A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

<p>Abstract</p> <p>Background</p> <p>Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene.</p> <p>Methods</p> <p>A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein.</p> <p>Results</p> <p>Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts.</p> <p>Conclusions</p> <p>In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.</p

Characterization of a fatal feline panleukopenia virus derived from giant panda with broad cell tropism and zoonotic potential

Represented by feline panleukopenia virus (FPV) and canine parvovirus (CPV), the species carnivore protoparvovirus 1 has a worldwide distribution through continuous ci13rculation in companion animals such as cats and dogs. Subsequently, both FPV and CPV had engaged in host-to-host transfer to other wild animal hosts of the order Carnivora. In the present study, we emphasized the significance of cross-species transmission of parvoviruses with the isolation and characterization of an FPV from giant panda displaying severe and fatal symptoms. The isolated virus, designated pFPV-sc, displayed similar morphology as FPV, while phylogenetic analysis indicated that the nucleotide sequence of pFPV-sc clades with Chinese FPV isolates. Despite pFPV-sc is seemingly an outcome of a spillover infection event from domestic cats to giant pandas, our study also provided serological evidence that FPV or other parvoviruses closely related to FPV could be already prevalent in giant pandas in 2011. Initiation of host transfer of pFPV-sc is likely with association to giant panda transferrin receptor (TfR), as TfR of giant panda shares high homology with feline TfR. Strikingly, our data also indicate that pFPV-sc can infect cell lines of other mammal species, including humans. To sum up, observations from this study shall promote future research of cross-host transmission and antiviral intervention of Carnivore protoparvovirus 1, and necessitate surveillance studies in thus far unacknowledged potential reservoirs

Development and efficacy evaluation of remodeled canine parvovirus-like particles displaying major antigenic epitopes of a giant panda derived canine distemper virus

Canine parvovirus (CPV) and Canine distemper virus (CDV) can cause fatal diseases in giant panda (Ailuropoda melanoleuca). The main capsid protein of CPV VP2 can be self-assembled to form virus-like particles (VLPs) in vitro, which is of great significance for potential vaccine development. In the present study, we remodeled the VP2 protein of a giant panda-derived CPV, where the major CDV F and N epitopes were incorporated in the N-terminal and loop2 region in two combinations to form chimeric VLPs. The reactivity ability and morphology of the recombinant proteins were confirmed by Western blot, hemagglutination (HA) test and electron microscopy. Subsequently, the immunogenicity of the VLPs was examined in vivo. Antigen-specific antibodies and neutralizing activity were measured by ELISA, hemagglutination inhibition (HI) test and serum neutralization test (SNT), respectively. In addition, antigen specific T cell activation were determined in splenic lymphocytes. The results indicated that the VLPs displayed good reaction with CDV/CPV antibodies, and the heterologous epitopes do not hamper solubility or activity. The VLPs showed decent HA activity, and resembled round-shaped particles with a diameter of 22–26 nm, which is identical to natural virions. VLPs could induce high levels of specific antibodies to CPV and CDV, shown by the indication of neutralizing antibodies in both VP2N and VP2L VLPs group. In addition, splenic lymphocytes of mice immunized with VLPs could proliferate rapidly after stimulation by specific antigen. Taken together, the CPV VP2 VLPs or chimeric VLPs are highly immunogenic, and henceforth could function as CPV/CDV vaccine candidates for giant pandas

The molecular diversity of transcriptional factor TfoX is a determinant in natural transformation in Glaesserella parasuis

Natural transformation is a mechanism by which a particular bacterial species takes up foreign DNA and integrates it into its genome. The swine pathogen Glaesserella parasuis (G. parasuis) is a naturally transformable bacterium. The regulation of competence, however, is not fully understood. In this study, the natural transformability of 99 strains was investigated. Only 44% of the strains were transformable under laboratory conditions. Through a high-resolution melting curve and phylogenetic analysis, we found that genetic differences in the core regulator of natural transformation, the tfoX gene, leads to two distinct natural transformation phenotypes. In the absence of the tfoX gene, the highly transformable strain SC1401 lost its natural transformability. In addition, when the SC1401 tfoX gene was replaced by the tfoX of SH0165, which has no natural transformability, competence was also lost. These results suggest that TfoX is a core regulator of natural transformation in G. parasuis, and that differences in tfoX can be used as a molecular indicator of natural transformability. Transcriptomic and proteomic analyses of the SC1401 wildtype strain, and a tfoX gene deletion strain showed that differential gene expression and protein synthesis is mainly centered on pathways related to glucose metabolism. The results suggest that tfoX may mediate natural transformation by regulating the metabolism of carbon sources. Our study provides evidence that tfoX plays an important role in the natural transformation of G. parasuis

Early cretaceous multi-stage extension in the North Dabie complex in the eastern central China: insights from structural and geochronological data

A dominantly NW-SE directed extensional tectonics in the Early Cretaceous significantly reworked the Late Permian-Triassic orogenic framework of the Dabie orogenic belt. The North Dabie complex (NDC) is the principal domain recording this tectonic event. However, the precise structure-kinematic architectures, particularly those observed in the ductile regime, along with the respective time scales for different extensional stages, have not been adequately established. This significantly impedes our comprehensive understanding of the extensional style and deformation history in the North Dabie complex. To better address these issues, we conducted a systematic structural study and LA-ICP-MS zircon U-Pb dating of the pre-, syn-, and post-kinematic intrusions and syn-kinematically metamorphosed high-grade gneisses/migmatites of the NDC. Our results demonstrate that the extensional deformation in the NDC may initiate at ca. 144 Ma, which is characterized by a pervasive NW-SE oriented coaxial plastic flow in the ductile regime of the middle-lower crust. A large-scale detachment processing zone subsequently started activating at ca. 140 Ma at the upper-middle level of the middle crust, and concentratedly accommodated the extensional strain by top-to-NW ductile shearing. Locally, there was uprising of sub-magmatic flow in the atatexite-diatexite from the deeper lower crust taking place in the manner of top-to-outward shearing as early as ca. 137 Ma. This composite process of extension manifests vertical strain partitioning across the ductile middle-lower crusts and progressive strain localization during the lithospheric thinning. The NW-SE orientation dominated extensional tectonics was strongly driven by the westward subduction of the Paleo-Pacific oceanic plate during the Late Mesozoic

