Assessment of the Impact of the Recombinant Porcine Reproductive and Respiratory Syndrome Virus Horsens Strain on the Reproductive Performance in Pregnant Sows.

This study assessed the impact of a PRRSV (porcine reproductive and respiratory syndrome virus) recombinant strain (Horsens strain) on the reproductive performance of naïve pregnant sows in the last third of gestation. Fifteen sows were included: four negative reproductive controls (NTX), five infected with a PRRSV-1 field strain (Olot/91, T01), and six infected with the recombinant PRRSV-1 strain (Horsens strain, T02). Piglets were monitored until weaning. Reproductive performance was the primary variable. In sows, viremia and nasal shedding (T01 and T02 groups), and, in piglets, viral load in blood and in lungs, as well as macroscopic lung lesions (T01 and T02 groups), were the secondary variables. The reproductive performance results were numerically different between the two challenged groups. Moreover, viral loads in blood were 1.83 × 106 ± 9.05 × 106 copies/mL at farrowing, 1.05 × 107 ± 2.21 × 107 copies/mL at weaning from piglets born from T01 animals and 1.64 × 103 ± 7.62 × 103 copies/mL at farrowing, 1.95 × 103 ± 1.17 × 104 copies/mL at weaning from piglets born from T02 sows. Overall, 68.8% of T01 piglets and 38.1% of T02 piglets presented mild lung lesions. In conclusion, the results suggest that Horsens strain is less virulent than the field strain Olot/91 under these experimental conditions.

Construction and efficacy evaluation of novel swine leukocyte antigen (SLA) class I and class II allele-specific poly-T cell epitope vaccines against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico. Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge.

With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) γ secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge.

Cytotoxic T lymphocyte epitopes identified from a contemporary strain of porcine reproductive and respiratory syndrome virus enhance CD4+CD8+ T, CD8+ T, and γδ T cell responses.

Immuno-stimulatory class I-restricted cytotoxic T lymphocytes (CTL) epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) are important for vaccine development. In this study we first determined the expression frequency of swine leukocyte antigen (SLA) class I alleles in commercial pigs in the United States. The SLA genotyping result allowed us to predict potential CTL epitopes from a contemporary strain of PRRSV (RFLP 1-7-4) by using bioinformatic tools. The predicted epitopes were then evaluated in an ex vivo stimulation assay with peripheral blood mononuclear cells isolated from pigs experimentally-infected with PRRSV. Using flow-cytometry analysis, we identified a number of immuno-stimulatory CTL epitopes, including two peptides from GP3 and two from Nsp9 that significantly improved both degranulation marker CD107a and IFN-γ production in cytotoxic CD4+CD8+ T cells, CD8+ T cells, and γδ T cells, and two peptides that inhibited IFN-γ production. These CTL epitopes will aid future vaccine development against PRRSV.

Immune response development after vaccination of 1-day-old naïve pigs with a Porcine Reproductive and Respiratory Syndrome 1-based modified live virus vaccine.

