Dissecting the potential role of hepatitis E virus ORF1 nonstructural gene in cross-species infection by using intergenotypic chimeric viruses.

Hepatitis E virus (HEV) infects humans and more than a dozen other animal species. We previously showed that open reading frame 2 (ORF2) and ORF3 are apparently not involved in HEV cross-species infection, which infers that the ORF1 may contribute to host tropism. In this study, we utilize the genomic backbone of HEV-1 which only infects humans to construct a panel of intergenotypic chimeras in which the entire ORF1 gene or its functional domains were swapped with the corresponding regions from HEV-3 that infects both humans and pigs. We demonstrated that the chimeric HEVs were replication competent in human liver cells. Subsequently, we intrahepatically inoculated the RNA transcripts of chimeras into pigs to determine if the swapped ORF1 regions confer the chimeras' ability to infect pigs. We showed that there was no evidence of infectivity in pigs for any of the chimeras. We also investigated the role of human ribosome protein sequence S17, which expanded host range in cultured cells, in HEV cross-species infection. We demonstrated that S17 insertion in HEV ORF1 did not abolish HEV replication competency in vitro, but also did not expand HEV host tropism in vivo. The results highlight the complexity of the underlying mechanism of HEV cross-species infection.

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions.

The causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10-20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10-65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.

Possible risks posed by single-stranded DNA viruses of pigs associated with xenotransplantation.

Routine large-scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state-of-the-art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross-species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single-stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross-species barriers. Pig-associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig-associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.

Application of imaging flow cytometry for characterization of acute inflammation in non-classical animal model systems.

Phagocytes display marked heterogeneity in their capacity to induce and control acute inflammation. This has a significant impact on the effectiveness of antimicrobial immune responses at different tissue sites as well as their predisposition for inflammation-associated pathology. Imaging flow cytometry provides novel opportunities for characterization of these phagocyte populations through high spatial resolution, statistical robustness, and a broad range of quantitative morphometric cell analysis tools. This study highlights an integrative approach that brings together new tools in imaging flow cytometry with conventional methodologies for characterization of phagocyte responses during acute inflammation. We focus on a comparative avian in vivo challenge model to showcase the added depth gained through these novel quantitative multiparametric approaches even in the absence of antibody-based cellular markers. Our characterization of acute inflammation in this model shows significant conservation of phagocytic capacity among avian phagocytes compared to other animal models. However, it also highlights evolutionary divergence with regards to phagocyte inflammation control mechanisms based on the internalization of apoptotic cells.

Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are three of the most economically important swine pathogens, causing immense economic losses to the global swine industry. Monovalent commercial vaccines against each of the three viruses are routinely used in pig farms worldwide. A trivalent vaccine against all three pathogens would greatly simplify the vaccination programme and reduce the financial burden to the swine industry. In this study, by using an attenuated strain of PRRSV (strain DS722) as a live virus vector, we generated a multi-component vaccine virus, DS722-SIV-PCV2, which expresses the protective antigens from SIV and PCV2. The DS722-SIV-PCV2 trivalent vaccine virus replicates well, and expresses PCV2 capsid and SIV HA proteins in vitro. A subsequent vaccination and challenge study in 48 pigs revealed that the DS722-SIV-PCV2-vaccinated pigs had significantly reduced lung lesions and viral RNA loads when challenged with PRRSV. Upon challenge with PCV2, the vaccinated pigs had partially reduced lymphoid lesions and viral DNA loads, and when challenged with SIV the vaccinated pigs had significantly reduced acute respiratory sign scores. The results from this study demonstrate the potential of DS722-SIV-PCV2 as a candidate trivalent vaccine, and also shed light on exploring PRRSV as a potential live virus vaccine vector.

Probiotics mediated gut microbiota diversity shifts are associated with reduction in histopathology and shedding of Lawsonia intracellularis.

