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Stress granules (SGs) are dynamic sites of cytosolic mRNA storage that are formed in response to stress conditions, including viral infection. SGs have been implicated in regulating several aspects of the host immune response to various pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically-important global swine pathogen, reportedly induced SGs during replication, although the underlying mechanisms are poorly defined. In this study, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism of bona fide SGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions.

Replication of most RNA viruses is closely associated with the endoplasmic reticulum (ER) within permissive cells. As such, viruses often induce tremendous amounts of stress on cells during viral replication. To cope with the stress, cells initiate the unfolded protein response (UPR) to promote cellular survival. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically important swine pathogen, was previously shown to induce cellular stress at late stages post-infection resulting in the formation of stress granules (SGs). Here in this study, we demonstrate that PRRSV also induces additional cellular response pathways, including the UPR. Confocal microscopy analysis demonstrated significant morphological changes in the ER of PRRSV-infected cells, indicative of pronounced ER stress. Further investigation revealed induction of all three branches of the UPR, including eukaryotic translation initiation factor 2-alpha kinase 3 (PERK), serine-threonine protein kinase/endoribonuclease (IRE1), and cyclic AMP-dependent transcription factor (ATF6). Activation of these sensors resulted in significant transcriptional upregulation of downstream UPR effectors. Additionally, UPR activation was shown to be detrimental to PRRSV replication, as treatment of cells with chemical ER stress inducers potently suppressed viral replication and RNA synthesis. Further investigation into the molecular mechanisms of UPR suppression of PRRSV replication revealed that PERK exacerbates the PRRSV-induced cytokine response. Collectively, these results demonstrate that PRRSV infection induces UPR activation through all three branches, and that UPR signaling may play a role in PRRSV pathogenesis. The results of this study further our understanding of the underlying molecular mechanisms of PRRSV replication and host-pathogen interactions.

Co-infection of pigs in the field with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is common and poses a major concern in effective control of PCV2 and PRRSV. We previously demonstrated that insertion of foreign epitope tags in the C-terminus of PCV2 ORF2 produced infectious virions that elicited humoral immune responses against both PCV2 capsid and inserted epitope tags. In this study, we aimed to determine whether the non-pathogenic chimeric virus PCV1-2a, which is the basis for the licensed PCV2 vaccine Fostera PCV, can express PRRSV antigenic epitopes, thus generating dual immunity as a potential bivalent vaccine against both PCV2 and PPRSV. Four different linear B-cell antigenic epitopes of PRRSV were inserted into the C-terminus of the capsid gene of the PCV1-2a vaccine virus. We showed that insertion of 12 (PRRSV-GP2 epitope II, PRRSV-GP3 epitope I, and PRRSV-GP5 epitope I), and 14 (PRRSV-GP5 epitope IV) amino acid residues did not impair the replication of the resulting PCV1-2a-PRRSVEPI chimeric viruses in vitro. The four chimeric PCV1-2a viruses expressing PRRSV B-cell linear epitopes were successfully rescued and characterized. An immunogenicity study in pigs revealed that two of the four chimeric viruses, PCV1-2a-PRRSVEPIGP3IG and PCV1-2a-PRRSVEPIEPIGP5IV, elicited neutralizing antibodies against PRRSV VR2385 as well as PCV2 (strains PCV2a, PCV2b, and mPCV2b). The results have important implications for exploring the potential use of PCV1-2a vaccine virus as a live virus vector to develop bivalent MLVs against both PCV2 and PRRSV.

