Highly-pathogenic PRRS virus (HP-PRRSV) belongs to type 2 genotype (North American, prototype strain VR-2332) of PRRSV, which is a member of the genus Arterivirus, family Arteriviridae. HP-PRRSV is characterized by high fever and high rates of morbidity and mortality [1Tian K, Yu X, Zhao T, et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS One 2007; 2: e526.
[http://dx.doi.org/10.1371/journal.pone.0000526] ]. This novel and highly virulent variant of PRRSV, which first emerged in China in 2006, has rapidly spread to most countries in South East Asia [2Ni J, Yang S, Bounlom D, et al. Emergence and pathogenicity of highly pathogenic Porcine reproductive and respiratory syndrome virus in Vientiane, Lao People's Democratic Republic. J Vet Diagn Invest 2012; 24: 349-54.
[http://dx.doi.org/10.1177/1040638711434111] ]. HP-PRRSV exhibits more tissue tropism than classic PRRSV [3He Y, Wang G, Liu Y, et al. Characterization of thymus atrophy in piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus. Vet Microbiol 2012; 160: 455-62.
[http://dx.doi.org/10.1016/j.vetmic.2012.05.040] ]. Besides lymphoid tissues, IHC examination showed that HP-PRRSV antigen can also be detected in other tissues including trachea, esophagus, liver, kidney, cerebellum, stomach, and intestine [4Li L, Zhao Q, Ge X, et al. Chinese highly pathogenic porcine reproductive and respiratory syndrome virus exhibits more extensive tissue tropism for pigs. Virol J 2012; 9: 203.
[http://dx.doi.org/10.1186/1743-422X-9-203] ].

Compared with the prototype of type 2 genotype strain VR-2332, HP-PRRSV can elicit strong immune responses by the evidence of a striking elevation in the level of cytokines associated with both innate and adaptive immunity in HP-PRRSV infected pigs [5Guo B, Lager KM, Henningson JN, et al. Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus. Virology 2013; 435: 372-84.
[http://dx.doi.org/10.1016/j.virol.2012.09.013] ]. In contrast, VR-2332, first isolated in 1987, only leads to mild clinical symptoms and does not circulate in the field any longer [6Collins JE, Benfield DA, Christianson WT, et al. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J Vet Diagn Invest 1992; 4: 117-26.
[http://dx.doi.org/10.1177/104063879200400201] ]. NADC-20 is a virulent North American PRRSV strain, which was first isolated in an “atypical PRRSV abortion storm” in 2001 [7Harms PA, Sorden SD, Halbur PG, et al. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus. Vet Pathol 2001; 38: 528-39.
[http://dx.doi.org/10.1354/vp.38-5-528] ]. It has been widely used for viral challenge to evaluate the efficacy of PRRSV vaccines in the U.S [8Calzada-Nova G, Husmann RJ, Schnitzlein WM, Zuckermann FA. Effect of the host cell line on the vaccine efficacy of an attenuated porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 2012; 148: 116-25.
[http://dx.doi.org/10.1016/j.vetimm.2012.05.008] ]. Compared with the other strains of PRRSV in the U.S., NADC-20 can lead to clinical fever (≥ 40°C) and more robust immune responses after infection of pigs [8Calzada-Nova G, Husmann RJ, Schnitzlein WM, Zuckermann FA. Effect of the host cell line on the vaccine efficacy of an attenuated porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 2012; 148: 116-25.
[http://dx.doi.org/10.1016/j.vetimm.2012.05.008] ]. Therefore, analysis of the host immune responses elicited by two virulent strains of PRRSV will contribute to better understanding of the pathogenesis of HP-PRRSV and facilitate more effective vaccine development. In this study, 7-week old pigs were infected with the HV-PRRSV (a HP-PRRSV isolate, GenBank accession no. JX317648), or NADC-20 strain of PRRSV and the clinical symptoms and the profiles of host immune response were compared.

