodies against the two serotypes of vesicular stomatitis virus measured by enzyme-linked immunosorbent assay: immunodominance of serotype-specific determinants and induction of asymmetrically cross-reactive antibodies.

The serological relationship between the two vesicular stomatitis virus (VSV) strains Indiana (VSV-Ind) and New Jersey (VSV-NJ) were analyzed by using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G responses, defined by their resistance to treatment with 2-mercaptoethanol, were assessed by ELISA by using sucrose gradient-purified VSV or purified VSV glycoproteins (G) as antigens. When low doses (10(6) PFU) of live VSV or 10(8) PFU of UV-inactivated virus were given intraperitoneally (i.p.), only non-cross-reactive antibody responses were observed in a primary immune response. However, when 10(6) PFU of live VSV were injected intravenously (i.v.), cross-reactive antibodies were generated; anti-VSV-NJ antibodies cross-reacted more against VSV-Ind than did anti-VSV-Ind antibodies against VSV-NJ. When 10(8) PFU of live VSV or UV-inactivated VSV mixed with complete Freund adjuvant was given i.p., high levels of cross-reactive antibodies detectable by ELISA were induced in primary and secondary responses. When purified G protein was used instead of purified whole virus in the ELISA, the cross-reactivity was found to be asymmetrical after immunization with live VSV given i.v. but not after i.p. inoculation; anti-VSV-NJ sera bound almost equally well to VSV-Ind G protein, whereas anti-VSV-Ind sera bound virtually exclusively to the G protein of the homologous serotype. The data suggest that immunization with VSV given i.p. results in a more specific, i.e., less cross-reactive, response than that either after i.v. infection or after the virus antigen is made available in great amounts or if it persists for prolonged periods when given i.p. together with complete Freund adjuvant. The unique determinants were immunodominant because they induced antibodies preferentially, whereas partially shared determinants induced antibody responses asymmetrically, more slowly, and with lower titers. Interestingly, the asymmetric cross-reactivity of anti-VSV antibodies, as measured by ELISA, against purified VSV G was opposite that observed for cytotoxic T cells.     

A dot-immunobinding assay on nitrocellulose with psoralen inactivated Herpesvirus simiae (B virus)

An enzyme-immunoassay performed with Herpesvirus simiae (B virus) and H. simplex antigens inactivated with a psoralen derivative and long-wavelength ultraviolet light irradiation is described. Although B virus is a known human pathogen requiring extreme care in its handling, the use of inactivated antigens in the test allows its performance without biosafety containment. The test utilizes nitrocellulose sheets dotted with antigen for the assay of antibody against B virus in nonhuman primate sera. Antigen-antibody complexes are detected visually as red dots by the use of enzyme-conjugated antiglobulin second antibody and a substrate that produces an insoluble product. The test is more rapid, sensitive and specific than the serum neutralization test it is intended to replace. Of 150 macaque monkey sera tested, 83 were negative by the enzyme and neutralization tests, 56 were positive by both tests and 11 were positive by enzyme-assay but negative by neutralization. Positive sera reacted with both simian and human viral antigens in the enzyme assay but with greater intensity against B virus. Absorption with H. simplex removes reactivity with this virus without reducing the B virus response.     

Neutralization of vesicular stomatitis virus (VSV) by human complement requires a natural IgM antibody present in human serum

Neutralization of VSV by human serum ws previously shown to involve C1, C2, C3, and C4 of the classical complement (C) pathway. All normal human sera tested were equivalently active in this regard. However, purified C1, C2, C3, and C4 were unable to mediate VSV neutralization. In the present studies an additional factor required for C-mediated neutralization was isolated from normal human serum and identified as a natural IgM antibody specific for a viral encoded antigen. Purified IgM bound to the virus and formed a complex that activated component C1. Normal serum concentrations of purified IgM, C1, C2, C3, C4 neutralized VSV to the same extent as normal serum. Purified IgM did not neutralize VSV alone or in conjunction with C1, C2, and C4. Inclusion of C3 resulted in full neutralization and C3b binding to the virus was demonstrated. Thus, normal human serum contains a natural antibody of the IgM class that is directed toward a viral antigen. The antibody facilitates neutralization by forming an immune complex that activates C1 and thus efficiently initiates the classical pathway at the viral surface. Neutralization occurs with C3b deposition on the viral envelope and probably results from a blanket of C protein that interferes with viral attachment to susceptible cells.     

Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus.

Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses. Titers obtained with Mengo virus as challenge were all lower than with VSV. With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold. With Mengo virus-induced interferon the reduction was much greater (about 17-fold). This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus. The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus. This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro.     

Antibody-independent neutralization of vesicular stomatitis virus by human complement. II. Formation of VSV-lipoprotein complexes in human serum and complement-dependent viral lysis.

Vesicular stomatitis virus (VSV) is efficiently neutralized by normal, nonimmune human serum without the participation of antibody. Neutralization is complement- (C) dependent and requires the early-acting components of the classical pathway, C1, C4, C2, and C3, but not later-acting C components. In further studies, normal human serum was found to markedly increase the density of a variable but significant proportion of virus-associated RNA and to markedly decrease the density of the remainder of virus-associated RNA. The RNA of increased density was found to be dense ribonucleocapsid cores released from VSV by C-dependent viral lysis mediated through the classical pathway. The released ribonucleocapsid cores found at the bottom of sucrose density gradient after incubation of VSV with human serum were resistant to degradation by proteolytic enzymes. The VSV-derived RNA found floating on the tops of sucrose density gradients performed on serum-treated VSV was infectious virus. The decreased density was due to binding of VSV to human serum lipoproteins (LP), primarily very low density lipoproteins (VLDL). Binding of VLDL to VSV required the presence of the viral envelope and the external glycoprotein, G. Despite the binding of LP to VSV, LP did not neutralize VSV, and LP-depleted sera were fully active in neutralizing VSV. Thus, LP do not represent an accessory factor for the C-dependent neutralization of VSV.