Assembly of pseudorabies virus genome-based transfer vehicle carrying major antigen sites of S gene of transmissible gastroenteritis virus: potential perspective for developing live vector vaccines.

Two severe porcine infectious diseases, pseudorabies (PR) and transmissible gastroenteritis (TGE) caused by pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV) respectively often result in serious economic loss in animal husbandry worldwide. Vaccination is the important prevention means against both infections. To achieve a PRV genome-based virus live vector, aiming at further TGEV/PRV bivalent vaccine development, a recombinant plasmid pUG was constructed via inserting partial PK and full-length gG genes of PRV strain Bartha K-61 amplified into pUC119 vector. In parallel, another recombinant pHS was generated by introducing a fragment designated S1 encoding the major antigen sites of S gene from TGEV strain TH-98 into a prokaryotic expression vector pP(RO)EX HTc. The SV40 polyA sequence was then inserted into the downstream of S1 fragment of pHS. The continuous region containing S1fragment, SV40 polyA and four single restriction enzyme sites digested from pHS was subcloned into the downstream of gG promoter of pUG. In addition, a LacZ reporter gene was introduced into the universal transfer vector named pUGS-LacZ. Subsequently, a PRV genome-based virus live vector was generated via homologous recombination. The functionally effective vector was purified and partially characterized. Moreover, the potential advantages of this system are discussed.     

ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a 6 x His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals.     

Insertions in the gG gene of pseudorabies virus reduce expression of the upstream Us3 protein and inhibit cell-to-cell spread of virus infection

The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is conserved in most viruses of the alphaherpesvirus subfamily. In the swine pathogen pseudorabies virus (PRV), mutant viruses with internal deletions and insertions in the gG gene have shown no discernible phenotypes. We report that insertions in the gG locus of the attenuated PRV strain Bartha show reduced virulence in vivo and are defective in their ability to spread from cell to cell in a cell-type-specific manner. Similar insertions in the gG locus of the wild-type PRV strain Becker had no effect on the ability of virus infection to spread between cells. Insertions in the gG locus of the virulent NIA-3 strain gave results similar to those found with the Bartha strain. To examine the role of gG in cell-to-cell spread, a nonsense mutation in the gG signal sequence was constructed and crossed into the Bartha strain. This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain. These data indicated that, while insertions in the gG locus result in decreased cell-to-cell spread, the phenotype was not due to loss of gG expression as first predicted. Analysis of gene expression upstream and downstream of gG revealed that expression of the upstream Us3 protein is reduced by insertion of lacZ or egfp at the gG locus. By contrast, expression of the gene immediately downstream of gG, Us6, which encodes glycoprotein gD, was not affected by insertions in gG. These data indicate that DNA insertions in gG have polar effects and suggest that the serine/threonine kinase encoded by the Us3 gene, and not gG, functions in the spread of viral infection between cells.     

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T gondii SAG1 gene-transfectants were established by using RMA.S (H-2b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).     

The attenuated pseudorabies virus strain Bartha fails to package the tegument proteins Us3 and VP22