The HP-PRRSV has been reported to induce high fever, loss of body weight, severe respiratory symptoms and high mortality. In our study, body temperature in HV-PRRSV infected pigs was higher than 40°C during the duration of the infection, which may partially contribute to dehydration and respiratory distress Fig. (1A). The HV-PRRSV infection led to significant pig body weight loss as compared with NADC-20 infected pigs. The HV-PRRSV infected pigs lost an average of 10% of their body weight at 3 and 6 DPI, however, body weight returned to original weight by 10 DPI Fig. (1C). The body weight of NADC-20 infected pigs increased consistently after infection, although it was significantly lower when compared with the control pigs at 6 and 10 DPI (Fig. 1C). Consistent with a previous report [14Hu SP, Zhang Z, Liu YG, et al. Pathogenicity and distribution of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs. Transbound Emerg Dis 2013; 60: 351-9.
[http://dx.doi.org/10.1111/j.1865-1682.2012.01354.x] , 15Wang G, Song T, Yu Y, et al. Immune responses in piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus. Vet Immunol Immunopathol 2011; 142: 170-8.
[http://dx.doi.org/10.1016/j.vetimm.2011.05.004] ], HV-PRRSV infected pigs also showed more severe clinical symptoms including cutaneous hemorrhages and cyanotic extremities on the edge of ears (“blue ear”) and higher mortality rate (3/5 pigs died).

HV-PRRSV led to significant thymus atrophy compared with NADC-20 infection. The ratio of thymus/total body weight was significantly lower in HV-PRRSV infected pigs as compared with NADC-20 infected pigs (Fig. 1C). Thymus is the primary lymphoid tissue, in which T-lymphocytes mature and constitute the peripheral T-cell repertoire responsible for directing many facets of the adaptive immune responses. The malfunction/atrophy of thymus leads to the depletion of T lymphocytes, which was consistent with the significant loss of total T lymphocytes in the lung analyzed by flow cytometry. In contrast, the ratio of lung/total body weight was significantly higher in HV-PRRSV infected pigs as compared with NADC-20 infected pigs (Fig. 2), which indicated more inflammatory responses after HV-PRRSV infection. Several T cell subpopulations which exert cytotoxic functions, such as CTLs (CD3⁺CD4^-CD8⁺) and Th/memory (CD3⁺CD4⁺CD8⁺), were significantly higher after HV-PRRSV infection when compared with NADC-20 infection.

It has been reported that HP-PRRSV has higher proliferation ability than classic PRRSV both in vitro and in vivo [5Guo B, Lager KM, Henningson JN, et al. Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus. Virology 2013; 435: 372-84.
[http://dx.doi.org/10.1016/j.virol.2012.09.013] ]. In this study, both HV-PRRSV and NADC-20 showed similar proliferation ability within the first 6 DPI. Interestingly, by 9 DPI the viremia in NADC-20 infected pigs declined while the viremia of HV-PRRSV infected pigs was still increasing (Fig. 3A). In a study by Guo et al. [5Guo B, Lager KM, Henningson JN, et al. Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus. Virology 2013; 435: 372-84.
[http://dx.doi.org/10.1016/j.virol.2012.09.013] ], the virus titer and virus load in the serum were significantly higher after rJXwn6 HP-PRRSV infection as compared with VR-2332 infection from 2 to 11 DPI. The discrepancy of the viremia level could be due to the different strains of PRRSV used in each study, and the NADC-20 used in our study is more virulent than VR-2332. However, the high proliferation ability of HV-PRRSV did not correlate with higher titer of PRRSV-specific IDEXX ELISA antibody response, in that the average antibody titer in NADC-20 infected pigs was significantly higher than HV-PRRSV infected pigs at 9 DPI (Fig. 3B). The IDEXX ELISA measures the antibody response against N proteins of PRRSV, which has no protective ability to the PRRSV infection although it has been widely used for field diagnosis [16Thanawongnuwech R, Suradhat S. Taming PRRSV: Revisiting the control strategies and vaccine design. Virus Res 2010; 154: 133-40.
[http://dx.doi.org/10.1016/j.virusres.2010.09.003] ]. The different abilities to induce PRRSV IDEXX ELISA Ab between HP-PRRSV and classic PRRSV may contribute to the pathogenesis of viruses, and need further exploration.