Swine hepatitis E virus (swine HEV) belongs to the species Orthohepevirus A within the genus Orthohepevirus in the family Hepeviridae. Four different genotypes of swine HEV within the species Orthohepevirus A have been identified so far from domesticated and wild swine population: genotypes 3 (HEV-3) and 4 (HEV-4) swine HEVs are zoonotic and infect humans, whereas HEV-5 and HEV-6 are only identified from swine. As a zoonotic agent, swine HEV is an emerging public health concern in many industrialized countries. Pigs are natural reservoir for HEV, consumption of raw or undercooked pork is an important route of foodborne HEV transmission. Occupational risks such as direct contact with infected pigs also increase the risk of HEV transmission in humans. Cross-species infection of HEV-3 and HEV-4 have been documented under experimental and natural conditions. Both swine HEV-3 and swine HEV-4 infect non-human primates, the surrogates of man. Swine HEV, predominantly HEV-3, can establish chronic infection in immunocompromised patients especially in solid organ transplant recipients. The zoonotic HEV-3, and to lesser extent HEV-4, have also been shown to cause neurological diseases and kidney injury. In this review, we focus on the epidemiology of swine HEV, host and viral determinants influencing cross-species HEV infection, zoonotic infection and its associated pork safety concern, as well as swine HEV-associated chronic infection and neurological diseases.

Highlights • Targeting PEDV spike protein domains to Langerin-expressing dendritic cells through a single chain antibody. • Transdermal Langerin-targeting of PEDV S antigen significantly enhanced CD4 T cell immunity. • Systemic Langerin-targeting of PEDV S antigen significantly augmented serum IgG and IgA responses., Porcine epidemic diarrhea virus (PEDV) first emerged in the United States in 2013 causing high mortality and morbidity in neonatal piglets with immense economic losses to the swine industry. PEDV is an alpha-coronavirus replicating primarily in porcine intestinal cells. PEDV vaccines are available in Asia and Europe, and conditionally-licensed vaccines recently became available in the United States but the efficacies of these vaccines in eliminating PEDV from swine populations are questionable. In this study, the immunogenicity of a subunit vaccine based on the spike protein of PEDV, which was directly targeted to porcine dendritic cells (DCs) expressing Langerin, was assessed. The PEDV S antigen was delivered to the dendritic cells through a single-chain antibody specific to Langerin and the targeted cells were stimulated with cholera toxin adjuvant. This approach, known as “dendritic cell targeting,” greatly improved PEDV S antigen-specific T cell interferon-γ responses in the CD4posCD8pos T cell compartment in pigs as early as 7 days upon transdermal administration. When the vaccine protein was targeted to Langerinpos DCs systemically through intramuscular vaccination, it induced higher serum IgG and IgA responses in pigs, though these responses require a booster dose, and the magnitude of T cell responses were lower as compared to transdermal vaccination. We conclude that PEDV spike protein domains targeting Langerin-expressing dendritic cells significantly increased CD4 T cell immune responses in pigs. The results indicate that the immunogenicity of protein subunit vaccines can be greatly enhanced by direct targeting of the vaccine antigens to desirable dendritic cell subsets in pigs.

Porcine circovirus type 2 (PCV2) is the causative agent of an economically significant collection of disease syndromes in pigs, now known as porcine circovirus associated diseases (PCVADs) in the United States or porcine circovirus diseases (PCVDs) in Europe. Inactivated and subunit vaccines based on PCV2a genotype are commercially available and have been shown to be effective at decreasing mortality and increasing growth parameters in commercial swine herds. Since 2003, there has been a drastic global shift in the predominant prevalence of PCV2b genotype in swine populations, concurrently in most but not all cases with increased severity of clinical disease. Although the current commercial vaccines based on PCV2a do confer cross-protection against PCV2b, novel experimental vaccines based on PCV2b genotype such as modified live-attenuated vaccines are being developed and may provide a superior protection and reduce vaccine costs. In this review, we discuss the current understanding of the impact of PCV2 infection on the host immune response, review the efficacy of the currently available commercial PCV2 vaccines in experimental and field conditions, and provide insight into novel experimental approaches that are useful in the development of next generation vaccines against PCV2.