BACKGROUND: The development of the innate and adaptive immune responses to Porcine reproductive and respiratory syndrome virus (PRRSV) after vaccination of 1 day-old pigs with a PRRSV-1 based modified live virus (MLV) vaccine by intramuscular (IM) and intranasal (IN) routes was characterised, before and after challenge with a heterologous PRRSV-1 isolate at 18 weeks post-vaccination. Twenty-five PRRSV-seronegative piglets were used. At 1 day of age, pigs were administered with a single dose of vaccine via the IM (n = 10) or the IN route (n = 10). Control group (n = 5) received saline solution. After vaccination, pigs were bled at days 3, 7, 28, 56, 83, 113 and 125. Levels of cytokines IL-10, IL-8, IFN-α (measured by ELISA tests of serum), TNF-α and IFN-γ (measured by ELISA and ELISPOT, respectively, from stimulated peripheral blood mononuclear cells), and serum neutralising antibodies (NA) to the vaccine strain, were measured. RESULTS: The induction of IL-10 was rare, indicating that IL-10 mediated immunomodulation/immune dysfunction was not a feature of this vaccine or of the challenge virus. IL-8 was detected in only two pigs following vaccination, but in the majority of pigs after challenge, indicating that their ability to produce an innate immune response was not impaired. TNF-α was not detected in any vaccinated pigs until day 83. After challenge, only a minority of pigs produced TNF-α. IFN-α was detected in all vaccinated pigs following vaccination, indicating the potential for development of an effective Th1 adaptive immune response. IFN-γ-secreting cells were detected in all vaccinated pigs after vaccination. NA to the vaccine strain were first detected at day 56 in pigs vaccinated by both routes, and remained at similar levels until challenge. After challenge, a boost in NA was observed. The efficacy of the vaccine was demonstrated by reduction of viraemia and nasal shedding after challenge. CONCLUSIONS: The administration of a PRRSV-1 based MLV vaccine to 1 day-old piglets was able to induce an immune response characterised by: (1) undetectable or low levels of IL-10, IL-8 and TNF-α, (2) an increase in IFN-α expression within the first seven days, (3) a gradual increase in the number of antigen-specific IFN-γ-secreting cells, and (4) induction of detectable NA. After challenge with a heterologous strain, there was a rapid boost in NA titres, indicating a priming effect of the vaccine.

Vaccination of 1-day-old pigs with a porcine reproductive and respiratory syndrome virus (PRRSV) modified live attenuated virus vaccine is able to overcome maternal immunity.

BACKGROUND: The objective of the study was to evaluate the influence of maternally derived antibodies (MDA) on the efficacy of a PRRSV-1 based attenuated vaccine, when administered in 1 day-old piglets by the intramuscular route. The protective immunity of the modified live virus vaccine was evaluated in pigs born from seropositive sows, vaccinated at 1 day of age, upon inoculation with a PRRSV-1 isolate. The animals were challenged when the levels of MDAs detected by seroneutralization test (SNT) in the non-vaccinated control group became undetectable (10 weeks after vaccination). RESULTS: A protective effect of vaccination was observed since a significant reduction of viral load in serum compared to the control group was detected in all sampling days after challenge; efficacy was supported by the significant reduction of nasal and oral shedding as well as in rectal temperatures. Clinical signs were not expected after the inoculation of a PRRSV-1 subtype 1 challenge strain. However, the challenge virus was able to develop fever in 61% of the control pigs. Vaccination had a positive impact on rectal temperatures since the percentage of pigs that had fever at least once after challenge was reduced to 31% in vaccinated animals, and control pigs had significantly higher rectal temperatures than vaccinated pigs 3 days post-challenge. The lack of a vaccination effect in body weight gain was probably due to the short evaluation period after challenge (10 days). In the vaccinated group, 9/16 pigs (56%) experienced an increase in ELISA S/P ratio from the day of vaccination to 67 days post-vaccination. All vaccinated pigs were seropositive before challenge, indicating the development of an antibody response following vaccination even in the face of MDAs. In contrast to ELISA results, only 2/16 vaccinated pigs developed neutralizing antibodies detectable by a SNT that used a subtype 1 MA-104 adapted strain. Even in the absence of SN antibodies, vaccinated pigs were protected from challenge with a heterologous strain. The role of cell-mediated immunity should be considered, if protection was not mediated by SN antibodies only. CONCLUSIONS: The efficacy of the attenuated PRRSV-1 vaccine in 1-day-old pigs seropositive to PRRSV prior to a PRRSV-1 challenge was demonstrated by improvement of clinical, virological and immunological variables. With the current experimental design, maternal immunity did not interfere with the development of a protective immune response against a PRRSV-1 challenge, after vaccination of 1 day-old pigs. Confirmation of these results under field conditions will be needed.

Vaccination against porcine reproductive and respiratory syndrome virus (PRRSV) reduces the magnitude and duration of viremia following challenge with a virulent heterologous field strain.