TNF-α is a pro-inflammatory cytokine, which plays a very important role in regulation of immune responses, fever development (inflammation), and cell apoptosis [17Salek-Ardakani S, Croft M. Tumor necrosis factor receptor/tumor necrosis factor family members in antiviral CD8 T-cell immunity. J Interferon Cytokine Res 2010; 30: 205-18.
[http://dx.doi.org/10.1089/jir.2010.0026] ]. Several studies showed that PRRSV down-regulated TNF-α production in the early stage of infection, which may be used by virus to circumvent infected cell apoptosis [18Lopez-Fuertes L, Campos E, Domenech N, et al. Porcine reproductive and respiratory syndrome (PRRS) virus down-modulates TNF-alpha production in infected macrophages. Virus Res 2000; 69: 41-6.
[http://dx.doi.org/10.1016/S0168-1702(00)00172-6] , 19Ait-Ali T, Wilson AD, Westcott DG, et al. Innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated Swine alveolar macrophages. Viral Immunol 2007; 20: 105-18.
[http://dx.doi.org/10.1089/vim.2006.0078] ]. At the late stage of PRRSV infection, the peak of both apoptotic cells and viral antigen expression were observed in lymph nodes and tonsils of infected animals [20Gomez-Laguna J, Salguero FJ, Fernandez de Marco M, et al. Type 2 porcine reproductive and respiratory syndrome virus infection mediated apoptosis in B- and T-cell areas in lymphoid organs of experimentally infected pigs. Transbound Emerg Dis 2013; 60: 273-8.
[http://dx.doi.org/10.1111/j.1865-1682.2012.01338.x] ]. In our study, HV-PRRSV induced significantly higher TNF-α in both serum and lung samples at 6 DPI, and the high level of TNF-α expression correlates with the high level of viremia. The coincidence between high expression of TNF-α and high level of viremia at the late stage of PRRSV infection may indicate that PRRSV induces TNF-α mediated cell apoptosis to release virion progeny to infect other vulnerable cells.

Previous studies have shown that infection with several classic strains of PRRSV virus induced delayed or failed production of detectable serum IFN-α [21Diaz I, Darwich L, Pappaterra G, Pujols J, Mateu E. Immune responses of pigs after experimental infection with a European strain of Porcine reproductive and respiratory syndrome virus. J Gen Virol 2005; 86: 1943-51.
[http://dx.doi.org/10.1099/vir.0.80959-0] -23Gomez-Laguna J, Salguero FJ, Pallares FJ, et al. Acute phase response in porcine reproductive and respiratory syndrome virus infection. Comp Immunol Microbiol Infect Dis 2010; 33: e51-8.
[http://dx.doi.org/10.1016/j.cimid.2009.11.003] ]. In contrast, HV-PRRSV infection induced significantly higher IFN-α in the serum of pigs but significantly lower levels in the lung samples. Working as a potent antiviral molecule, IFN-α was reported to significantly inhibit PRRSV replication and enhance cellular-mediated immunity (IFN-γ responses) [24Royaee AR, Husmann RJ, Dawson HD, et al. Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination. Vet Immunol Immunopathol 2004; 102: 199-216.
[http://dx.doi.org/10.1016/j.vetimm.2004.09.018] , 25Albina E, Carrat C, Charley B. Interferon-alpha response to swine arterivirus (PoAV), the porcine reproductive and respiratory syndrome virus. J Interferon Cytokine Res 1998; 18: 485-90.
[http://dx.doi.org/10.1089/jir.1998.18.485] ]. However, the elevated serum IFN-α has no effect on virus clearance by the evidence of high level of viremia in HV-PRRSV infected pigs at 9 DPI (Figs. 3A and 4A). Also, the low level of IFN-α expression in the lung tissue after HV-PRRSV infection did not lead to decreased IFN-γ production as compared with NADC-20 infected pigs. Therefore, the role of IFN-α in the pathogenesis of PRRSV and host immunity to combat PRRSV needs to be further explored.

