Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity.

A cDNA fragment covering the genomic region that encodes the structural proteins of hog cholera virus (HCV) was inserted into the tk gene of vaccinia virus. Expression studies with vaccinia virus/HCV recombinants led to identification of HCV-specific proteins. The putative HCV core protein p23 was demonstrated for the first time by using an antiserum against a bacterial fusion protein. The glycoproteins expressed by vaccinia virus/HCV recombinant migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. A disulfide-linked heterodimer between gp55 and gp33 previously detected in HCV-infected cells was also demonstrated after infection with the recombinant virus. The vaccinia virus system allowed us to identify, in addition to the heterodimer, a disulfide-linked homodimer of HCV gp55. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.     

A second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus.

Several monoclonal antibodies (MAbs) raised against hog cholera virus (HCV) reacted with the HCV structural glycoprotein gp44/48 and neutralized the virus. The presence of HCV gp44/48 on the viral surface was directly demonstrated by immunogold electron microscopy. Eight anti-HCV gp44/48 MAbs were tested by immunoperoxidase assay against a panel of pestivirus strains. Each MAb showed a distinct pattern of reactivity with HCV strains. It is suggested that the MAbs are well suited for epidemiological investigations of HCV outbreaks.     

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.     

Differences in pathogenicity and antigenicity among hog cholera virus strains.

Using antiserum against a particular strain of bovine viral diarrhea virus, the strains of hog cholera virus were divided into two groups, H and B, on the basis of the difference in the degree of neutralization. Group H consisted of strains reacting poorly in neutralization, and group B Consisted of strains reacting well with bovine viral diarrhea antiserum. Most of the strains of group H induced a typical clinical form of hog cholera in experimentally infected pigs. Inoculation of pigs with a strain of group B, however, resulted in a chronic type of illness. When immunized with bovine viral diarrhea virus, pigs succumbed to challenge with group H virus after showing clinical signs of hog cholera, but survived challenge with group B virus without manifesting any clinical sign.     

Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera.

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.     

猪瘟病毒兔化弱毒株cDNA片段的克隆及序列分析

猪瘟是猪最重要的传染病之一,往往给养猪业造成重大经济损失,猪瘟的病原为猪瘟病毒（ＨＣＶ）,属黄病毒科,瘟病毒属成员,其基因组为单股正链ＲＮＡ,长度为１２３ｋｂ,仅含有一个大的开放阅读框架,编码一个含３８９８个氨基酸残基（ＡＡ）的多聚前体蛋白［１,２］。目前已经定位的蛋白有５种,即Ｎｐｒｏ、Ｃ、Ｅ０、Ｅ１和Ｅ２,它们均由ＨＣＶＲＮＡ５′端所编码,除Ｎｐｒｏ外,其它４种均为ＨＣＶ的结构蛋白［３］。Ｎｐｒｏ为具有自我催化功能的蛋白水解酶,也是多聚蛋白Ｎ端的第一个蛋白水解酶,分子量为２３ｋＤ,Ｃ为构成…     

Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen.

T-cell responses of pigs to hog cholera virus (HCV) have reportedly been absent or difficult to detect. Therefore, little is known about cellular immunity to HCV. In this study, we used an attenuated strain of pseudorabies virus expressing the envelope glycoprotein E1 of HCV and purified recombinant E1 to examine whether the E1 protein is a target antigen recognized by the T cells of HCV-immune pigs. We were unable to identify the E1 protein as a major target antigen recognized by the T cells of HCV-immune animals. However, such cells proliferated in vitro upon stimulation with viable HCV antigen. The lymphoproliferative response to HCV was strictly time and dose dependent and could be induced upon stimulation by live but not by UV light-inactivated HCV. Depletion studies demonstrated that lymphoproliferation depended on the presence of CD2+CD8bright+ lymphocytes, but CD2+CD4+ cells also contributed to the lymphoproliferative response. The primary lymphoproliferative response in animals inoculated with 10(7) 50% tissue culture infective doses of strain Brescia 2.1.1 was stronger than that observed in animals inoculated with 10(3) 50% tissue culture infective doses of the Cedipest strain. A remarkable finding was the increase in non-antigen-specific lymphoproliferation upon inoculation of the animals with HCV strains. This immunological phenomenon may mask a specific T-cell response to the virus.     

Replacement of the pseudorabies virus glycoprotein gIII gene with its postulated homolog, the glycoprotein gC gene of herpes simplex virus type 1.

gIII, the major envelope glycoprotein of pseudorabies virus (PRV), shares approximately 20% amino acid similarity with glycoprotein gC of herpes simplex virus type 1 (HSV-1) and HSV-2. We describe here our first experiments on the potential conservation of function between these two genes and gene products. We constructed PRV recombinants in which the gIII gene and regulatory sequences have been replaced with the entire HSV-1 gC gene and its regulatory sequences. The gC promoter functions in the PRV genome, and authentic HSV-1 gC protein is produced, albeit at a low level, in infected cells. The gC protein is present at the cell surface but cannot be detected in the PRV envelope.     