The Bartha strain of pseudorabies virus has several recognized mutations, including a deletion in the unique short region encompassing the glycoprotein I (gI), gE, Us9, and Us2 genes and point mutations in the gC, gM, and UL21 genes. We have determined that Bartha has mutations in the serine/threonine kinase encoded by the Us3 gene relative to the wild-type Becker strain. Our analysis revealed that Becker virions contain the Us3 protein, whereas Bartha virions do not. To test whether the mutations in the Bartha Us3 protein were responsible for this observation, we constructed a recombinant Bartha strain, PRV632, which expresses the Becker Us3 protein. PRV632 failed to package Us3 into the tegument, indicating that mutations other than those in the Us3 primary amino acid sequence were responsible for the failure of Bartha to package its Us3 protein. A recombinant Becker strain, PRV634, which expresses the Bartha Us3 protein, was constructed to test whether it was capable of being packaged into virions. The Bartha Us3 protein was not incorporated into PRV634 virions efficiently, suggesting that the primary sequence of the Bartha Us3 protein affects packaging into the tegument. To determine whether the packaging of other tegument proteins was affected in the Bartha strain, we examined VP22. Whereas Becker packaged VP22 into virions, Bartha had a severe deficiency in VP22 incorporation. Analysis of VP22 expression in Bartha-infected cells revealed that Bartha VP22 had a slower mobility on sodium dodecyl sulfate-polyacrylamide gels, indicating either primary sequence differences and/or different posttranslational modifications relative to Becker VP22. Taken together, these data indicate that, while the primary sequence of the Us3 protein does affect its incorporation into the tegument, other factors are involved. Furthermore, our data suggest that one or more of the gI, gE, Us9, or Us2 genes influences the localization of the Us3 protein in infected cells, and this effect may be important for the proper incorporation of Us3 into virions.     

Genetic basis of the neurovirulence of pseudorabies virus.

Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (US) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessary for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-]) revealed the following. None of the recombinant plaque isolates that were either TK- or which had a deletion in the US was virulent. Not all recombinant plaque isolates which were both TK+ and had an intact US were virulent. These results indicate that both thymidine kinase activity and an intact US were necessary but not sufficient for the expression of virulence. Marker rescue experiments involving cotransfection of the Bartha strain DNA and a restriction fragment spanning the region of the genome that was missing from the Bartha strain resulted in the isolation of virions to which an intact US had been restored. These virions were not virulent but had an improved ability to replicate in the brains of chicks compared with that of the parental nonrescued Bartha strain. Our results show that genes in the US region, which are missing from the Bartha strain, were necessary for virulence but that this strain was also defective in other genes required for the expression of virulence. Thus, the virulence of pseudorabies virus, as measured by intracerebral inoculation into chicks, appears to be controlled multigenically.     

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.     

共表达PCV2 ORF2和猪IL-18基因的重组猪伪狂犬病毒对小鼠的免疫原性

【目的】研制猪伪狂犬病毒(PRV)和猪圆环病毒2型(PCV2)的二联活疫苗,并用猪IL-18作为免疫佐剂。【方法】将猪IL-18基因插入到质粒p GO中,获得的重组转移质粒p GO18与猪PRV弱毒HB98株DNA共转染ST细胞,并进行空斑筛选和纯化;RT-PCR和Western blot分别从转录和蛋白水平鉴定其表达情况。将重组病毒PGO18和PGO、PRV弱毒株HB98、PCV2灭活商品苗和1640细胞培养基分别免疫6周龄雌性昆明小鼠,4周后二次免疫,二免后4周用PCV2 DF强毒和PRV Min/A强毒接种小鼠。通过ELISA、血清中和试验和流式细胞术及攻毒保护试验评价重组病毒的免疫原性。【结果】获得了重组病毒PGO18,并且可在ST细胞内表达;PGO18可诱导小鼠机体产生PCV2的ELISA和PRV的中和抗体水平,刺激CD3+、CD4+、CD8+T细胞亚群的增殖,且能有效抵抗PCV2和PRV强毒攻击。【结论】IL-18基因可增强重组病毒的免疫效果,使重组病毒具有良好的免疫原性,有望成为防治PCV2和PRV的候选疫苗株。     

Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.     

伪狂犬病病毒TK-/gG-缺失株的构建及其生物学特性研究

将伪狂犬病病毒TK^-／gG^-／LacZ^ 突变株的基因组DNA与含有缺失的gG基因的转移质粒pUSKBB共转染猪肾传代细胞PK-15,待完全病变后收获病毒进行空斑试验,用PCR筛选gG缺失的重组病毒。空斑纯化3次后,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的TK^-／gG^-缺失株。遗传稳定性试验表明该重组病毒能在PK-15细胞上稳定遗传,动物试验表明该缺失株对Balb／c小鼠极为安全且能保护Balb／c小鼠抵抗致死量PRV强毒的攻击。该突变株的获得为我国伪狂犬病的控制和根除奠定了基础。     