BACKGROUND: Clinical intervention during bacterial infections in farm animals such as pigs commonly includes the use of antimicrobials. With the rise of antimicrobial resistance and the attempts to reduce the use of antibiotics in food animals, effective alternatives are urgently needed to reduce or even remove pathogens and disease risks. Improving clinical outcomes and overall pig health by using probiotics appears attractive. However, reliable data sets on the efficacy of probiotics are scarce. The obligate intracellular bacterium Lawsonia intracellularis is widespread in pigs and associated with severe enteropathy, mainly in the ileum, commonly resulting in substantial reduction in weight gain. The impact of three in-feed probiotics and a commercial live L. intracellularis vaccine was compared in a pig challenge model. Probiotic treatment was associated with reduced L. intracellularis fecal shedding and reduced gut lesions. Here, the bacterial microbiota of the ileum of these pigs was characterized with 16S rRNA gene sequencing and was subsequently analyzed with bioinformatics tools. RESULTS: The greatest microbial richness was observed in the probiotic treated group T03-LAW, which accounted for 87% of richness observed in the study. Treatment had a significant impact on both the microbiota structure and taxonomic profile in the ileum, explaining between 26 and 36% of the structural variation, with the strongest association in the T03-LAW group. Overall, the largest changes were observed for the pigs treated with in-feed Bacillus pumilus; the microbiota of these pigs had the greatest diversity and highest richness. We also observed depleted and enriched core microbiota amongst the groups; however, there was no correlation with clinical characteristics. The results suggest that an increased diversity of the ileal microbiota is associated with a reduction in shedding, i.e. a unit increase in Shannon diversity index resulted in 2.8 log reduction in shedding. CONCLUSIONS: Probiotic supplementation of a base feed ration increased ileum microbiota diversity leading to a mitigation of the effects of a pathogenic L. intracellularis challenge. An even and diverse microbiota community benefits pigs infected with L. intracellularis, however, investigations are needed to determine if this is also true for other pathogens. The study unambiguously demonstrates the usefulness of probiotic supplementation in reducing the impact of enteric pathogens and pathogen shedding rates in food animals without the use of antimicrobials.

Future perspectives on swine viral vaccines: where are we headed?

Deliberate infection of humans with smallpox, also known as variolation, was a common practice in Asia and dates back to the fifteenth century. The world's first human vaccination was administered in 1796 by Edward Jenner, a British physician. One of the first pig vaccines, which targeted the bacterium Erysipelothrix rhusiopathiae, was introduced in 1883 in France by Louis Pasteur. Since then vaccination has become an essential part of pig production, and viral vaccines in particular are essential tools for pig producers and veterinarians to manage pig herd health. Traditionally, viral vaccines for pigs are either based on attenuated-live virus strains or inactivated viral antigens. With the advent of genomic sequencing and molecular engineering, novel vaccine strategies and tools, including subunit and nucleic acid vaccines, became available and are being increasingly used in pigs. This review aims to summarize recent trends and technologies available for the production and use of vaccines targeting pig viruses.

Porcine circoviruses: current status, knowledge gaps and challenges.

Circoviruses (CV) include some of the smallest viruses known. They were named after their circularly arranged single-stranded DNA genome with a gene encoding a conserved replicase protein on the sense strand. Circoviruses are widely distributed in mammals, fish, avian species and even insects. In pigs, four different CVs have been identified and named with consecutive numbers based on the order of their discovery: Porcine circovirus 1 (PCV1), Porcine circovirus 2 (PCV2), Porcine circovirus 3 (PCV3) and most recently Porcine circovirus 4 (PCV4). PCVs are ubiquitous in global pig populations and uninfected herds are rarely found. It is generally accepted that PCV1 is non-pathogenic. In contrast, PCV2 is considered an important, economically challenging pathogen on a global scale with comprehensive vaccination schemes in place. The role of PCV3 is still controversial several years after its discovery. Propagation of PCV3 appears to be challenging and only one successful experimental infection model has been published to date. Similarly to PCV2, PCV3 is widespread and found in many pigs regardless of their health history, including high health herds. PCV4 has only recently been discovered and further information on this virus is required to understand its potential impact. This review summarizes current knowledge on CVs in pigs and aims to contrast and compare known facts on PCVs.

Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge.

With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) γ secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge.

A prime-boost concept using a T-cell epitope-driven DNA vaccine followed by a whole virus vaccine effectively protected pigs in the pandemic H1N1 pig challenge model.

