Parapoxviruses: potential alternative vectors for directing the immune response in permissive and non-permissive hosts.

Parapoxvirus (PPV) represents a genus of the poxviridae, and particularly PPV ovis (Orf virus, OV) seems to offer several potential advantages for the use of vector vaccine. Therefore, we started to investigate the genome of the highly attenuated OV strain D1701, which was only poorly characterised until now. Due to recombination of non-homologous sequences, part of the right hand end of the D1701 genome was duplicated and translocated to the opposite end of the genome. As a consequence gene deletion had occurred and the inverted terminal repeat region is increased. Results are described to identify viral genes, which are non-essential for virus replication and potentially influence viral pathogenesis, virulence, and host immunity. In more detail, we analysed the expression and functional activity of the OV-specific vascular endothelial growth factor (VEGF) gene homologue. Finally the construction and production of a D1701 mutant lacking the VEGF gene homologue is reported.     

CpG Distribution and Methylation Pattern in Porcine Parvovirus

Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can’t methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome.     

Restriction analysis of the genome of swine abortion parvovirus and cloning of its PstI-EcoRI fragment

The cultured pig kidney cells infected by the porcine parvovirus (PPV) produced the virions and viral DNA. The latter was used as a matrix to synthesize the double stranded DNA. The obtained preparation is more homogenic than the natural replicative form and was used for restriction analysis of porcine parvovirus genome and for molecular cloning of its fragment. The isolated recombinant plasmids contained the PstI-EcoRI fragment of PPV DNA, containing 70% of the viral genome. The restriction analysis of replicative PPV DNA isolated from the infected cells has resulted in finding of the replicative form containing a 300 bp deletion in the 5'-region of PPV genome.     

Coexistence of multiple strains of porcine parvovirus 2 in pig farms

The porcine parvovirus 2 (PPV2) genome was first identified in 2001 in Myanmar. Recently, the PPV2 genome has been found in several other countries. In this study, the prevalence of PPV2 in Japanese domestic pigs was investigated and found to be 58% (69/120) in healthy domestic pigs and 100% (69/69) in sick domestic pigs. Sequencing and phylogenetic analysis of the PCR products of the VP1 gene and an almost full length PPV2 clone indicated that diverged PPV2 strains exist in Japan. Clearly distinct strains of PPV2 were detected in 7 of the 10 pig farms.     

小反刍兽疫病毒研究进展

小反刍兽疫（PPR）是由小反刍兽疫病毒（PPRV）引起的一种主要感染小反刍动物的急性、烈性、接触性A类传染病,患病率、死亡率高。本文就世界PPR流行状况、PPRV基因组及病毒结构蛋白、PPRV检测方法、最新的药物及疫苗、存在的问题等方面做了简要综述。     

猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究

将猪细小病毒VP2基因克隆至pCI-neo真核表达载体中,构建了pCIneo-VP2重组质粒,转染至PK-15细胞中,利用免疫荧光方法检测在体外表达情况;并以小鼠为动物模型,将pCIneo-VP2、pCI-neo重组质粒、猪细小病毒活疫苗和对照组通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-VP2质粒 1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-VP2能够有效诱导机体产生体液免疫和细胞免疫,为研制出高效、新型猪细小病毒疫苗提供了科学依据和试验依据。     

