Enhancement of innate immunity with granulocyte colony-stimulating factor did not mitigate disease in pigs infected with a highly pathogenic Chinese PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for one of the most economically important diseases in swine worldwide. It causes reproductive failure in sows and pneumonia in pigs that predisposes them to secondary bacterial infections. Methods to control PRRSV and/or limit secondary bacterial infections are desired to reduce the impact of this virus on animal health. Neutrophils play a major role in combatting infection; they can act as phagocytes as well as produce and release lytic enzymes that have potent antimicrobial effects leading to the destruction and clearance of bacterial pathogens. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that controls the production, differentiation and function of granulocytes (including neutrophils) from the bone marrow. Recent work from our laboratory has shown that encoding porcine G-CSF in a replication-defective adenovirus (Ad5-G-CSF) and delivering a single dose to pigs induced a neutrophilia lasting more than two weeks. As secondary bacterial infection is a common occurrence following PRRSV infection, particularly following challenge with highly pathogenic (HP)-PRRSV, the aim of the current study was to evaluate the effectiveness of a single prophylactic dose of adenovirus-encoded G-CSF to mitigate secondary bacterial disease associated with HP-PRRSV infection. Administration of Ad5-G-CSF induced a significant neutrophilia as expected. However, between 1 and 2days following HP-PRRSV challenge the number of circulating neutrophils decreased dramatically in the HP-PRRSV infected group, but not the non-infected Ad5-G-CSF group. Ad5-G-CSF administration induced monocytosis as well, which was also reduced by HP-PRRSV challenge. There was no difference in the progression of disease between the Ad5-G-CSF and Ad5-empty groups following HP-PRRSV challenge, with pneumonia and systemic bacterial infection occurring in both treatment groups. Given the impact of HP-PRRSV infection on the neutrophilia induced by the Ad5-G-CSF administration, additional studies are warranted to evaluate the timing of Ad5-G-CSF induced neutrophilia and multiple G-CSF inoculations on protection against secondary bacterial infection following PRRSV infection. Nevertheless, this study may provide insight into the pathogenesis of HP-PRRSV.

Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a ubiquitous and costly virus that exhibits substantial sequence and virulence disparity among diverse isolates. In this study, we compared the whole genomic sequence and virulence of 4 Type 2 PRRSV isolates. Among the 4 isolates, SDSU73, MN184, and NADC30 were all clearly more virulent than NADC31, and among the 3 more virulent isolates, there were subtle differences based on viral replication, lung lesions, lymphadenopathy, febrile response, decreased weight gains, and cytokine responses in the lung. Lesions consistent with bacterial bronchopneumonia were present to varying degrees in pigs infected with PRRSV, and bacteria typically associated with the porcine respiratory disease complex were isolated from the lung of these pigs. Genomic sequence evaluation indicates that SDSU73 is most similar to the nucleotide sequence of JA142, the parental strain of Ingelvac(®) PRRS ATP, while the nucleotide sequences of NADC30 and NADC31 are more similar to strain MN184. Both the NADC30 and NADC31 isolates of PRRSV, isolated in 2008, maintain the nonstructural protein 2 (nsp2) deletion seen in MN184 that was isolated in 2001, but NADC31 has two additional 15 and 36 nucleotide deletions, and these strains are 8-14% different on a nucleotide basis from the MN184 strain. These results indicate that newer U.S. Type 2 strains still exhibit variability in sequence and pathogenicity and although PRRSV strains appear to be reducing the size of the nsp2 over time, this does not necessarily mean that the strain is more virulent.

Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine.

Fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolate genomes were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200, and 403 amino acid deletions in the second hypervariable region of nsp2, were found to be stable in the nsp2 coding region after in vivo infection (Faaberg et al., 2010). Furthermore, 3 of 4 mutants achieved replication kinetics similar to wt virus by study end. We hypothesized that other mutations elsewhere in the virus may have contributed to their replication fitness in swine. To further assess the stability of the engineered viruses, all sequenced genomes were compared and contrasted. No specific mutations occurred in all nsp2 deletion mutant genomes that were not also seen in the parent genome of Type 2 PRRSV strain VR-2332. Second site (non-nsp2) deletions and/or insertions were not evident after replication in swine. The number of point mutations seen increased slightly with deletion size, but even the largest deletion (403 aa) had very few consensus mutations. Thus, our findings provide further substantiation that the nsp2 deletion mutant genomes were genetically stable after in vivo passage.

Strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus.

The primary objective of the study was to determine strain specificity of the immune response of pigs following vaccination with selected strains of porcine reproductive and respiratory syndrome virus (PRRSV). The experimental design included five groups (I through V, six pigs per group) free of antibody for PRRSV at the beginning of the experiment (day 0). On day 0, groups III, IV, and V were vaccinated with attenuated versions of PRRSV strains 8, 9, and 14, respectively. On day 21, the immunity of group II (non-vaccinated/challenged controls) and groups III, IV, and V was challenged by exposing each pig to a composite of the virulent versions of these same three strains. On day 35, all pigs, including non-vaccinated/non-challenged pigs of group I, were necropsied. Lungs and selected lymph nodes were examined for lesions. Serum samples obtained at weekly intervals throughout the study and lung lavage fluids obtained at necropsy were tested for the presence of PRRSV and its strain identity. Before challenge the strain of PRRSV identified in the sera of vaccinated pigs was always that with which the particular pig had been vaccinated (i.e. homologous strain), whereas, with one exception, only heterologous strains were identified after challenge. This apparent strain exclusion as a result of vaccination was statistically significant (P = 0.004). The tendency for heterologous strains to predominate after challenge suggests that a pig's immune response to PRRSV has some degree of strain specificity. Whether this finding has any clinical relevance in regard to immunoprophylaxis remains to be determined.

Comparative safety and efficacy of attenuated single-strain and multi-strain vaccines for porcine reproductive and respiratory syndrome.

The objective of this study was to compare the efficacy and safety of single-strain and multi-strain vaccines for the prevention of the respiratory facet of porcine reproductive and respiratory syndrome. The study comprised six groups of pigs (A through F, eight pigs per group). At the beginning of the study (Day 0) Groups C and D were vaccinated with a single-strain vaccine, and Groups E and F were vaccinated with a multi-strain vaccine. The multi-strain vaccine contained five attenuated strains of PRRSV including the strain used as the single-strain vaccine. On Day 28 Groups B (nonvaccinated/challenged control), D, and F were challenged with a highly virulent field strain of PRRSV that was unrelated to any of the strains used for vaccination. Group A was kept as a nonvaccinated/nonchallenged control. On Day 42 all pigs were necropsied. Their lungs and lymph nodes were examined for virus-induced changes. Serum samples obtained at weekly intervals during the study and lung lavage fluids obtained at necropsy were tested for the presence and titer of PRRSV. Serum samples were also tested for antibody. The presence and severity of clinical signs and lesions were the primary means by which vaccine efficacy and safety were evaluated. Both vaccines provided a high level of protective immunity to challenge. However, appreciable lymph node enlargement in pigs vaccinated with multi-strain vaccine, with or without subsequent challenge, raised a question as to its safety. Collectively these results indicate that both single-strain and multi-strain attenuated PRRSV vaccines can be effective immunogens, but additional studies in regard to safety are needed before multi-strain vaccines can be recommended for routine field use.