Influenza A virus (IAV) vaccines in pigs generally provide homosubtypic protection but fail to prevent heterologous infections. In this pilot study, the efficacy of an intradermal pDNA vaccine composed of conserved SLA class I and class II T cell epitopes (EPITOPE) against a homosubtypic challenge was compared to an intramuscular commercial inactivated whole virus vaccine (INACT) and a heterologous prime boost approach using both vaccines. Thirty-nine IAV-free, 3-week-old pigs were randomly assigned to one of five groups including NEG-CONTROL (unvaccinated, sham-challenged), INACT-INACT-IAV (vaccinated with FluSure XP® at 4 and 7 weeks, pH1N1 challenged), EPITOPE-INACT-IAV (vaccinated with PigMatrix EDV at 4 and FluSure XP® at 7 weeks, pH1N1 challenged), EPITOPE-EPITOPE-IAV (vaccinated with PigMatrix EDV at 4 and 7 weeks, pH1N1 challenged), and a POS-CONTROL group (unvaccinated, pH1N1 challenged). The challenge was done at 9 weeks of age and pigs were necropsied at day post challenge (dpc) 5. At the time of challenge, all INACT-INACT-IAV pigs, and by dpc 5 all EPITOPE-INACT-IAV pigs were IAV seropositive. IFNγ secreting cells, recognizing vaccine epitope-specific peptides and pH1N1 challenge virus were highest in the EPITOPE-INACT-IAV pigs at challenge. Macroscopic lung lesion scores were reduced in all EPITOPE-INACT-IAV pigs while INACT-INACT-IAV pigs exhibited a bimodal distribution of low and high scores akin to naïve challenged animals. No IAV antigen in lung tissues was detected at necropsy in the EPITOPE-INACT-IAV group, which was similar to naïve unchallenged pigs and different from all other challenged groups. Results suggest that the heterologous prime boost approach using an epitope-driven DNA vaccine followed by an inactivated vaccine was effective against a homosubtypic challenge, and further exploration of this vaccine approach as a practical control measure against heterosubtypic IAV infections is warranted.

Lawsonia intracellularis: Revisiting the Disease Ecology and Control of This Fastidious Pathogen in Pigs.

Lawsonia intracellularis is an anaerobic obligate intracellular bacterium infecting the small intestine and infrequently also the large intestine of pigs and other animals including hamsters and horses. The infection is characterized by proliferation, hemorrhage, necrosis, or any combination commonly referred to as "ileitis," affecting the health and production efficacy of farmed pigs. Despite decades of research on this pathogen, the pathogenesis and virulence factors of this organism are not clearly known. In pigs, prophylaxis against L. intracellularis infection is achieved by either administration of subtherapeutic levels of in-feed antibiotic growth promoters or vaccination. While the former approach is considered to be effective in L. intracellularis control, potential regulations on subtherapeutic antibiotics in many countries in the near future may necessitate alternative approaches. The potential of manipulating the gut microbiome of pigs with feed ingredients or supplements to control L. intracellularis disease burden is promising based on the current understanding of the porcine gut microbiome in general, as well as preliminary insights into the disease ecology of L. intracellularis infection accrued over the last 30 years.

Porcine Circovirus Type 2 (PCV2) Vaccines in the Context of Current Molecular Epidemiology.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen and, although small, it has the highest evolution rate among DNA viruses. Since the discovery of PCV2 in the late 1990s, this minimalistic virus with a 1.7 kb single-stranded DNA genome and two indispensable genes has become one of the most important porcine pathogens, and presently is subjected to the highest volume of prophylactic intervention in the form of vaccines in global swine production. PCV2 can currently be divided into five different genotypes, PCV2a through PCV2e. It is well documented that PCV2 continues to evolve, which is reflected by changes in the prevalence of genotypes. During 2006, commercial vaccines for PCV2 were introduced on a large scale in a pig population mainly infected with PCV2b. Since 2012, the PCV2d genotype has essentially replaced the previously predominant PCV2b genotype in North America and similar trends are also documented in other geographic regions such as China and South Korea. This is the second major PCV2 genotype shift since the discovery of the virus. The potential increase in virulence of the emergent PCV2 genotype and the efficacy of the current vaccines derived from PCV2a genotype against the PCV2d genotype viruses has received considerable attention. This review attempts to synthesize the understanding of PCV2 biology, experimental studies on the antigenic variability, and molecular epidemiological analysis of the evolution of PCV2 genotypes.

A novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response.

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating epizootic of porcine species. Current vaccines are inadequate to control the disease burden and outbreaks in the field. We report a novel baculovirus vaccine vector with White spot syndrome virus immediate early 1 shuttle promoter, with strong activity in both insect cells and mammalian cells, for immunization against PRRSV. The insect cell cultured baculovirus vector produces PRRSV envelope glycoproteins ORF2a, ORF3, ORF4 and ORF5, which are similar to the antigens in the infectious PRRS virion, and these antigens are stably incorporated on the surface of the baculovirus. Further, the baculovirus vector efficiently transduces these antigens in cells of porcine origin, thereby simulating a live infection. The baculovirus vectored PRRSV antigens, upon inoculation in mice, elicits robust neutralizing antibodies against the infective PRRS virus. Further, the experiments indicate that hitherto under emphasized ORF2a and ORF4 are important target antigens for neutralizing PRRSV infectivity.