展示生长抑素的猪细小病毒样颗粒构建及其免疫原性

为了获得既可预防猪细小病毒感染又能促进生长的嵌合病毒样颗粒疫苗,以PPV NJ-a株基因组DNA为模板扩增VP2基因片段,在VP2基因N端融合人工合成的4拷贝生长抑素基因,构建杆状病毒转移载体pFast-SS4-VP2。通过转化DH10Bac感受态细胞,pFast-SS4-VP2与穿梭载体Bacmid重组,获得重组Bacmid,命名为rBacmid-SS4-VP2。rBacmid-SS4-VP2转染Sf-9细胞,获得重组病毒rBac-SS4-VP2。SDS-PAGE与Western blotting鉴定可见约68 kDa的rSS4-VP2条带;rBac-SS4-VP2感染细胞IFA检测产生很强的特异性绿色荧光;感染细胞超薄切片电镜观察到大量特征性病毒样颗粒。将重组蛋白分别辅以铝胶、IMS和白油不同佐剂免疫小鼠,通过检测免疫小鼠VP2特异性ELISA抗体、PPV特异性中和抗体、生长抑素的抗体水平及生长激素水平来评价嵌合病毒样颗粒的免疫原性。结果表明,辅以铝胶与IMS佐剂重组蛋白组均产生了与PPV全毒组相似的ELISA抗体与中和抗体反应;重组蛋白免疫组均产生较好的针对生长抑素的抗体反应;免疫小鼠体内生长激素的水平明显升高;其中以铝胶佐剂组产生的各抗体水平最高,白油佐剂组各抗体水平最低。为以后生产安全、有效的颗粒化亚单位疫苗提供了一个新的设计思路,又为应用病毒样颗粒递呈外源肽,从而生产多联亚单位疫苗奠定了基础。     

Randomized,Single Blind,Controlled Trial to Evaluate the Prime-Boost Strategy for Pneumococcal Vaccination in Renal Transplant Recipients

Renal transplant recipients are at increased risk of developing invasive pneumococcal diseases but may have poor response to the 23-valent pneumococcal polysaccharide vaccine (PPV). It may be possible to enhance immunogenicity by priming with 7-valent pneumococcal conjugate vaccine (7vPnC) and boosting with PPV 1 year later. In a randomized single-blind, controlled study, adult recipients of renal transplants received either 7nPVC or PPV followed by PPV 1 year later. The vaccine response was defined as 2-fold increase in antibody concentration from baseline and an absolute post-vaccination values ≥1 µg/ml. The primary endpoint was vaccine response of the primed group (7vPnC/PPV) compared with single PPV vaccination. Antibody concentrations for 10 serotypes were measured at baseline, 8 weeks after first vaccination, before second vaccination, and 8 weeks after second vaccination. Of 320 screened patients, 80 patients were randomized and 62 completed the study. Revaccination with PPV achieved no significant increase of immune response in the 7vPnC/PPV group compared with the single PPV recipients A response to at least 1 serotype was seen in 77.1% of patients who received 7vPnC and 93.1% of patients who received PPV (P = 0.046). After second vaccination response to at least 1 serotype was seen in 87.5% patients of 7vPnC/PPV group and 87.1% patients of PPV group (non significant p). The median number of serotypes eliciting a response was 3.5 (95% CI 2.5–4.5) in the 7vPnC/PPV group versus 5 (95% CI 3.9–6.1) in the PPV group (non-significant p). Immunogenicity of pneumococcal vaccination was not enhanced by the prime–boost strategy compared with vaccination with PPV alone. Administration of a single dose of PPV should continue to be the standard of care for adult recipients of renal transplants.

Trial Registration

EudraCT 2007-004590-25.     

The immune enhancement of propolis adjuvant on inactivated porcine parvovirus vaccine in guinea pig

Two experiments were carried out. In immune response test, the immune enhancement of propolis, oilemulsion and aluminium salt were compared in guinea pig vaccinated with inactivated porcine parvovirus (PPV) vaccine. The result showed that three adjuvants could enhance antibody titer, T lymphocyte proliferation, IL-2 and IL-4 secretion of splenic lymphocyte. The action of propolis was similar to that of oilemulsion and superior to that of aluminium salt, especially in early period of vaccination propolis could accelerate antibody production. In immune protection test, the effects of three adjuvants on PPV infection were compared in guinea pig vaccinated with PPV vaccine then challenged with PPV. The result showed that propolis and oilemulsion could enhance the antibody titer, IL-2 and IL-4 content in serum and decrease the PPV content in blood and viscera. In the effect of improving cellular immune response, the propolis was the best. These results indicated that propolis possessed better immune enhancement and would be exploited into a effective adjuvant of inactivated vaccine.     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