Hepatitis E virus (HEV) is an important but extremely understudied pathogen. The mechanisms of HEV replication and pathogenesis are poorly understood, and a vaccine against HEV is not yet available. HEV is classified in the family Hepeviridae consisting of at least four recognized major genotypes. Genotypes 1 and 2 HEV are restricted to humans and associated with epidemics in developing countries, whereas genotypes 3 and 4 HEV are zoonotic and responsible for sporadic cases worldwide. The identification and characterization of a number of animal strains of HEV from pigs, chickens, rabbits, rats, mongoose, deer, and possibly cattle and sheep have significantly broadened the host range and diversity of HEV. The demonstrated ability of cross-species infection by some animal strains of HEV raises public health concerns for zoonotic HEV infection. Pigs are a recognized reservoir for HEV, and pig handlers are at increased risk of zoonotic HEV infection. Sporadic cases of hepatitis E have been definitively linked to the consumption of raw or undercooked animal meats such as pig livers, sausages, and deer meats. In addition, since large amounts of viruses excreted in feces, animal manure land application and runoffs can contaminate irrigation and drinking water with concomitant contamination of produce or shellfish. HEV RNA of swine origin has been detected in swine manure, sewage water and oysters, and consumption of contaminated shellfish has also been implicated in sporadic cases of hepatitis E. Therefore, the animal strains of HEV pose not only a zoonotic risk but also food and environmental safety concerns.

A highly pneumovirulent strain of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was isolated from a pig exhibiting typical PRRS in the early 90s. While passaging the virus in monkey kidney cells, we identified a large spontaneous deletion of a 435-bp in the nsp2 gene. To assess the biological significance of this spontaneous deletion, we first determined the full-length genomic sequence of this virus and established a DNA-launched infectious clone of the passage 14 virus containing the 435-bp nsp2 deletion (designated as pIR-VR2385-CA). The full-length viral genome engineered with two ribozyme elements at both ends was placed under the control of the eukaryotic CMV promoter. The infectious virus was successfully rescued from pIR-VR2385-CA DNA-transfected BHK-21 cells. To characterize the biological and pathological significance of this large nsp2 deletion, we subsequently constructed another DNA-launched infectious clone, pIR-VR2385-R, in which we restored the deleted 435-bp nsp2 sequence back to the pIR-VR2385-CA backbone. The growth characteristics of the two rescued viruses (VR2385-CA and VR2385-R) were compared, and the results showed that the VR2385-CA virus with the nsp2 deletion replicated more efficiently in vitro (1.0–1.5 log titer higher) than the VR2385-R virus with the restored nsp2 sequence but the VR2385-CA virus exhibited a significantly reduced serum viral RNA load in vivo . A comparative pathogenicity study in pigs ( n = 10) revealed that the nsp2 deletion had no effect on virus virulence, and the restored nsp2 sequence in the VR2385-R virus remains stable during virus replication in pigs. The results from this study indicates that the spontaneous nsp2 deletion plays a role for enhanced PRRSV replication in vitro but has no effect on the pathogenicity of the virus.

Hepatitis E virus (HEV) is an important human pathogen. At least four recognized and two putative genotypes of mammalian HEV have been reported: genotypes 1 and 2 are restricted to humans whereas genotypes 3 and 4 are zoonotic. The current experimental vaccines are all based on a single strain of HEV, even though multiple genotypes of HEV are co-circulating in some countries and thus an individual may be exposed to more than one genotype. Genotypes 3 and 4 swine HEV is widespread in pigs and known to infect humans. Therefore, it is important to know if prior infection with a genotype 3 swine HEV will confer protective immunity against subsequent exposure to genotypes 3 and 4 human and swine HEV. In this study, specific-pathogen-free pigs were divided into 4 groups of 6 each. Pigs in the three treatment groups were each inoculated with a genotype 3 swine HEV, and 12 weeks later, challenged with the same genotype 3 swine HEV, a genotype 3 human HEV, and a genotype 4 human HEV, respectively. The control group was inoculated and challenged with PBS buffer. Weekly sera from all pigs were tested for HEV RNA and IgG anti-HEV, and weekly fecal samples were also tested for HEV RNA. The pigs inoculated with swine HEV became infected as evidenced by fecal virus shedding and viremia, and the majority of pigs also developed IgG anti-HEV prior to challenge at 12 weeks post-inoculation. After challenge, viremia was not detected and only two pigs challenged with swine HEV had 1-week fecal virus shedding, suggesting that prior infection with a genotype 3 swine HEV prevented pigs from developing viremia and fecal virus shedding after challenges with homologous and heterologous genotypes 3 and 4 HEV. The results from this study have important implications for future development of an effective HEV vaccine.