Forty PRRS-negative, three week-old weaned pigs were randomized into two groups in separate rooms and inoculated with a modified live PRRS vaccine (Fostera® PRRS) or control (PBS). Four weeks after vaccination pigs were rehoused in a single room and challenged intranasally and intramuscularly with virulent PRRSV strain NADC20. Timed serum samples were collected and titrated for PRRS virus and anti-PRRS virus antibodies. The study concluded when ≥80% of the pigs in the control group were determined to be virus negative (27days post-challenge). Mean duration of viremia was significantly lower (p=0.0327) for vaccinated pigs compared to non-vaccinated pigs. A significant reduction (p≤0.0053) in mean post-challenge viremia titer was seen in vaccinates compared to non-vaccinates from days 8 through 22 post-challenge. At the individual pig level, no pigs in the vaccinated group had detectible PRRSV in serum at the end of the study (27days post-challenge), while 15% of non-vaccinated pigs remained positive for virus.

Chimeric porcine reproductive and respiratory syndrome virus containing shuffled multiple envelope genes confers cross-protection in pigs.

The extensive genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains is a major obstacle for vaccine development. We previously demonstrated that chimeric PRRSVs in which a single envelope gene (ORF3, ORF4, ORF5 or ORF6) was shuffled via DNA shuffling had an improved heterologous cross-neutralizing ability. In this study, we incorporate all of the individually-shuffled envelope genes together in different combinations into an infectious clone backbone of PRRSV MLV Fostera(®) PRRS. Five viable progeny chimeric viruses were rescued, and their growth characteristics were characterized in vitro. In a pilot pig study, two chimeric viruses (FV-SPDS-VR2,FV-SPDS-VR5) were found to induce cross-neutralizing antibodies against heterologous strains. A subsequent vaccination/challenge study in 72 pigs revealed that chimeric virus FV-SPDS-VR2 and parental virus conferred partial cross-protection when challenged with heterologous strains NADC20 or MN184B. The results have important implications for future development of an effective PRRSV vaccine that confers heterologous protection.

Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) was reported in the late 1980s. PRRS still is a huge economic concern to the global pig industry with a current annual loss estimated at one billion US dollars in North America alone. It has been 20 years since the first modified live-attenuated PRRSV vaccine (PRRSV-MLV) became commercially available. PRRSV-MLVs provide homologous protection and help in reducing shedding of heterologous viruses, but they do not completely protect pigs against heterologous field strains. There have been many advances in understanding the biology and ecology of PRRSV; however, the complexities of virus-host interaction and PRRSV vaccinology are not yet completely understood leaving a significant gap for improving breadth of immunity against diverse PRRS isolates. This review provides insights on immunization efforts using infectious PRRSV-based vaccines since the 1990s, beginning with live PRRSV immunization, development and commercialization of PRRSV-MLV, and strategies to overcome the deficiencies of PRRSV-MLV through use of replicating viral vectors expressing multiple PRRSV membrane proteins. Finally, powerful reverse genetics systems (infectious cDNA clones) generated from more than 20 PRRSV isolates of both genotypes 1 and 2 viruses have provided a great resource for exploring many innovative strategies to improve the safety and cross-protective efficacy of live PRRSV vaccines. Examples include vaccines with diminished ability to down-regulate the immune system, positive and negative marker vaccines, multivalent vaccines incorporating antigens from other porcine pathogens, vaccines that carry their own cytokine adjuvants, and chimeric vaccine viruses with the potential for broad cross-protection against heterologous strains. To combat this devastating pig disease in the future, evaluation and commercialization of such improved live PRRSV vaccines is a shared goal among PRRSV researchers, pork producers and biologics companies.

Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction.

