Phosphoproteins, structural components of rhabdoviruses

Polypeptides derived from purified rabies, vesicular stomatitis, and Kern Canyon viruses, which were labeled with ³H-amino acids and ³²P-phosphate, were subjected to electrophoretic fractionation in order to determine which of the structural proteins is phosphorylated. In the rabies virion, the nucleocapsid protein (N protein) was found to be phosphorylated, whereas the other three virus proteins were not. N protein of free viral nucleocapsids isolated from rabies virus-infected cells was also phosphorylated. The phosphorylation of the N protein of rabies virus is confined to a relatively small terminal segment of the polypeptide chain which can be specifically cleaved off by treatment of the nucleocapsid with trypsin. In vesicular stomatitis virus the minor NS protein component is the only phosphoprotein. In Kern Canyon virus the envelope glycoprotein and the N protein are both phosphorylated, but to a lesser extent than either the N protein of rabies virus or the NS protein of vesicular stomatitis virus. All three rhabdoviruses contain a virion-bound protein kinase. Free nucleocapsids derived from rabies virus-infected cells and purified by equilibrium centrifugation in CsCl solution also contain a protein kinase. Intracellular nucleocapsids of vesicular stomatitis or Kern Canyon viruses do not exhibit protein kinase activity.     

Host gene influence on interferon action in adult mouse hepatocytes: specificity for influenza virus

Primary monolayer cultures of hepatocytes isolated from adult mice either genetically resistant (bearing the autosomal dominant allele Mx) or genetically susceptible toward influenza virus infection were tested for susceptibility to three viruses: a hepatotropic influenza A virus, vesicular stomatitis virus, and herpes simplex type I virus. Hepatocytes of both genotypes spontaneously released equal amounts of interferon during the first day of culture. Spontaneous interferon was sufficient to protect the cells markedly against vesicular stomatitis virus and marginally against herpesvirus infection. With respect to influenza virus, only Mx-bearing hepatocytes lost their initial susceptibility. Treatment with anti-interferon antibodies during the first 24 hr of culture rendered Mx-bearing cells as permissive for all three viruses as non-Mx-bearing cells. In such anti-interferon-treated cultures the antiviral resistance could be restored with highly purified mouse type I interferon. The antiviral state induced by exogenous interferon displayed the same characteristics as that induced by spontaneous interferon: it was highly efficient in inhibiting influenza A virus replication in Mx-bearing cells and inefficient in non-Mx-bearing cells. Inhibition of vesicular stomatitis virus or herpesvirus was independent of the Mx genotype.     

Isolation of the matrix (membrane) protein of vesicular stomatitis virus by gel filtration in guanidine hydrochloride.

The L, N and M proteins of vesicular stomatitis virus (VSV) were resolved from each other by gel filtration in the presence of 6 m-guanidine hydrochloride. Amino acid analysis for purified M protein of VSV showed that its chemical composition differed from those of influenza and SV5 M proteins.     

Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase

Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2 μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.     

A novel approach for the production of monoclonal antibodies using infectious vaccinia virus recombinants

We describe a novel approach of producing monoclonal antibodies (MABs) to one specific protein of a virus or other agent consisting of several proteins, without the use of purified antigen in either the immunization or screening phase of the procedure. This method has general application in the production of MABs when the antigen cannot be obtained in a pure form, but the gene is available. We illustrate this application by producing MAB specific to the nucleocapsid protein (N) of vesicular stomatitis virus serotype Indiana (VSV-IN) from BALB/c mice immunized with an infectious vaccinia virus recombinant vector (v38) that expresses the N gene of VSV-IN. This novel method of immunization obviates the need for initial purification of the protein antigen and injection of adjuvants with the isolated protein as is done in traditional MAB production.     

Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies

Splenic lymphocytes from BALB/c mice immunized with the TC-83 vaccine strain of Venezuelan equine encephalomyelitis virus (VEE) were fused to the nonsecreting myeloma cell line Sp2/0-Ag 14. Fusion products were screened for antibody synthesis using an enzyme-linked immunosorbent assay (ELISA) with purified TC-83 virus. Antigenic specificity of cloned hybridoma cells was determined by ELISA or radioimmune precipitation using purified E2 (gp56), El (gp50), and capsid. Antibodies were charcterized by chromatography on protein A-Sepharose and by isoelectric focusing. Analysis of antigenic epitopes by cross-reactivity panels using closely related VEE strains and competitive binding assays indicated the presence of three epitopes on the gp56 and four epitopes on the gp50. Close spatial arrangement of gp56 and gp50 in the native virion could be demonstrated. The biologic functions of hemagglutination and virus neutralization were primarily associated with only one antigenic epitope present on the gp56.     

Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.     

Sensitive radioimmune assay for measuring Aleutian disease virus antigen and antibody

A solid-phase, one-step radioimmune assay was developed which could detect as little as 0.02 microliter of a standard Aleutian disease virus antigen preparation, approximately 3.2 ng of viral protein. Virus antigen was measured in different mink organs and cell types during experimental intraperitoneal infection. The gut and kidney were the first organs in which virus antigen could be detected (day 3 to 6 after infection). On day 6 or later virus antigen was found in spleen, liver, kidney, lymph nodes, peritoneal exudate, and bone marrow cells. With inhibition of antigen binding, a radioimmune assay was developed for antibody detection. Viral antibodies could be detected as early as 3 days after infection. Antibody titers from 1/10(5) to more than 1/10(6) were found in plasmacytotic mink. When the sensitivity of the antibody radioimmune assay was compared with that of other known methods for anti-Aleutian disease virus quantitation, the radioimmune assay was considerably more sensitive, detecting as little as 5 ng of antibody.     

马传染性贫血病毒囊膜蛋白GP90的表达及其免疫效果研究

目的：探索马传染性贫血病毒（EIAV）野毒株（强毒，LN）和疫苗毒株（弱毒，DLV）的囊膜蛋白GP90作为鉴别诊断试剂的效果及基因工程疫苗的可能性。方法：将中国EIAV疫苗株（DLV）及其亲本毒株辽毒株（LN）接种于驴外周血白细胞培养物，提取前病毒DNA，并以其为模板，经聚合酶链法（PCR）扩增出LN和DLV的GP90片段，在Bac-to-Bac杆状病毒表达系统表达。表达的GP90蛋白经金属离子亲和层析（IMAC）纯化后免疫小鼠，ELISA法检测抗EIAV抗体，中和试验检测中和抗体活性。同时对DLV和LN GP90的核苷酸与氨基酸进行比较。结果：DLV和LN GP90核苷酸序列的同源性为95．3％，氨基酸序列的同源性为87.7％。未加佐剂组抗体滴度为800，佐剂组抗体滴度达1600;不加佐剂组的中和抗体滴度在40－80之间，加佐剂组中和抗体滴度在80－160之间。结论：成功构建了分别表达EIAV野毒株LN和疫苗毒株DLV囊膜蛋白GP90的重组杆状病毒，大量表达的蛋白纯化后纯度较好，该纯化产物在小鼠体内可激发良好的体液免疫应答。     

In vivo interference in vesicular stomatitis virus infection.

Inactivated defective interfering and complete particles of vesicular stomatitis virus given intracerebrally to adult mice protect them against challenge with homologous virus whether this is given at the same time or several days later. Two separate protective processes appear to be involved. The first, which comes into operation immediately after inoculation, is also effective against heterologous strains of vesicular stomatitis virus, rabies (another rhabdovirus), and a neurotropic strain of foot-and-mouth disease virus. The second, later effect, which is strain specific, appears to be correlated with the appearance of circulating neutralizing antibody. Our results suggest that the protective effect that Holland and his colleagues described using defective interfering particles of vesicular stomatitis virus may also be accounted for by an immunological mechanism rather than one involving interference.     

Separation and purification of the mRNAs for vesicular stomatitis virus NS and M proteins.

All five vesicular stomatitis virus mRNAs were separated and purified by acid-urea agarose gel electrophoresis. The identities of the NS and M mRNAs were established by their in vitro translation.     