Genotypes 1 and 2 hepatitis E virus (HEV) infect only humans whereas genotypes 3 and 4 HEV infect both humans and pigs. To evaluate the mechanism of cross-species HEV infection between humans and swine, in this study we constructed five intergenotypic chimeric viruses and tested for their infectivity in vitro and in pigs. We demonstrated that chimeric viruses containing the ORF2 capsid gene either alone or in combination with its adjacent 5' junction region (JR) and 3' noncoding region (NCR) from a genotype 4 human HEV in the backbone of a genotype 3 swine HEV are replication-competent in Huh7 cells and infectious in HepG2/C3A cells and in pigs, and thus supporting the hypothesis that genotypes 3 and 4 human HEV are of swine origin. However, chimeric viruses containing the JR+ORF2+3' NCR of genotypes 3 or 4 HEV in the backbone of genotype 1 human HEV failed to infect pigs, suggesting that other genomic regions such as 5' NCR and ORF1 may also be involved in HEV cross-species infection. The results from this study provide the first experimental evidence of the exchangeability of the capsid gene between genotype 3 swine HEV and genotype 4 human HEV, and have important implications for understanding the mechanism of HEV cross-species infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important swine pathogen. Since its discovery in the early 1990 s, tremendous progresses have been made in understanding the molecular biology and pathogenesis of PRRSV. Although modified live-attenuated vaccines (MLVs) and inactivated vaccines against PRRSV have been available for more than a decade, the disease remains difficult to control. The efficacies of these vaccines especially against heterologous strains remain questionable: the MLVs were generally effective against homologous strains but variable in success against heterologous strains, and the outcomes of inactivated vaccines in the field are not very promising. With the development of PRRSV reverse genetics systems and the acquisition of new understanding on anti-PRRSV immunity, rational design of the next generation of PRRSV vaccines can now be explored. In this review, we discussed the recent advances in anti-PRRSV immunity and vaccinology, the recent progresses in PRRSV vaccine development particularly the reverse genetics system-based vaccine development, and provided a perspective on potential novel strategies and approaches that may be applicable to the development of the next generation of PRRSV vaccines.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD) in pigs. The open reading frame (ORF) 3 of PCV2 reportedly induces apoptosis and is associated with PCV2 pathogenicity. In this study, we first created an ORF3-null PCV2 mutant (muPCV2) by site-directed mutagenesis and demonstrated that the dimerized plasmid DNA of muPCV2 clone is infectious when injected intramuscularly (I.M.) into pigs. Subsequently, by using a well-characterized pig model we compared the pathogenicity of the muPCV2 and the wildtype PCV2. Thirty-one pigs were divided into 3 groups of 11, 10, and 10 each: group 1 pigs were each inoculated I.M. with PBS buffer as negative controls, group 2 pigs each with 200 microg of muPCV2 infectious DNA clone, and group 3 pigs each with 200 microg of wildtype PCV2 infectious DNA clone. Blood was collected prior to inoculation and weekly thereafter, and tested for PCV2 antibodies by ELISA and serum viral DNA loads by quantitative PCR. All pigs were necropsied at 35 days post-inoculation. The results showed that pigs inoculated with muPCV2 had a delayed seroconversion and lower serum viral load. However, there was no significant difference in the average scores of the histological or gross lesions or the amount of PCV2-specific antigen in tissues between wildtype PCV2- and muPCV2-inoculated groups. Thus, the data from this study do not fully support the conclusion of a previous report regarding PCV2 attenuation by abrogation of ORF3 although the results did show that ORF3 is dispensable for PCV2 replication in pigs.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen. It has been shown that treatment of PCV2-infected cells with interferon gamma (INF-gamma) or alpha (IFN-alpha) increases PCV2 replication in vitro, and that an interferon-stimulated response element (ISRE)-like sequence was identified in the PCV2 genome. To determine if the ISRE is involved in viral response to IFNs, several ISRE mutants of PCV2 were created by serial mutations of the ISRE sequence. Treatment of ISRE mutants-infected cells with IFN-gamma or IFN-alpha showed a progressive diminishment in the enhanced viral replication in correlation with increasing alterations to the ISRE sequence. To determine if the ISRE was necessary and sufficient for IFN-mediated enhancement of PCV2 replication, DNA fragments spanning the ISRE-containing promoter region of the rep gene from wildtype and ISRE-mutant PCV2 were tested in a luciferase reporter gene system. 3D4/31 cells transfected with reporter gene constructs were treated with IFN-gamma or IFN-alpha, respectively. The results showed that the untreated controls for both ISRE-mutant and wildtype PCV2 had higher levels of luciferase reporter activity than IFN-treated samples, indicating that, when removed from the context of whole viral genome, the ISRE-like activity of the sequence was lost. Furthermore, treatment with IFNs diminished the promoter activity regardless of the mutation, suggesting that other cis elements in the viral genome may be required for regulating the ISRE-mediated gene transcription. In conclusion, the PCV2 ISRE, when present in the context of intact virus but not in isolation, influences the interferon-mediated enhancement of PCV2 replication in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease, and has a complicated virus–host immunomodulation that often leads to a weak Th2 immune response and viral persistence. In this study, we identified a Src homology 3 (SH3) binding motif, PxxPxxP, that is conserved within the N protein of PRRSV strains. Subsequently, we identified five host cellular proteins [signal transducing adaptor molecule (STAM)I, TXK tyrosine kinase (TXK), protein tyrosine kinase fyn (Fyn), hematopoietic cell kinase (Hck), and cortactin] that interact with this SH3 motif. We demonstrated that binding of SH3 proteins with PRRSV N protein depends on at least one intact PxxP motif as disruption of P53 within the motif significantly reduced interaction of each of the 5 proteins. The first PxxP motif appears to be more important for STAMI–N protein interactions whereas the second PxxP motif was more important for Hck interaction. Both STAMI and Hck interactions with PRRSV N protein required an unhindered C-terminal domain as the interaction was only observed with STAMI and Hck proteins with N-terminal but not C-terminal fluorescent tags. We showed that the P56 residue within the SH3 motif is critical for virus lifecycle as mutation resulted in a loss of virus infectivity, however the P50 and P53 mutations did not abolish virus infectivity suggesting that these highly conserved proline residues within the SH3 motif may provide a selective growth advantage through interactions with the host rather than a vital functional element. These results have important implications in understanding PRRSV–host interactions.