Antimicrobial resistance and multilocus sequence typing analysis of Vibrio parahaemolyticus from seafood in Chengdu

Objective To find out the contamination, antimicrobial resistance, virulence gene distribution and genotyping of Vibrio parahaemolyticus in different kinds of marine products in Chengdu. Increase the basic data on the prevalence and risk assessment of foodborne Vibrio parahaemolyticus in Chengdu. Methods According to GB 4789.7-2013, suspected strains of Vibrio parahaemolyticus were isolated from different marine products and accurately identified by biochemical tests and 16S rDNA sequencing. The antimicrobial resistance test of the isolated strains was carried out by Kirby-Bauer disk diffusion method. Two virulence genes related to its pathogenicity were detected by polymerase chain reaction (PCR), and then the isolated strains were typed by multilocus sequence typing. Results A total of 104 strains of Vibrio parahaemolyticus were isolated from 380 seafood samples collected, with an overall detection rate of 27.4%. Antimicrobial resistance test showed that 97.1% (101/104) of the isolates had resistance, of which the drug resistance rate to ampicillin was the highest (95.2%, 99/104). The harboring rate of trh gene among the isolated strains was 12.5% (13/104), and was 0.1% (1/104) for tdh gene.104 isolates were divided into 38 ST types, of which ST1801, ST392 and ST413 were at high level. No epidemic clone group was found in the isolates. Conclusion There were differences in the contamination rate, antimicrobial resistance, and virulence genes distribution of Vibrio parahaemolyticus in different kinds of marine products, which might be related to the breeding environment and transportation conditions

Construction and immunogenicity of a ∆apxIC/ompP2 mutant of <i>Actinobacillus pleuropneumoniae</i> and <i>Haemophilus parasuis</i>

The apxIC genes of the Actinobacillus pleuropneumoniae serovar 5 (SC-1), encoding the ApxIactivating proteins, was deleted by a method involving sucrose counter-selection. In this study, a mutant strain of A. pleuropneumoniae (SC-1) was constructed and named DapxIC/ ompP2. The mutant strain contained foreign DNA in the deletion site of ompP2 gene of Haemophilus parasuis. It showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. The LD50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (LD50 of the mutant strain and parent strain in mice were determined to be 1.0 × 107 CFU and 3.5 × 105 CFU respectively). The mutant strain that was attenuated could secrete inactivated ApxIA RTX toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of A. pleuropneumoniae and H. parasuis.</em

Prevalence and seroepidemiology of Haemophilus parasuis in Sichuan province, China

Haemophilus parasuis, the causative agent of Glässer’s disease, has been reported as widespread, but little is known about its epidemiology in the Sichuan province of China. The goal of our research is to reveal the prevalence and distribution of H. parasuis in this area. Sampling and isolation were performed across Sichuan; isolates were processed using serotyping multiplex PCR (serotyping-mPCR) and agar gel diffusion (AGD) for confirmation of serovar identity. This study was carried out from January 2014 to May 2016 and 254 H. parasuis field strains were isolated from 576 clinical samples collected from pigs displaying clinical symptoms. The isolation frequency was 44.10%. Statistically very significant differences of infection incidence were found in three age groups (P < 0.01) and different seasons (P < 0.01). Serovars 5 (25.98%) and 4 (23.62%) were the most prevalent, however, non-typeable isolates accounted for nearly 7.87%. In terms of geographical distribution, serovars 5 and 4 were mostly prevalent in west and east Sichuan. The results confirmed that the combined approach was dependable and revealed the diversity and distribution of serovars in Sichuan province, which is vital for efforts aimed at developing vaccine candidates allowing for the prevention or control of H. parasuis outbreaks

One of two TolC-like proteins is involved in antibiotic resistance and biofilm formation of Actinobacillus pleuropneumoniae clinical isolate SC1516

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a significant disease that causes serious economic losses to the swine industry worldwide. Persistent infections caused by bacterial biofilms are recalcitrant to treat because of the particular drug resistance of biofilm-dwelling cells. TolC, a key component of multidrug efflux pumps, are responsible for multidrug resistance in many Gram-negative bacteria. In this study, we identified two TolC-like proteins, TolC1 and TolC2, in A. pleuropneumoniae. Deletion of tolC1, but not tolC2, caused a significant reduction in biofilm formation, as well as increased drug sensitivity of both planktonic and biofilm cells. The genetic-complementation of the tolC1 mutation restored the competent biofilm and drug resistance. Besides, biofilm formation was inhibited and drug sensitivity was increased by the addition of phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux pump inhibitor (EPI), suggesting a role for EPI in antibacterial strategies towards drug tolerance of A. pleuropneumoniae. Taken together, TolC1 is required for biofilm formation and is a part of the multidrug resistance machinery of both planktonic and biofilm cells, which could supplement therapeutic strategies for resistant bacteria and biofilm-related infections of A. pleuropneumoniae clinical isolate SC1516