An immunoenzyme quantitative assay for the antiviral effect of interferons

A technique is described for measurement of the antiviral activity of interferon by an immunoenzymatic assay for viral proteins. Cells treated by tested samples of interferon (IFN) are infected with vesicular stomatitis virus (VSV) and following the development of viral cytopathy are lysed by the addition of deoxycholate and then transferred into ELISA microplates. The viral proteins bind effectively to the microplates proportionally to their level in the culture and may be measured by incubating the plates sequentially with (1) rabbit antiserum against VSV, (2) a conjugate of alkaline phosphatase either to protein A or to an antibody against rabbit IgG and (3) p-nitrophenylphosphate. This procedure may be further simplified by using antibodies against VSV to which alkaline phosphatase has been directly conjugated. We found this immunoenzyme assay to be superior to the 'cytopathic effect inhibition' assay in precision and sensitivity and in being independent of the effectiveness of viral cytopathy.     

Validation of a recombinant integrin αvβ6/monoclonal antibody based antigen ELISA for the diagnosis of foot-and-mouth disease

A sandwich ELISA using recombinant integrin αvβ6 as a capture ligand and serotype-specific monoclonal antibodies (Mabs) as detecting reagents has been compared with a polyclonal antibody based ELISA (using type-specific rabbit antibodies as capture and guinea pig antibodies as detectors), which is employed routinely at the FAO World Reference Laboratory for Foot-and-Mouth Disease (FMD), for the identification and serotyping of FMD virus (FMDV). The study used cell culture grown antigens (1351 FMDV positive) derived from suspected cases of vesicular disease collected from 86 countries between 1924 and 2011, those positive for the other vesicular diseases of swine vesicular disease (n = 25) and vesicular stomatitis (n = 45) and negative samples collected from uninfected cell cultures (n = 36). The diagnostic sensitivity of the assays was similar at 98.1% (polyclonal ELISA) compared to 97.9% (integrin/Mab ELISA) but the serotypic-specificity of the latter was vastly superior (96%) to that of the former (61.5%). Reactions with the viruses of swine vesicular disease and vesicular stomatitis, which produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The integrin/Mab ELISA recognized FMDV strains of wide antigenic and molecular diversity of all seven serotypes and although some FMDV isolates were not detected, the greater specificity of the assay, while retaining test sensitivity comparable to the conventional assay, warrants its consideration for adoption for routine diagnostic use.     

Studies on the in vitro adenylation of RNA by vesicular stomatitis virus

Polyadenylate sequences present in RNA synthesized in vitro by purified vesicular stomatitis virus (VSV) are located at the 3′-termini of the RNA chains. These sequences are heterogeneous in length, averaging 140 bases, and are composed entirely of AMP. VSV purified from different cell lines synthesizes RNA containing poly(A) and complete removal of the viral envelope proteins G and M does not affect its synthesis. No poly(A) synthesis was observed with ATP as the only substrate either in the presence or absence of exogenous RNA primers. Cordycepin triphosphate did not inhibit in vitro RNA synthesis or substitute for ATP in the reaction. Poly(A) sequences are added to the product RNAs during or after their release from the genome RNA template. A possible mechanism of the VSV adenylation process is discussed.     

The interaction of antibody with the major surface glycoprotein of vesicular stornatitis virus II. Monoclonal antibodies to nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes

Eleven monoclonal antibodies reactive with the major surface glycoprotein (G-protein) of vesicular stomatitis virus serotypes Indiana and New Jersey (VSV-Ind, VSV-NJ) were used to map antigenic sites found on one or both serotypes. The antibodies used were unable to neutralize infectivity of the virus in vitro although they were able to bind to the G-protein. Six of the antibodies bound to the G-proteins of both serotypes and delineated three nonoverlapping epitopes as determined by a competitive binding assay. In addition, one monoclonal antibody bound to both serotypes and could neutralize infectivity in vitro of only VSV-Ind. This antibody could compete with several cross-reactive nonneutralizing antibodies which could not neutralize either VSV-Ind or VSV-NJ. Three monoclonal antibodies were serotype specific for VSV-NJ and exhibited no overlap among themselves or with the cross-reactive antibodies. One VSV-Ind serotype-specific antibody was isolated which could compete with a cross-reactive antibody. Enhancement of antibody binding by the binding of a second antibody was observed in some cases. This phenomenon appeared to be due to an increase in availability of antigenic sites caused by allosteric modifications.