Eric Martin Vaughn

Age-dependent resistance to Porcine reproductive and respiratory syndrome virus replication in swine

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) causes a prolonged, economically devastating infection in pigs, and immune resistance to infection appears variable. Since the porcine adaptive immune system is not fully competent at birth, we hypothesized that age influences the dynamics of PRRSV infection. Thus, young piglets, growing 16-20-week-old finisher pigs, and mature third parity sows were infected with virulent or attenuated PRRSV, and the dynamics of viral infection, disease, and immune response were monitored over time.ResultsVirulent PRRSV infection and disease were markedly more severe and prolonged in young piglets than in finishers or sows. Attenuated PRRSV in piglets also produced a prolonged viremia that was delayed and reduced in magnitude, and in finishers and sows, about half the animals showed no viremia. Despite marked differences in infection, antibody responses were observed in all animals irrespective of age, with older pigs tending to seroconvert sooner and achieve higher antibody levels than 3-week-old animals. Interferon γ (IFN γ) secreting peripheral blood mononuclear cells were more abundant in sows but not specifically increased by PRRSV infection in any age group, and interleukin-10 (IL-10) levels in blood were not correlated with PRRSV infection status.ConclusionThese findings show that animal age, perhaps due to increased innate immune resistance, strongly influences the outcome of acute PRRSV infection, whereas an antibody response is triggered at a low threshold of infection that is independent of age. Prolonged infection was not due to IL-10-mediated immunosuppression, and PRRSV did not elicit a specific IFN γ response, especially in non-adult animals. Equivalent antibody responses were elicited in response to virulent and attenuated viruses, indicating that the antigenic mass necessary for an immune response is produced at a low level of infection, and is not predicted by viremic status. Thus, viral replication was occurring in lung or lymphoid tissues even though viremia was not always observed.

Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: Correlation between TTSuV2 viral load and IgG antibody level in pigs

Porcine Torque teno virus (TTV) has a single-stranded circular DNA genome and is currently classified into a new genus Iotatorquevirus with two species in a newly established family Anelloviridae. Viral DNA of both porcine TTV species (TTSuV1 and TTSuV2) has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States and in Europe. However, the prevalence of specific TTSuV antibodies in pigs remains unknown. In this study, the putative ORF1 capsid protein from TTSuV2 isolate PTTV2c-VA was expressed in Escherichia coli. The purified recombinant ORF1 protein was used as the antigen for the development of Western blot and indirect ELISA to detect TTSuV2-specific IgG antibodies in pig sera. The results revealed a relatively high rate of seropositivity to TTSuV2 in conventional pigs from different sources but not in gnotobiotic pigs. Overall, pigs with undetectable TTSuV2 viral load were more likely to have a lower anti-TTSuV2 antibody level. An analysis of 10 conventional pigs during a 2-month period showed that decreased viral loads or presumed virus clearance were associated with elevated anti-ORF1 IgG antibody levels. Interestingly, porcine circovirus associated disease (PCVAD)-affected pigs had a significantly lower level of TTSuV2 antibody than PCVAD-unaffected pigs (p<0.01). This is the first study to establish essential serodiagnostic tools for investigation of TTSuV seroprevalence and infection dynamics, which will help elucidate the potential pathogenicity of TTSuV infection in pigs.

Serological Profile of Torque Teno Sus Virus Species 1 (TTSuV1) in Pigs and Antigenic Relationships between Two TTSuV1 Genotypes (1a and 1b), between Two Species (TTSuV1 and -2), and between Porcine and Human Anelloviruses

ABSTRACT The family Anelloviridae includes human and animal torque teno viruses (TTVs) with extensive genetic diversity. The antigenic diversity among anelloviruses has never been assessed. Using torque teno sus virus (TTSuV) as a model, we describe here the first investigation of the antigenic relationships among different anelloviruses. Using a TTSuV genotype 1a (TTSuV1a) or TTSuV1b enzyme-linked immunosorbent assay (ELISA) based on the respective putative ORF1 capsid antigen and TTSuV1-specific real-time PCR, the combined serological and virological profile of TTSuV1 infection in pigs was determined and compared with that of TTSuV2. TTSuV1 is likely not associated with porcine circovirus-associated disease (PCVAD), because both the viral loads and antibody levels were not different between affected and unaffected pigs and because there was no synergistic effect of concurrent PCV2/TTSuV1 infections. We did observe a higher correlation of IgG antibody levels between anti-TTSuV1a and -TTSuV1b than between anti-TTSuV1a or -1b and anti-TTSuV2 antibodies in these sera, implying potential antigenic cross-reactivity. To confirm this, rabbit antisera against the putative capsid proteins of TTSuV1a, TTSuV1b, or TTSuV2 were generated, and the antigenic relationships among these TTSuVs were analyzed by an ELISA and by an immunofluorescence assay (IFA) using PK-15 cells transfected with one of the three TTSuV ORF1 constructs. The results demonstrate antigenic cross-reactivity between the two genotypes TTSuV1a and TTSuV1b but not between the two species TTSuV1a or -1b and TTSuV2. Furthermore, an anti-genogroup 1 human TTV antiserum did not react with any of the three TTSuV antigens. These results have important implications for an understanding of the diversity of anelloviruses as well as for the classification and vaccine development of TTSuVs.

