The recent emergence of a unique group of North American type 1 porcine reproductive and respiratory syndrome virus (PRRSV) in the United States presents new disease control problems for a swine industry that has already been impacted seriously by North American type 2 PRRSV. In this study, a full-length cDNA infectious clone was generated from a low-virulence North American type 1 PRRSV isolate, SD01-08. In vitro studies demonstrated that the cloned virus maintained growth properties similar to those of the parental virus. Virological, pathological, and immunological observations from animals challenged with cloned viruses were similar to those from animals challenged with the parental virus and a modified live virus vaccine. To further explore the potential use as a viral backbone for expressing foreign genes, the green fluorescent protein (GFP) was inserted into a unique deletion site located at amino acid positions 348 and 349 of the predicted Nsp2 region in the virus, and expression of the Nsp2-GFP fusion protein was visualized by fluorescent microscopy. The availability of this North American type 1 infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of this group of type 1 PRRSV in the United States.
Porcine reproductive and respiratory syndrome (PRRS) was a disease first described to occur in the United States in 1987 (12) and in Europe in 1991 (34). Its current economic impact is estimated at $560 million annually (23). The etiologic agent, PRRS virus (PRRSV), was first isolated in The Netherlands in 1991 and is represented by the European prototypic strain, Lelystad virus (LV) (34). In the United States, PRRSV was first isolated in 1992, and the prototypic strain was designated VR-2332 (2, 5). PRRSV is classified in the family Arteriviridae, which includes equine arteritis virus, lactate dehydrogenase-elevating virus, and simian hemorrhagic fever virus (28).
PRRSV is a small, enveloped virus containing a single positive-stranded RNA genome. The genome is about 15 kb in length and contains nine open reading frames. The protease and replicase-associated genes, ORF1a and ORF1b, situated at the 5′ end of the genome, represent nearly 75% of the viral genome. The ORF1ab polyprotein of PRRSV is predicted to be cleaved into 13 nonstructural protein products, Nsp1α, Nsp1β, and Nsp2 to Nsp12 (6, 29, 31, 33). The 3′ end of the genome encodes four membrane-associated glycoproteins (GP2a, GP3, GP4, and GP5, encoded by subgenomic [sg] mRNAs 2 to 5), two unglycosylated membrane proteins (2b and M, encoded by sg mRNAs 2 and 6), and a nucleocapsid protein (N, encoded by sg mRNA 7) (1, 13, 14, 16, 17, 20, 22, 35).
PRRSV consists of two major genotypes, the European genotype (type 1) and the North American genotype (type 2), formerly located on different continents. More recently, type 1 PRRSV isolates (North American type 1) have been identified in U.S. swine herds. Previous studies (7, 26) indicate that this group of viruses possesses unique antigenic and genetic characteristics that are distinct from those of typical North American- and European-type PRRSV. A unique 51-bp deletion has been identified in the immunodominant region of Nsp2. Furthermore, the rapid appearance of this group of viruses since 1999 and the relatively large genetic diversity within the group indicate that this group of viruses is rapidly evolving and becoming well adapted in U.S. swine herds (7).
Several infectious clones have been produced for North American type 2 PRRSV isolates (3, 4, 24, 30). The first infectious clone of type 1 PRRSV was developed using the European prototypic strain, LV, isolated in 1991 in The Netherlands (15). Here, we report an infectious clone of North American type 1 PRRSV, pSD01-08. Compared to the LV infectious clone, pSD01-08 possesses several distinct biological properties: (i) the pSD01-08 infectious clone was derived from a parental strain isolated in the United States in 2001, which represents the most recently characterized North American type 1 PRRSV, (ii) the parental strain SD01-08 was isolated from a group of 8-week-old pigs showing no clinical signs, and (iii) SD01-08 possesses a unique 51-bp deletion in the immunodominant region of Nsp2 (7).