Within a few years of its emergence in the late 1980s, the PRRS virus had spread globally to become the foremost infectious disease concern for the pork industry. Since 1994, modified live-attenuated vaccines against porcine reproductive and respiratory syndrome virus (PRRSV-MLV) have been widely used, but have failed to provide complete protection against emerging and heterologous field strains of the virus. Moreover, like many other MLVs, PRRSV-MLVs have safety concerns including vertical and horizontal transmission of the vaccine virus and several documented incidences of reversion to virulence. Thus, the development of efficacious inactivated vaccines is warranted for the control and eradication of PRRS. Since the early 1990s, researchers have been attempting to develop inactivated PRRSV vaccines, but most of the candidates have failed to elicit protective immunity even against homologous virus challenge. Recent research findings relating to both inactivated and subunit candidate PRRSV vaccines have shown promise, but they need to be pursued further to improve their heterologous efficacy and cost-effectiveness before considering commercialization. In this comprehensive review, we provide information on attempts to develop PRRSV inactivated and subunit vaccines. These includes various virus inactivation strategies, adjuvants, nanoparticle-based vaccine delivery systems, DNA vaccines, and recombinant subunit vaccines produced using baculovirus, plant, and replication-deficient viruses as vector vaccines. Finally, future directions for the development of innovative non-infectious PRRSV vaccines are suggested. Undoubtedly there remains a need for novel PRRSV vaccine strategies targeted to deliver cross-protective, non-infectious vaccines for the control and eradication of PRRS.

A single amino acid deletion in the matrix protein of porcine reproductive and respiratory syndrome virus confers resistance to a polyclonal swine antibody with broadly neutralizing activity.

Assessment of virus neutralization (VN) activity in 176 pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) identified one pig with broadly neutralizing activity. A Tyr-10 deletion in the matrix protein provided escape from broad neutralization without affecting homologous neutralizing activity. The role of the Tyr-10 deletion was confirmed through an infectious clone with a Tyr-10 deletion. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection.

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. FINDINGS: We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. CONCLUSION: Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

Interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus.

Innate immunity is the first line of defense against viral infection, and in turn, viruses have evolved to evade host immune surveillance. As a result, viruses may persist in host and develop chronic infections. Type I interferons (IFN-α/ß) are among the most potent antiviral cytokines triggered by viral infections. Porcine reproductive and respiratory syndrome (PRRS) is a disease of pigs that is characterized by negligible induction of type I IFNs and viral persistence for an extended period. For IFN production, RIG-I/MDA5 and JAK-STAT pathways are two major signaling pathways, and recent studies indicate that PRRS virus is armed to modulate type I IFN responses during infection. This review describes the viral strategies for modulation of type I IFN responses. At least three non-structural proteins (Nsp1, Nsp2, and Nsp11) and a structural protein (N nucleocapsid protein) have been identified and characterized to play roles in the IFN suppression and NF-κB pathways. Nsp's are early proteins while N is a late protein, suggesting that additional signaling pathways may be involved in addition to the IFN pathway. The understanding of molecular bases for virus-mediated modulation of host innate immune signaling will help us design new generation vaccines and control PRRS.

A brief review of CD163 and its role in PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) infects fully differentiated cells of the monocyte/macrophage lineage. Recently, CD163 was shown to be a cellular receptor capable of mediating infection of otherwise PRRSV non-permissive cell lines. CD163 is a macrophage differentiation antigen belonging to the scavenger receptor cysteine-rich (SRCR) family of membrane proteins. We provide a brief review of current knowledge regarding CD163 in relation to PRRSV infection, and propose a structure-based prediction of amino acid sequences involved in PRRSV interaction.