Rescue of a porcine anellovirus (torque teno sus virus 2) from cloned genomic DNA in pigs.

ABSTRACT Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.

Development of SYBR green-based real-time PCR and duplex nested PCR assays for quantitation and differential detection of species- or type-specific porcine Torque teno viruses.

Porcine Torque teno virus (TTV), a single-stranded circular DNA virus, has been incriminated in swine diseases recently. Multiple infection with porcine TTV species 1 (PTTV1) and species 2 (PTTV2), each consisting of two types (PTTV1a and 1b) or subtypes (PTTV2b and 2c), in a single pig had been reported by our group previously. The present study described three novel assays for quantitation and differential detection of porcine TTV. First, we developed two SYBR green-based real-time PCR assays to quantify viral loads of two porcine TTV species, respectively. The PTTV1- and PTTV2-specific real-time PCR primer sequences were selected to target conserved regions identified by multiple alignments of ten available porcine TTV full-length genomes. Furthermore, by coupling the two singleplex PCR assays, a duplex real-time PCR assay followed by melting curve analysis was established for simultaneous detection and differentiation of PTTV1 and PTTV2. In addition, a type-specific duplex nested PCR was also developed to simultaneously detect and distinguish between the two types, PTTV1a and 1b, in PTTV1 species. These assays provide rapid and practical tools for molecular diagnosis of species- or type-specific porcine TTV.

Particle and subunit-based hemagglutinin vaccines provide protective efficacy against H1N1 influenza in pigs.

The increasing diversity of influenza strains circulating in swine herds escalates the potential for the emergence of novel pandemic viruses and highlights the need for swift development of new vaccines. Baculovirus has proven to be a flexible platform for the generation of recombinant forms of hemagglutinin (HA) including subunit, VLP-displayed, and baculovirus-displayed antigens. These presentations have been shown to be efficacious in mouse, chicken, and ferret models but little is known about their immunogenicity in pigs. To assess the utility of these HA presentations in swine, Baculovirus constructs expressing HA fused to swine IgG2a Fc, displayed in a FeLV gag VLP, or displayed in the baculoviral envelope were generated. Vaccines formulated with these antigens wer The e administered to groups of pigs who were subsequently challenged with H1α cluster H1N1 swine influenza virus (SIV) A/Swine/Indiana/1726/88. Our results demonstrate that vaccination with any of these three vaccines elicits robust hemagglutinin inhibition titers in the serum and decreased the severity of SIV-associated lung lesions after challenge when compared to placebo-vaccinated controls. In addition, the number of pigs with virus detected in the lungs and nasal passages was reduced. Taken together, the results demonstrate that these recombinant approaches expressed with the baculovirus expression vector system may be viable options for development of SIV vaccines for swine.

Replication and Expression Analysis of PRRSV Defective RNA

family Arteriviridae, which was recently grouped together with the coronaviruses and the toroviruses in the newly established order of the Nidovirales. PRRSV is a spherical, enveloped virus with a diameter of 50 to 60 nm and a positive-stranded RNA genome of 15.0–5.5 kilobases. Nidovirus defective RNAs have been well documented and studied, especially for coronaviruses and equine arterivirus (EAV). Defective RNAs are truncated, and in some cases, rearranged genomes that have usually lost the potential to replicate autonomously due to deletions in the viral replicase gene(s), the replication of which depends on the replicase encoded by the helper virus. Defective RNAs have retained all replication signals and, frequently, also the sequences required for RNA encapsidation. However, the generation of many of nidovirus defective RNAs requires serial undiluted

Pathogenicity of three isolates of porcine respiratory coronavirus in the USA

The pathogenicity of three isolates of porcine respiratory coronavirus (AR310, LEPP and 1894) from the USA was assessed in specific pathogen-free pigs. Pigs inoculated with 1894 developed mild respiratory disease and pigs inoculated with AR31o and LEPP developed moderate respiratory disease from four to 10 days after they were inoculated, but all the pigs recovered fully by 14 days after inoculation. Gross and microscopic examination revealed mild (1894) to moderate (AR31o and LEPP) multifocal bronchointerstitial pneumonia from four to 10 days after inoculation. The lesions were characterised by necrotising bronchiolitis, septal infiltration with mononuclear cells, and a mixed alveolar exudate. No clinical signs or microscopic lesions were observed in control pigs that had not been inoculated.

A genetically engineered H5 protein expressed in insect cells confers protection against different clades of H5N1 highly pathogenic avian influenza viruses in chickens

ABSTRACT The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition titres were observed for the recH5 vaccine antigen, and lower haemagglutination inhibition titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 days PV and 42 days PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by reverse transcription followed by a quantitative polymerase chain reaction in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a differentiation infected from vaccinated animals (DIVA) approach, based on the detection of nucleoprotein antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.