Protective antiviral immune responses to pseudorabies virus induced by DNA vaccination using dimethyldioctadecylammonium bromide as an adjuvant

To enhance the efficacy of a DNA vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of plasmids coding for gamma interferon or interleukin-12, of CpG immunostimulatory motifs, and of the conventional adjuvants dimethyldioctadecylammonium bromide in water (DDA) and sulfolipo-cyclodextrin in squalene in water. We demonstrate that a DNA vaccine combined with DDA, but not with the other adjuvants, induced significantly stronger immune responses than plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection. This is the first study to demonstrate that DDA, a conventional adjuvant, enhances DNA vaccine-induced antiviral immunity.     

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.     

Production of Aujeszky's disease (pseudorabies) virus envelope glycoproteins gB and gC as recombinant polyhedra in baculovirus-infected silkworm larvae

The expression of viral antigens in baculovirus-infected insect cells is often ineffective. As an alternative approach, therefore, we developed the recombinant polyhedra technology, which is an efficient strategy for the production of viral subunit vaccine. Here, we report a strategy for the large-scale production of a pseudorabies virus (PRV) gB or gC in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the epitope regions of PRV gB or heparin-binding domains of PRV gC. Recombinant BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae expressed native polyhedrin and fusion protein that was detected using both anti-polyhedrin and anti-PRV gB or anti-PRV-gC antibodies. Electron and confocal microscopy demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin with a normal morphology and that the recombinant polyhedra contained PRV gB or gC. The yield of gB or gC antigen produced in BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae reached 0.69 or 0.46 mg per larva, respectively, at 6 days post-infection. These results demonstrate that the recombinant polyhedra strategy can be used for the large-scale production of PRV gB or gC antigen.     

表达猪繁殖与呼吸综合征病毒GP3基因的重组伪狂犬病病毒的构建

猪繁殖与呼吸综合征 (porcinereproductiveandrespiratorysyndrome ,PRRS)是引起怀孕母猪早产、流产、死胎及仔猪呼吸系统疾病的一种新发现的病毒性传染病[1] .该病毒的基因组为单股正链RNA ,约15kb ,含有 8个开放阅读框架 (ORFs) ,ORF1编码病毒非结构蛋白 (依赖RNA的RNA聚合酶 ) ,ORF2 ORF7编码病毒的结构蛋白 .其中ORF3含有 2 6 5个氨基酸 ,编码的GP3蛋白为高度糖基化的结构蛋白 ,有 7个糖基化位点 ,具有免疫原性[2 ,3 ] .目前 ,用于预防PRRS的疫苗主要是弱毒苗和灭活苗 ,虽然都有一定的免疫效果 ,但由于PRRS抗体依赖性…     

Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

A recombinant pseudorabies virus co-expressing capsid proteins precursor P1-2A of FMDV and VP2 protein of porcine parvovirus: a trivalent vaccine candidate

Pseudorabies (PR), foot-and-mouth disease (FMD), and porcine parvovirus disease are three important infectious diseases in swine worldwide. The gene-deleted pseudorabies virus (PRV) has been used as a live-viral vector to develop multivalent genetic engineering vaccine. In this study, a recombinant PRV, which could co-express protein precursor P1-2A of FMDV and VP2 protein of PPV, was constructed using PRV TK⁻/gE⁻/LacZ⁺ mutant as the vector. After homologous recombination and plaque purification, recombinant virus PRV TK⁻/gE⁻/P1-2A-VP2 was acquired and identified. Immunogenicity, safety of the recombinant PRV and its protection against PRV were confirmed in a mouse model by indirect ELISA and serum neutralization test. The results show that the recombinant PRV is a candidate vaccine strain to develop a novel trivalent vaccine against PRV, FMDV and PPV in swine.     