猪细小病毒结构蛋白VP1和VP2的基因免疫研究

利用真核表达载体pCIneo和pcDNA3.1( )分别构建了含有猪细小病毒VP1基因的pCIneo VP1和含有VP2基因的pCIneo VP2与pcDNA VP2三种真核表达质粒。将上述三种真核表达质粒分别转染ＩＢＲＳ－２细胞,利用间接ELISA检测表达情况,结果表明上述三种质粒均能在ＩＢＲＳ－２细胞表达,表达产物位于细胞中。在此基础上,利用这三种质粒分别以肌内注射的方式,间隔2周2次免疫小鼠,结果发现所有表达质粒均能诱导产生明显的细胞免疫和体液免疫,其中pCIneo VP1质粒诱导的体液免疫最强,与猪细小病毒灭活疫苗免疫组相当,pCIneo VP2诱导的细胞免疫应答强于PPV灭活组,pCIneo VP1和pCIneo VP2联合免疫并没有加强作用。     

Efficacy of an Inactivated Porcine Parvovirus (PPV) Vaccine under Field Conditions

Dose response experiments were carried out by vaccinating groups of seronegative gilts (7 months of age) and groups of gilts with residual maternal serum antibodies to PPV (5 months of age). PPV vaccines containing different amounts of inactivated virions were used. Vaccinations were carried out twice with 3 weeks intervals. It was demonstrated, that a single vaccination even with a vaccine with low antigen content elicited an antibody response to PPV in seronegative gilts. The titer values increased after the 2nd vaccination. When the gilts had residual maternal serum antibodies at the time of vaccination the antibody response was generally lower. In some animals the titer values decreased after the 1st vaccination, but except for 2 gilts vaccinated with vaccines with a low antigen content an increase of titer values followed after the 2nd vaccination. During a field trial performed in a herd with enzootic PPV infection all the gilts were vaccinated with PPV vaccine before mating. Blood samples were examined for HI antibodies before and after vaccination and at weaning time of each of the resulting 5 litters. In total 24 batches of gilts comprising 326 animals mated during a 2 year period were examined. It was demonstrated, that the applied vaccine preparations used under field conditions gave a relatively high and long lasting antibody response, even when the gilts were vaccinated as early as at about 5 months of age.     

The efficiency of RNA interference for conferring stable resistance to plum pox virus

Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to plum pox virus (PPV) infection through the specific process of RNA silencing involving both small interfering-RNA (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host. Plums inoculated with the five major PPV strains, three widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy. PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that was unable to infect P. domestica. This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation, through a recovery reaction. Regardless PPV strain variability based on geography, host species, epidemiology and serotypes of the CP protein and substitutions of nucleotides at the NH₂-terminus of CP of the major five PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards.     

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.     

Induction of immune responses in mice after intragastric administration of Lactobacillus casei producing porcine parvovirus VP2 protein

Lactobacillus casei ATCC 393 was selected as an antigen delivery vehicle for mucosal immunization against porcine parvovirus (PPV) infection. A 64-kDa fragment of PPV major protective antigen VP2 protein was used as the parvovirus antigen model. A recombinant Lactobacillus expressing VP2 protein was constructed with plasmid pPG611.1, where expression and localization of the VP2 protein from recombinant Lc393-rPPV-VP2 was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immunofluorescence. Both local mucosal and systemic immune responses against PPV were induced in BALB/c mice immunized orally with the recombinant Lactobacillus expressing VP2 protein. The induced antibodies demonstrated neutralizing effects on PPV infection. These data indicated that the use of recombinant lactobacilli could be a valuable strategy for future vaccine development of PPV.     

Genome organization of the Kresse strain of porcine parvovirus: identification of the allotropic determinant and comparison with those of NADL-2 and field isolates.

