The main goal of this study was to investigate the pathogenic characteristics of PRRSV field isolates. When PRRSV-positive supernatants of field strains were inoculated into MARC-145 cells, CPE appeared at the first passage, indicating that the isolated viruses were initially highly pathogenic in these cells. These isolates easily adapted to MARC-145 cells but were different from viruses isolated before 2005
, which had to be adapted to porcine alveolar macrophages (PAMs) for several passages before successful propagation in MARC-145 cells. While the isolates we examined in this study replicated as well as HP-PRRSV strains JXA1 and HUB1 isolated from 2006–2007, there were some differences in pathogenicity among HN-1/2008, YN-1/2008 and GX-1/2008. The times at which 75% CPE was observed for HN-1/2008, YN-1/2008 and GX-1/2008 were 84, 92 and 100 h, respectively. Phylogenetic trees based upon ORF5 and ORF7 indicated that these strains were all genotype II PRRSV, and clustered on the same branch as JXA1 and HUB1. Similarities among the protein homologs for ORF7 among these five strains were 100%. Similarities between highly pathogenic and two typical strains (CH-1a/2001 and VR-2332/2000) were 94.3%. This highlighted that highly pathogenic strains have become predominant among epidemic PRRSV strains in China.
To confirm the pathogenicity of HN-1/2008, YN-1/2008 and GX-1/2008 in susceptible animals, we inoculated piglets in groups B–D with 106 TCID50 of HN-1/2008, YN-1/2008 and GX-1/2008 after five passages in cells. Piglets in group E were inoculated with 104 TCID50 of GX-1/2008F. Approximately 2–6 days post-challenge, all inoculated animals presented with clinical signs, but no animals died. The pathogenic characteristics of the isolates in this study were consistent with those of JXA1and HUB1. However, mortality rates were low, suggesting that the virulence of highly pathogenic strains prevailing in China have abated. Additionally, the pathogenicity of the field sample was significantly different to the isolates that were passaged in cell cultures. Although clinical signs induced by HN-1/2008 and YN-1/2008 presented earlier than those of the field isolate, the pathological changes that were observed in all infected piglets were similar. The extent of these changes, along with the maximal rectal temperatures caused by HN-1/2008 and YN-1/2008, were not as apparent as those caused by GX-1/2008F.
The clinical signs and pathological changes induced by GX-1/2008 and GX-1/2008F were very different, even though the dose of the isolate passaged in cells was higher than that of the field sample. This indicates that virulence and pathogenicity of the field isolate became attenuated after passage in MARC-145 cells. There have been some reports regarding the loss of infectivity of isolates in susceptible animals after propagation in MARC-145 cells. The reason for this phenomenon has not been fully elucidated. Zuckermann
 developed a PAM cell line, ZMAC-3, which was shown to efficiently support the replication of a number of PRRSV isolates. They injected piglets with an equivalent dose of the Prime Pac vaccine grown in either ZMAC-3 or MARC-145 cells. After 4 weeks, all vaccinated animals were challenged with an “atypical PRRS abortion storm” virus isolate (NADC-20). It was found that the virus used in vaccines and grown in ZMAC-3 cells was significantly more effective than that derived from MARC-145 cells at reducing viremia and eliminating virus from the lungs. The effectiveness of a PRRS modified-live virus vaccine is not only determined by its genetic similarity to the challenge virus, but is also influenced by how it is produced. At present, three PRRSV receptors have been identified on PAMs: heparan sulfate (HS); sialoadhesin (Sn, also known as CD169); and CD163. Early attachment of PRRSV is mediated mainly via interaction with HS, while Sn is required for PRRSV attachment and internalization. Although HS is not essential for Sn-mediated internalization, it has been shown to enhance internalization and infection
. It is possible that binding Sn is a necessary first step in a pathway leading to uncoating and release of viral RNA into the cytoplasm
. HS, CD163, and CD151 have been identified or proposed as cellular receptors, co-receptors or mediators for in vitro PRRSV infection in MARC-145 cells. Because MARC-145 cells do not express Sn, it has been suggested that simian vimentin can play the role of Sn in that cell line
. We concluded that the attenuation, or loss of pathogenicity, of field isolates following passage in MARC-145 cells may be related to changes in conformation of PRRSV proteins responsible for attachment, infection or propagation.