Porcine reproductive and respiratory syndrome virus as a vector: immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which is characterized by late-term abortions in sows and respiratory disease in young pigs. Using an infectious cDNA clone of North American PRRSV strain P129, the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6 (TRS6) were inserted between ORFs 1b and 2a, yielding a general purpose expression vector. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Upon transfection of cells with this plasmid, PRRSV infection was initiated and progeny virus "P129-GFP" was obtained. Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial in vitro passages. Subsequently, a capsid (C) protein gene was cloned from porcine circovirus type 2 (PCV2) recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS) and inserted into the PRRSV infectious clone vector, generating virus "P129-PCV". To determine the immunogenicity of the recombinant viruses, pigs were immunized intramuscularly with P129-WT (wild-type), P129-GFP, or P129-PCV2. By 5 weeks post-infection, specific antibody responses to GFP and PCV2 capsid were elicited. This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.

Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity.

Developing a vaccine that can differentiate infected and vaccinated animals (DIVA) is a new challenge in the design of a vaccine for porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein 2 (nsp2) is the single largest viral product, and it has multiple roles in polypeptide processing and replication complex formation. Using reverse genetics and an infectious PRRSV cDNA clone, we constructed several deletion mutants in the non-essential region of nsp2. One mutant, which has a 131 amino acid deletion within a relatively conserved region of nsp2, was recovered and found to produce a viable virus. The deleted region was replaced with a peptide tag encoding eight amino acids. A recombinant virus containing the 131 amino acid deletion was found to produce normal virus yields in MARC-145 cells and porcine alveolar macrophages (PAM); however, gross and micro-histopathology showed that the virus was less virulent in pigs. The 131 amino acid peptide was expressed as a recombinant protein and used to coat enzyme-linked immunosorbent assay (ELISA) plates. This peptide was recognized by sera from pigs infected with wild-type virus, but not by sera from pigs infected with the deletion mutant. The results from this study show that nsp2 is an important target for the development of marker vaccines and for virus attenuation.

Functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association.

PRRSV (porcine reproductive and respiratory syndrome virus) nucleocapsid (N) protein is the most abundant structural protein of the virus. During infection, the N protein is specifically localized to the nucleus and nucleolus in addition to its normal cytoplasmic distribution. Previously, a nuclear localization signal (NLS, 41-PGKK(N/S)KKKN)-null mutant virus (41-PGGGNKKKN) showed reduced viremia and increased production of neutralizing antibodies in infected pigs. However, the mutagenized NLS underwent strong selection pressure in the pig that resulted in partial or complete reversion and reacquisition of NLS function, and thus the biological effect of the NLS-null mutation needed further investigation. In the present study, a total of 9 "reversion resistant" mutants were generated by amino acid deletions and substitutions using an infectious cDNA clone. Two mutant clones (PG--SKKKS and PG--S-KKS) that produced progeny viruses were genetically stable for at least 20 passages in cell culture. Infection of pigs with those mutants induced neutralizing antibodies to higher titers than with wild-type virus. Both mutant viruses induced viremia of lower titer and of shorter duration than wild-type virus. RT-PCR from tonsils showed that both mutants persisted at a reduced level. Virus transmission to contact pigs was also lower in the mutant virus infected groups. No reversion to functional NLS was detected in either mutant from any pig. These data demonstrate that N protein nuclear localization is indeed associated with viral pathogenesis and host response to PRRS.

Expression and stability of foreign tags inserted into nsp2 of porcine reproductive and respiratory syndrome virus (PRRSV).

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the single largest protein produced during infection. The cDNA of the pCMV-129 infectious PRRSV clone was modified for accepting foreign tags by first creating unique Mlu I and SgrA I restrictions sites at nucleotide (nt) positions 3219 and 3614, respectively, within the C-terminal region of nsp2. cDNAs encoding oligo- and polypeptide tags, including FLAG, enhanced green fluorescent protein (EGFP) and luciferase were inserted into the newly created restriction sites. The results showed that only the EGFP-containing genomes were properly expressed and produced virus. EGFP fluorescence, but not EGFP immunoreactivity, was lost during passage of recombinant EGFP viruses in culture. Sequencing of a fluorescent-negative EGFP virus showed that the EGFP remained intact, except for the appearance of arginine to cysteine mutation at position 96, which may interfere with chromophore formation or function.

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses.

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.