Proteins specified by the short unique region of the genome of pseudorabies virus play a role in the release of virions from certain cells.

Two pseudorabies virus vaccine strains (Bartha and Norden) that have a similar deletion in the short unique (Us) region of the genome have been identified previously (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). These strains do not code for the glycoprotein gI, a glycoprotein that has been mapped on the wild type virus genome by T. C. Mettenleiter, N. Lukacs, and H. J. Rziha (J. Virol. 53:52-57, 1985) to the sequences deleted from the vaccine strain. Restoration of these deleted sequences to the Bartha strain genome restores to the virus the ability to specify the gI glycoprotein. The Bartha vaccine strain grows as well as wild-type virus in pig kidney and in rabbit kidney (RK) cells, but is not released efficiently from and forms small plaques in RK cells. The rescued Bartha 43/25a strain (which has an intact Us) is released considerably more efficiently than the Bartha vaccine strain, but less efficiently than wild-type virus from RK cells; it also forms larger plaques on RK cells than does the parental Bartha vaccine strain. The Norden vaccine strain, which has a deletion in the Us, is released better from RK cells than is the Bartha strain, but not as well as is wild-type virus. We conclude that whereas the sequences in the Us that are deleted from the Bartha and Norden strain genomes specify functions that play a role in the release of virions from some cell types, at least one other function (which is defective in the Bartha strain but not in the Norden strain) also affects release of virus from these cells. Since restoration to the Bartha strain of an intact Us restores to the virus both the ability to grow in chicken brains (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, J. Virol. 52:198-205, 1984) and to be released from RK cells, the possibility that the lack of virulence of the Bartha vaccine strain may be related to its limited release from some target cells is discussed.     

Toxoplasma gondii: chimeric Dr fimbriae as a recombinant vaccine against toxoplasmosis

Toxoplasma gondii is responsible for fetopathy in farm animals and humans and severe disease in immunocompromised individuals (i.e. AIDS patients). Effective vaccines, inducing protective and long-lasting immunity to this global parasite, are still desired. In the work, we evaluated the immunogenic and immunoprotective activity of Escherichia coli chimeric Dr fimbriae bearing selected antigenic epitopes of three T. gondii antigens (SAG1, GRA1 and MAG1), in comparison with conventional recombinant antigens obtained in E. coli expression system. Our data demonstrate a very high protective efficacy of recombinant antigens supplemented with Freund's adjuvants, whereas chimeric Dr fimbriae as a vaccine proved non-protective. The recombinant antigen vaccine induced a strong specific antibody response and prevented the brain cysts formation by 89%. The results are promising and should be confirmed in further study on farm animals by use of less aggressive than Freund's adjuvant preparations.     

Quantitative proteomics study of host response to virulent and attenuated pseudorabies virus infection in mouse brain

Bartha, the pseudorabies virus (PRV) vaccine strain, is widely used in studies of neuronal circuit-tracing, due to its attenuated virulence and retrograde spreading. However, we know little regarding the molecular mechanisms of PRV infection and spreading between structurally connected neurons. In this study, we systematically analyzed the host brain proteomes after acute infection with PRV, attempting to identified the proteins involved in the processes. Mice were injected with PRV-Bartha and PRV-Becker (PRV-Bartha's wild-type parent strain) in the olfactory system, the proteomes of the brain and synaptosome were analyzed and compared at various infection intervals using mass spectrometry-based proteomics techniques. In all, we identified > 100 PRV-infection regulated proteins at the whole-tissue level and the synaptosome level. While at whole-tissue level, bioinformatics analyses mapped most of the regulations to the inflammation pathways, at the synaptosome level, most of those to synaptic transmission, cargo transport and cytoskeleton organization. We established regulated protein networks demonstrating distinct cellular regulation pattern between the global and the synaptosome levels. Moreover, we identified a series of potentially PRV-strain-specific regulated proteins with diverse biological functions. This study may provide new clues for molecular mechanisms for PRV infection and spread.