The Kresse strain of porcine parvovirus (PPV) was cloned into pUC19, and independent infectious clones were sequenced. The PPV Kresse and NADL-2 strains, which have different pathogenicities, shared an identical genomic organization and a high degree of sequence identity. Partial genomes (1.5 or 1.6 kb) of 15 field isolates were also amplified by PCR in regions with significant sequence differences between the laboratory strains. Five amino acid differences were consistently present within the VP1/VP2 coding region of the Kresse strain and virulent field isolates. A number of inconsistent point mutations were also found throughout the genomes of field isolates. In addition, among those with the vaccine amino acid profile, all but one isolate (IAF-3) contained a 127-bp noncoding direct repeat downstream of the capsid protein gene. The one exception was also the only vaccine-type PPV obtained from a mummified fetus. In order to identify genetic elements responsible for the distinct tropism (and possibly the pathology) of the Kresse strain, in vitro cell systems which differentiated the virulent from the vaccinal strains were established. Subsequently, chimeric infectious clones of the Kresse and NADL-2 strains were used to identify the allotropic determinant located in the VP1/VP2 region. The transfer of the BglII fragment of the Kresse genome, containing three amino acid differences, into the NADL-2 background, or the opposite construct, caused the phenotype of the target genome to revert to that of the parent strain of the BglII fragment. Prediction of the localization of amino acid differences on the basis of canine parvovirus capsid structure indicates that each is located on or near the outer surface of the virion. In particular, the position of one mutation (S-436-->P) maps by analogy to the threefold spike, the most accessible region of the capsid.     

Complete genome sequences of Newcastle disease virus strains circulating in chicken populations of Indonesia

Eight highly virulent Newcastle disease virus (NDV) strains were isolated from vaccinated commercial chickens in Indonesia during outbreaks in 2009 and 2010. The complete genome sequences of two NDV strains and the sequences of the surface protein genes (F and HN) of six other strains were determined. Phylogenetic analysis classified them into two new subgroups of genotype VII in the class II cluster that were genetically distinct from vaccine strains. This is the first report of complete genome sequences of NDV strains isolated from chickens in Indonesia.     

Induction of Immune Responses in Mice after Intragastric Administration of Lactobacillus casei Producing Porcine Parvovirus VP2 Protein

Lactobacillus casei ATCC 393 was selected as an antigen delivery vehicle for mucosal immunization against porcine parvovirus (PPV) infection. A 64-kDa fragment of PPV major protective antigen VP2 protein was used as the parvovirus antigen model. A recombinant Lactobacillus expressing VP2 protein was constructed with plasmid pPG611.1, where expression and localization of the VP2 protein from recombinant Lc393-rPPV-VP2 was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immunofluorescence. Both local mucosal and systemic immune responses against PPV were induced in BALB/c mice immunized orally with the recombinant Lactobacillus expressing VP2 protein. The induced antibodies demonstrated neutralizing effects on PPV infection. These data indicated that the use of recombinant lactobacilli could be a valuable strategy for future vaccine development of PPV.     

Identification of Plum pox virus pathogenicity determinants in herbaceous and woody hosts

Plum pox virus (PPV) is a member of the genus Potyvirus that is able to infect a large variety of plant species, including trees of the genus Prunus, its natural host. When some PPV isolates are propagated for an extended time in herbaceous plants, their ability to infect trees is reduced. The molecular basis of this change in host infectivity is poorly understood. We report the construction of hybrid viruses from cDNA clones of two D-strain isolates of PPV, PPV-D and PPV-R, which differ in their host range. PPV-D can infect GF305 peach seedlings efficiently, however, it is unable to infect Nicotiana clevelandii plants. Conversely, PPV-R infects N. clevelandii, but not GF305 peach seedlings. The analyses of the hybrid viruses showed that, although determinants of PPV pathogenicity are extensively spread throughout the PPV genome, the 3' terminal region of the PPV-R genome, including the 3' noncoding region and the coding regions for the coat protein (CP), NIb, and part of NIa protein, is sufficient to confer infectivity of N. clevelandii in a PPV-D background. Our data demonstrate a high concentration of amino acid substitutions in the CP and a host-specific effect of a deletion at the N terminus of this protein in PPV pathogenicity in peach and N. clevelandii infectivity experiments. These results suggest that relevant host specificity determinants are located in the N-terminal region of the CP. The analyses of the PPV-R and PPV-D chimeras also showed that key host-specific pathogenicity determinants lie in the 5' terminal third of the PPV genome, a region that spans proteins P1, HCPro, and P3. The selection of mutations in only a few specific residues in proteins P1, P3, and 6K1 after partial adaptation of a chimeric virus (BD-GFP) to N. clevelandii further suggests a relevant role for these proteins in host adaptation.