猪圆环病毒2型的PCR检测方法应用

本研究登录Genbank对猪圆环病毒2型基因的序列进行分析,用Primer 5.0设计了ORF2基因的扩增引物,试图选择一种比较合理的PCR方法检查PCV2感染的病原,以期这种PCR方法可以有效分析猪综合征障碍病毒(PRRSV)、猪细小病毒(PPV)、猪瘟病毒的扩增(HCV)、猪伪狂犬病毒(PRV)等比较常见的病原。研究结果表明,在PCV2进行扩增阳性样品检测中,发现一条异常447 bp的DNA条带,对产物测序结果进行扩增分析,证明其是PCV ORF2基因序列。敏感性检验分析表明检测样本DNA浓度时达到了9.8×10^-4ng/μL。PCR实验具有良好的稳定性和重复性。根据对66例临床病猪的分析表明,在PCV2的检测中阳性感染率是27.26%,这可能受到HCV、PRRSV、PRV、PPV等多种病毒的感染,其中混合感染的比例达到了72.23%。     

A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice

The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. And the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.     

Use of the agar diffusion precipitation test in the diagnosis of hog cholera

The application of the agar diffusion precipitation (ADP) test for diagnosing hog cholera was investigated. The test used as antigen, pancreatic tissue from 272 pigs that had been inoculated with hog cholera virus. The test was positive for 13.5% of the animals that were sick for 4 days or less, 40% of those sick for 5 days, and 77% of those sick for 6 days or more. The test was positive for 13.5% of all animals that had been vaccinated with crystal violet-glycerol hog cholera vaccine and had been sick for at least 6 days after challenge inoculation. Titration of the virus in ADP-test-negative and ADP-test-positive pancreatic suspensions did not show a direct correlation between the infective virus particle and the precipitating antigen.     

Characterization of hog cholera virus. I. Determination of buoyant density

Horzinek, Marian (Tier?rztliche Hochschule, Hannover, West Germany). Characterization of hog cholera virus. I. Determination of buoyant density. J. Bacteriol. 92:1723-1726. 1966.-Hog cholera virus was subjected to cesium chloride density gradient centrifugation. Most of the infectious activity was detected in fractions with densities between 1.15 and 1.20 g/ml, with a peak at 1.16 g/ml. Infectivity was assayed by use of either the exaltation of Newcastle disease virus method or the hemagglutination exaltation and inhibition of cytopathic effect method.     

Efficacy of intracerebral immunization against pseudorabies virus in mice

To evaluate the efficacy of intracerebral (IC) immunization, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SC) or IC injection, and then 10(6) plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional immunization via both the SC and IC routes, but was higher after IC immunization. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-immunized and 80% of the subcutaneously immunized mice died after developing neurological signs. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of PRV than SC immunization.     

Pathogenesis of classical swine fever: B-lymphocyte deficiency caused by hog cholera virus.

Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus. It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues. Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure. A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera.     

Hog cholera virus: molecular composition of virions from a pestivirus.

Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.     

Correlation between precolonization of trigeminal ganglia by attenuated strains of pseudorabies virus and resistance to wild-type virus latency.

We compared the levels of latent pseudorabies virus (PRV) DNA in trigeminal ganglia (TG) of pigs after intranasal inoculation of different PRV strains by using quantitative DNA PCR. The extent of colonization attained in each case varied significantly according to the type of strain and inoculum dose, wild-type (WT) PRV being the most efficient strain in colonizing TG. When groups of pigs representing different levels of precolonization of TG with an attenuated PRV strain were challenged with WT PRV, it became evident that there is a statistically significant inverse correlation between the extent of precolonization attained by an attenuated PRV strain in TG and the level of establishment of latency by superinfecting WT PRV. The protection against WT PRV latency did not correlate with the extent of WT PRV replication at the portal of entry.     

Characterization and mapping of a nonessential pseudorabies virus glycoprotein.

Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoprotein. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by marker rescue of a gIII- mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. (J. Mol. Appl. Gen. 2:485-496, 1984). Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.     

Characteristics of the porcine kidney cell line IB-RS-2 clone D10 (IB-RS-2 D10) which is free of hog cholera virus

Summary A Brazilian stock of clone C17 of the IB-RS-2 porcine kidney cell line which was contaminated with hog cholera virus (HCV) was cloned. One clone designated IB-RS-2 D10 was determined to be free of HCV, 20 other viruses, andMycoplasma. IB-RS-2 D10 cells possessed the same viral susceptibility pattern as the contaminated parent cells to the viruses of foot-and-mouth disease, swine vesicular disease, vesicular exanthema of swine, transmissible gastroenteritis, and several other viruses. The IB-RS-2 D10 cells had a median chromosome count of 34, were morphologically epithelioid cells, and were resistant to HCV infection. Freedom from HCV affords advantages for vaccine production and avoids laboratory contamination. Mention of a trademark, propriety product, or source does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may be suitable.