Highlights • The genetic variability of PRRSV during serial passage in PCV2-infected pigs was investigated. • PRRSV structural genes ORF6 and ORF7 mutated at various degrees over time in vivo. • A significantly higher mutation rate was observed when pigs were co-infected with PCV2., Porcine reproductive and respiratory syndrome virus (PRRSV) has a high degree of genetic and antigenic variability. The purpose of this study was to determine if porcine circovirus type 2 (PCV2) infection increases genetic variability of PRRSV during serial passages in pigs and to determine if there is a difference in the PRRSV mutation rate between pigs concurrently infected with PCV2a or PCV2b. After 8 consecutive passages of PRRSV alone (group 1), PRRSV with PCV2a (group 2), or PCV2b (group 3) in pigs, the sequences of PRRSV structural genes for open reading frame (ORF) 5, ORF6, ORF7 and the partial non-structural protein gene (Nsp) 2 were determined. The total number of identified amino acid mutations in ORF5, ORF6, ORF7 and Nsp2 sequences was 30 for PRRSV infection only, 63 for PRRSV/PCV2a concurrent infection, and 77 for PRRSV/PCV2b concurrent infection when compared with the original VR2385 virus used to infect the passage 1 pigs. Compared to what occurred in pigs infected with PRRSV only, the mutation rates in ORF5 and ORF6 were significantly higher for concurrent PRRSV/PCV2b infected pigs. The PRRSV/PCV2a pigs had a significantly higher mutation rate in ORF7. The results from this study indicated that, besides ORF5 and Nsp2, the PRRSV structural genes ORF6 and ORF7 were shown to mutate at various degrees when the PRRSV was passaged over time in vivo. Furthermore, a significantly higher mutation rate of PRRSV was observed when pigs were co-infected with PCV2 highlighting the importance of concurrent infections on PRRSV evolution and control.