The main gross lesions caused by PRRSV generally consist of a multifocal interstitial pneumonia with mottled areas and hyperplasia of lymph nodes
. The pathological changes caused by the highly pathogenic strains in this study were similar to those previously reported. Pathological changes were found in almost in every tissue examined in this study. Previous studies of piglets with acute viremia after PRRSV infection did not report inflammation in the stomach or the small intestine
, although Gao et al. discovered that piglets challenged with the VR-2332 strain exhibited hyperemia of the stomach and small intestine, but no epithelial shedding was observed.
The tropism of highly pathogenic strains examined in this study had altered compared with those previously studied. PAMs are the preferred target cell of PRRSV infection. In this study, a comparison of the number of PAMs between control and infected animals showed no decrease in their numbers. However, we did observe some virus inclusion bodies and autophagosomes in those cells. As reported by Xiao et al., we found that only a small proportion of PAMs were infected. Xiao et al. found that less than 2% of lung macrophages were PRRSV-positive during an acute infection, without causing a significant decline in local macrophage frequency in tissues. They deduced that this phenomenon may be due to the rapid replacement of destroyed macrophages; however, the majority of macrophages might not be permissive to PRRSV infection. Therefore, the elimination of permissive cells would limit the extent of infection. In our work, the presence of autophagosomes in alveolar macrophages indicated that infected macrophages were eliminated by normal macrophages, strongly supporting the findings of Xiao et al. Additionally, the numbers of types І and II alveolar cells were decreased and increased, respectively, but were all damaged. Type І alveolar cells reside in the respiratory tissue. Type II alveolar cells are the precursors of type І alveolar cells. We propose that the number of type II alveolar cells increase when type І alveolar cells are damaged. Respiratory obstruction occurred when types I and II alveolar cells were damaged, weakening the function of the respiratory system. Damage to cellular organelles such as the rough endoplasmic reticulum and mitochondria in plasmacytes and lymphocytes showed that infection with PRRSV induced immunosuppression.
Using ELISA, development of specific antibodies in groups B and C animals could be observed as early as 7 DPI. Pigs in group E were seropositive at 11 DPI. Although piglets in group D displayed clinical signs, and analysis by nPCR showed that tissues were PRRSV-positive, these animals remained seronegative throughout the experiment. It is unclear why group D animals did not seroconvert, but has been previously reported by Hermann et al.. They found that virus could be isolated from an animal exposed to 105.2 TCID50/ml at 7, 14 and 21 DPI, even though it was seronegative at all sampling points (0, 4, 14 and 21 DPI). They were unable to detect other clinical diseases or other agents of disease. Tests for the detection of PPV, PCV and CSFV antigens were also negative. No animals in group D had detectable viremia. However, the majority of piglets in other groups were viremic, but virus could only be detected after using a highly-sensitive nPCR assay. These results suggest that seroconversion and clinical manifestations were related to the presence of virus in the blood.
Innate immunity plays a crucial role in the defense against viral infections. It is characterized by rapid induction upon infection, and by lacking memory and specificity
. The response of animals infected with RNA viruses involves the production of type I interferons (IFN-α and/or -β), and can often be accompanied by secretion of tumor necrosis factors (TNFs) and IL-1
. In our study, the levels of IFN-α and IL-1 in all infected and control animals were not significantly different. The ability of the three isolates to induce IFN-α was similar, suggesting that PRRSV infection inhibited or suppressed the innate immune responses in these animals. IFN-γ plays a key role in cell-mediated immune responses against a variety of cytopathic viral infections in animals. Several reports have shown that IFN-γ can inhibit PRRSV infection
. We investigated the kinetics of PRRSV-specific IFN-γ levels in serum samples during an acute infection. An increase in the expression of IFN-γ levels was observed at 7 DPI after infection with GX-1/2008F, HN-1/2008 and YN-1/2008. Elevation in IFN-γ levels was not observed for group D animals. IFN-γ is known to protect macrophages against PRRSV replication in vitro
. However, viral replication still occurred following the induction of IFN-γ. We concluded that in our animal study the temporary IFN-γ response was not efficient to eliminate PRRSV.
IL-1β is known to induce IL-6 production. It has been demonstrated that the appearance of IL-1 and −6 are accompanied with the onset of clinical respiratory disease and increased body temperature
. In the present study, IL-6 levels in groups B, C, and E were elevated at 7, 7, and 14 DPI, respectively. At these time points, pneumonia and other clinical signs, especially fever, were apparent. Our results showed that the changes in expression for these cytokines likely reflect the level of pathology induced by the virus.