HV-PRRSV also elicited a significant elevation of adaptive immunity cytokines in the serum samples, such as IL-10 and IFN-γ, and significantly higher IL-10 in the lung samples (Figs. 4A, B). Induction of IL-10 following PRRSV infection is believed to be a focal mechanism leading to the unique immunological outcomes and interference of PRRSV vaccine efficacy. The production of IL-10 in the early stage of PRRSV infection is associated with a wide array of PRRSV-induced immunomodulatory activities [24Royaee AR, Husmann RJ, Dawson HD, et al. Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination. Vet Immunol Immunopathol 2004; 102: 199-216.
[http://dx.doi.org/10.1016/j.vetimm.2004.09.018] , 26Suradhat S, Thanawongnuwech R, Poovorawan Y. Upregulation of IL-10 gene expression in porcine peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus. J Gen Virol 2003; 84: 453-9.
[http://dx.doi.org/10.1099/vir.0.18698-0] ]. Consistent with previous studies, the expression of IL-10 in the serum and lung samples was significantly higher in HV-PRRSV infected pigs when compared with NADC-20 infected pigs [5Guo B, Lager KM, Henningson JN, et al. Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus. Virology 2013; 435: 372-84.
[http://dx.doi.org/10.1016/j.virol.2012.09.013] ]. The high level expression of IL-10 correlates with high titer of viremia in this study and PRRSV antigen gene expression in the lungs and tonsils of PRRSV infected pigs in previous studies [22Gomez-Laguna J, Salguero FJ, Barranco I, et al. Cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus. J Comp Pathol 2010; 142: 51-60.
[http://dx.doi.org/10.1016/j.jcpa.2009.07.004] ]. Some strains of modified live PRRSV vaccines also induced IL-10 production in vaccinated pigs, which may partially contribute to the failure of PRRSV vaccination [24Royaee AR, Husmann RJ, Dawson HD, et al. Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination. Vet Immunol Immunopathol 2004; 102: 199-216.
[http://dx.doi.org/10.1016/j.vetimm.2004.09.018] ]. Therefore, circumventing the inhibitory effect of IL-10 in the early stage of PRRVS vaccination/infection could be a challenge for PRRSV vaccine development.

IFN-γ is a key cytokine associated with host cell-mediated immunity (CMI) response, and is secreted by natural killer cells and several different T cell subpopulations. Significantly higher levels of IFN-γ in the serum was found in pigs infected with HV-PRRSV as compared with NADC-20 infected pigs (Fig. 4A), which was associated with a significantly higher percentage of NK cells in the blood (Fig. 5A). The coincidence of high levels of IFN-γ expression and the high percentage of NK cells may indicate that the production of IFN-γ at this stage might be a result of the innate immune response, most likely from antigen-stimulated NK cells [27Wesley RD, Lager KM, Kehrli ME Jr. Infection with Porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs. Can J Vet Res 2006; 70: 176-82.]. However, the higher level of IFN-γ in the serum did not lead to lower level of viremia in these pigs. The level of viremia in HV-PRRSV infected pigs was significantly higher than that in NADC-20 infected pigs. Therefore, the role IFN-γ plays in the protection to PRRSV infection at this stage is questionable.

In conclusion, by comparing immune responses in pigs infected with two virulent PRRSV strains: Chinese HV-PRRSV and U.S. NADC-20 strain, we found that the high proliferation ability of HV-PRRSV did not enhance the early production of PRRSV-specific ELISA antibodies (Ab). HV-PRRSV induced higher levels of TNF-α and IL-10 in both serum and the lung and higher levels of IFN-α and IFN-γ in the serum than did NADC-20. Flow cytometry analysis showed that HV-PRRSV infected pigs generated significantly higher frequencies of NK cells in the peripheral blood and Th/memory, CTLs, and T-reg cells in the lung tissue as compared with NADC-20 infected pigs. Thus, this study demonstrates that different immunity profiles were elicited by HV-PRRSV and NADC-20, and these differences may contribute to the better understanding of the pathogenesis of HP-PRRSV.