Natural compounds inhibiting the replication of Porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogenic virus in the swine production. Current vaccines against PRRSV do not induce sterile immunity and the virus evolves at a rapid rate with frequent appearances of new strains. In this study, we screened a library of 502 highly purified natural product compounds to identify specific inhibitors of PRRSV replication cycle. Our observations showed that many of the inhibitory compounds identified have activity on the cellular ion transport mechanisms. We identified for the first time, four compounds which inhibit the PRRSV replication cycle at micro molar concentration or less, namely, 12-deoxyphorbol 13-phenylacetate 20-acetate, ouabain, bufalin and valinomycin. Further, we have identified 15 other compounds which can inhibit the PRRSV replication at the concentration of 8µM. This study provides a basis for further development of pharmacological agents to inhibit PRRSV replication.

ORF3 of porcine circovirus 2 enhances the in vitro and in vivo spread of the of the virus.

The ORF3 protein of the pathogenic porcine circovirus 2 (PCV2) causes apoptosis of the virus-infected cells. In PCV2-infected piglets, ORF3 induces B and CD4 T lymphocyte depletion and lymphoid organ destruction and the ORF3-deficient PCV2 is attenuated in its pathogenicity (Virology, 383 (2009), 338). In addition to its role in causing the apoptosis of the immune cells, characteristic of the PCV2 infection associated disease conditions, the ORF3 also plays a role in the systemic dissemination of the PCV2 infection. Our experiments here show that ORF3 expedites the spread of the virus by inducing the early release of the virus from the infected cells. Further, in PCV2-infected mice, the ORF3-induced apoptosis also aids in recruiting macrophages to phagocytize the infected apoptotic cells leading to the systemic dissemination of the infection. The apoptotic activity of the ORF3 of PCV2 hence lends advantage to the spread of the virus.

Porcine circovirus type 2 ORF3 protein competes with p53 in binding to Pirh2 and mediates the deregulation of p53 homeostasis.

The ORF3 protein of porcine circovirus type 2 (PCV2) causes apoptosis in virus-infected cells and is not essential for virus replication. The ORF3 protein plays an important role in the pathogenesis of the PCV2 infection in mouse models and SPF piglets. The ORF3 protein interacts with the porcine homologue of Pirh2 (pPirh2), a p53-induced ubiquitin-protein E3 ligase, which regulates p53 ubiquitination. Here, we present our study analyzing the details of the molecular interaction between these three factors. Our experiments, in vitro and in vivo, show that ORF3 protein competes with p53 in binding to pPirh2. The amino acid residues 20 to 65 of the ORF3 protein are essential in this competitive interaction of ORF3 protein with pPirh2 over p53. The interaction of ORF3 protein with pPirh2 also leads to an alteration in the physiological cellular localization of pPirh2 and a significant reduction in the stability of pPirh2. These events contribute to the deregulation of p53 by pPirh2, leading to increased p53 levels and apoptosis of the infected cells.

Characterization and epitope mapping of monoclonal antibodies recognizing N-terminus of Rep of porcine circovirus type 2.

Two genotypes of porcine circovirus (PCV) have been described, the non-pathogenic PCV1 and the pathogenic PCV2 in pigs. PCV-ORF1 encodes Rep and Rep' proteins which have identical N-terminal sequence (RepN) within each PCV strain, but RepN has only 88% similarity between PCV1 and PCV2. Purified RepN of PCV2 was used as an immunogen to produce monoclonal antibodies (mAbs). 11 mAbs were screened out and established, and they were divided into two groups according to Western blot and IFA results. One group, including 1C1, bound only PCV2-RepN, while the other, including 3D10, had cross reactivity with RepN of both PCV1 and PCV2. Epitope mapping indicated that 1C1 and 3D10 recognized the linear epitopes L(39)FDYFIVG(46) and K(99)EGNLLIE(106) in PCV2-RepN, respectively. Protein sequence alignment of RepN indicated L(39)FDYFIVG(46) is conserved in all PCV2 in NCBI database, whereas the PCV1 has amino acid substitutions V(41)C(42) in this region. mAb 3D10 could recognize all PCV because all natural mutations in its epitope did not affect its binding. The information about characteristics and epitope of monoclonal antibodies may be useful for the development of diagnostic methods for PCV2 and for analyzing the function of Rep and Rep' of PCV.

Attenuation of porcine circovirus 2 in SPF piglets by abrogation of ORF3 function.