The discovery of porcine circovirus type 2 (PCV2) in 1998 initiated intensive research on arguably the most economically important pathogen facing the global swine industry today. PCV2 infection is now widespread worldwide, and increasing numbers of disease conditions have been linked to PCV2 infection in pigs. In this special issue of Virus Research, leading experts in the field review the history, epidemiology, transmission, clinical and pathological features, immunology, pathogenesis, molecular biology and vaccine development of PCV2 and porcine circovirus associated disease (PCVAD). In addition, circovirus-like DNA sequences recently identified from humans and other animal species and their biological significances are also reviewed. The articles in this special issue identify gaps in current PCV2 research and offer insights for the direction of future research.

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

The pathogenesis of HEV infection responsible for liver pathology and clinical disease is not well understood. The main target for the virus is the hepatocyte, where it replicates and is released to bile and gastrointestinal tract. Viremia is regularly seen during the virus replication. The exact mechanism of hepatocytic death is uncertain. In experimentally infected non-human primates, the peak of liver lesions, measured by alanine aminotransferase activity elevation, is concordant with the virus disappearance from stool at the time of dynamic humoral immune response; the role of cellular immunity has not been researched adequately, especially HEV-specific immune response in the liver. Non-human primates (chimpanzees, rhesus and cynomolgus macaques) are most widely used animal models for the study of HEV infection, its pathogenesis and vaccine trials. Several other animal models including pigs, rabbits and chickens have recently been established for the study of various aspects of HEV infection. Infectivity studies in susceptible primates were of significance in molecular studies of the virus itself. Preclinical vaccine trials with the use of various recombinant HEV capsid proteins and viral DNA established basic platform for formulation of HEV vaccine applied in HEV-endemic regions (China, Nepal).

In this study, an improved DNA-launched (plasmid DNA transfection-based) reverse genetics system with reduced cost and labor was developed for porcine reproductive and respiratory syndrome virus (PRRSV) by introduction of ribozyme elements at both termini of the viral genomic cDNA that were placed under the control of a eukaryotic hybrid promoter. The rescue efficacy of PRRSV with this system was approximately 10-50-fold higher than the in vitro-transcribed RNA-based system and the traditional DNA-launched system without the engineered ribozyme elements, as determined by reporter GFP level in transfected cells and the peak titer of the recovery virus. By using this new reverse genetics system, we identified and characterized a porcine monocytic cell line, 3D4/31, capable of supporting PRRSV replication, progeny virion production, and attachment on the cell surface. The establishment of this improved reverse genetic system and the identification of a porcine monocytic cell line supporting PRRSV replication will aid future studies of host-virus interaction of PRRSV.