Porcine circovirus 2 (PCV2), open reading frame 3 (ORF3) codes a 105 amino acid protein that causes apoptosis of PCV2 infected cells. In infected cells, the ORF3 causes the accumulation of p53 by interacting with pPirh2 and possibly by disrupting the association of p53 and pPirh2 (J.Virol.81(2007)9560). Mutant PCV2 lacking the expression of ORF3 are infectious and replicate in cells in vitro, but do not cause apoptosis of the infected cells. The ORF3 of PCV2 has been shown to be involved in pathogenesis of the virus in mice model (J. Virol. 80(2006)5065). Here we report the experimental inoculation of ORF3 deficient PCV2 in its natural host, the piglets. The pathogenicity of the ORF3 deficient virus is attenuated in the piglets. The mutant virus did not cause any observable disease or perturbation of the lymphocyte count in the inoculated piglets and elicited an efficient immune response. When compared with the wildtype virus infection, the mutant virus infection was characterized by mild viremia and absence of pathological lesions. The findings highlight the role of ORF3 in the pathogenesis of PCV2 infection in its host.

Protective immunity against influenza H5N1 virus challenge in mice by intranasal co-administration of baculovirus surface-displayed HA and recombinant CTB as an adjuvant.

The increasing number of recent outbreaks of HPAI H5N1 in birds and humans brings out an urgent need to develop potent H5N1 vaccine regimens. Here we present a study on the intranasal vaccination of recombinant baculovirus surface-displayed hemagglutinin (BacHA) or inactivated whole H5N1 viral (IWV) vaccine with a recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant in a BALB/c mouse model. Two groups of mice were vaccinated with different doses (HA titer of log 2(4) or log 2(8)) of either HA surface-displayed baculovirus or inactivated whole viral vaccine virus adjuvanted with different doses (2 mug or 10 mug) of rCTB. The vaccinations were repeated after 28 days. HA specific serum IgG and mucosal IgA antibodies were quantified by indirect ELISA, and serum neutralizing antibody titer were estimated by hemagglutination inhibition (HI) assay and virus neutralization titer assay. Functional protective efficacy of the vaccine was assessed by host challenge against HPAI H5N1 strains. The results revealed that mice co-administered with log 2(8) HA titer of BacHA vaccine and adjuvanted with 10 mug of rCTB had a significantly enhanced serum IgG and mucosal IgA immune response and serum microneutralization titer compared with mice administered with unadjuvanted log 2(4) or log 2(8) HA titer of BacHA alone. Also vaccination with 10 mug of rCTB and log 2(8) HA titer of BacHA elicited higher HA specific serum and mucosal antibody levels and serum HI titer than vaccination with log 2(8) HA titer of inactivated H5N1 virus adjuvanted with the same dose of rCTB. The host challenge study also showed that 10 mug rCTB combined with log 2(8) HA titer of BacHA provided 100% protection against 10MLD(50) of homologous and heterologous H5N1 strains. The study shows that the combination of rH5 HA expressed on baculovirus surface and rCTB mucosal adjuvant form an effective mucosal vaccine against H5N1 infection. This baculovirus surface-displayed vaccine is more efficacious than inactivated H5N1 influenza vaccine when administered by intranasal route and has no biosafety concerns associated with isolation, purification and production of the latter vaccine.

Enhanced replication of porcine circovirus type 2 (PCV2) in a homogeneous subpopulation of PK15 cell line.

Post-weaning multi-systemic wasting syndrome (PMWS) has emerged as a major disease that poses a significant threat to the economics of global swine industry. Porcine circovirus type 2 (PCV2) is the causal agent of PMWS in pigs. Currently, the prevention of PCV2 infection based on vaccines is limited, and the available vaccines are either killed viral vaccines or recombinant protein based vaccines and not cost effective. The PK-15 cells, which is widely used for PCV2 propagation, is not efficient and heterogeneous in terms of permissivity to viral infection. In order to acquire a homogeneous porcine kidney cell line that can reliably produce PCV2 in high titers, cell clones that show high- (PK15-C1) or low-permissive (PK15-A2) phenotype to PCV2 infection were derived from heterogeneous PK15 parent cells by limiting dilution and cell cloning. Maximum virus titers in PK15-C1, PK15-A2 and PK15 parent cells were 10(8), 10(2) and 10(5) tissue culture infective dose 50 (TCID 50)/ml, respectively. The viral proteins of PCV2 were produced and accumulated faster in PK15-C1 cells than those in PK15 parent cells. These results indicate that PK15-C1 cell clone is more permissive to PCV2 infection than PK15 parent cells and thus will be useful for PCV2 replication in vitro, as well as, vaccines, diagnostic and research applications on PCV2.