With the availability of this North American type 1 PRRSV infectious clone, we further explored its potential for the expression of foreign genes. The green fluorescent protein (GFP) was engineered into the unique 51-bp deletion site of the Nsp2 region, and the in vitro growth properties of these recombinant marker viruses were evaluated. This GFP-expressing infectious clone provides a system for studying type 1 PRRSV replication and pathogenic mechanisms in living cells.
MATERIALS AND METHODS Virus and cells.A North American type 1 PRRSV isolate, SD01-08, was originally isolated in 2001 in the United States from a group of 8-week-old pigs which were showing no clinical signs of PRRS. BHK-21 cells were used for initial transfection for recovery of virus from in vitro-transcribed RNA. MARC-145 cells were used for virus rescue and subsequent experiments. Porcine alveolar macrophages (PAMs) were obtained by lung lavage of specific-pathogen-free piglets free of PRRSV (36). Cells were maintained in the appropriate medium and incubation conditions were as we described previously (26).
RNA extraction, RT-PCR, and sequencing.MARC-145 cells were infected with plaque-purified viruses at a multiplicity of infection (MOI) of approximately 0.1. After 3 days, the culture supernatant was layered onto a 0.5 M sucrose cushion and centrifuged at 100,000 × g for 14 h in an SW41 rotor (Beckman). RNA was extracted from the pellet by use of a QIAamp viral RNA kit (QIAGEN). To obtain the full-length genome sequence of the parental virus, SD01-08, reverse transcription-PCR (RT-PCR) was performed using primers that we described previously (26). Each RT-PCR product was directly sequenced at least two times from both directions to obtain the consensus sequences. To construct the infectious clone, nine overlapping fragments (Fig. 1) covering the full-length viral genome flanked by unique restriction enzyme sites were amplified by RT-PCR. The forward and reverse oligonucleotides for the RT-PCR amplification were initially designed based on the sequence of LV (GenBank accession number M96262 [18]) and later modified to match the SD01-08 sequence (Table 1). RT-PCR was performed using a method we described before (7). These RT-PCR-amplified fragments were gel purified and cloned in the PCR-Blunt II-Topo vector (Invitrogen). Three clones of each fragment were sequenced, and the clone containing the consensus sequence was used for infectious clone assembly.
FIG. 1.Assembly of the full-length cDNA clone of a European-like PRRSV isolate, SD01-08. In the top scheme, the organization of the viral genome is shown, as are the positions of the unique restriction enzyme sites used for cloning purposes. The numbers 1A, 1B, and 2 through 7 indicate the PRRSV open reading frames. 5′ indicates the 5′ leader, and 3′ indicates the 3′ nontranslated region. The complete viral genome is divided into nine fragments (fragments a to i) flanked by unique restriction enzyme sites, represented by the horizontal lines. As shown in the bottom scheme, these fragments were individually cloned into the pACYC177 vector in alphabetical order. Prior to viral genome assembly, pACYC177 was prepared by inserting a stuffer fragment containing all of the matched restriction sites shown in the top scheme.
TABLE 1.Primers used for RT-PCR amplification
Primer name Sequencea Genome positionb in SD01-08 Fragment a E4849F 5′ GCA TGG CTC TTA AGG CAG AC 4849-4868 E7227R 5′ CAG CTT CAA GGC AGT TGT CA 7208-7227 Fragment b E7139F 5′ TGT TGT GAT CGG CGG TAT TA 7139-7158 E8297R 5′ CGG CGC GGG CAC ACA TTT CGT CAA TTT 8271-8297 Fragment c E8090F 5′ TAC GAC CTA TCC ACC CAA GG 8090-8109 E10275R 5′ GAA TCT ATG GTT ATC GCA GAG C 10254-10275 Fragment d E9946F 5′ CCT CGA TGA GGC TGG ATA TT 9946-9965 E12929R 5′ GCA CCA ACC AGG AGG AAA AAA GC 12907-12929 Fragment e E3173F 5′ CAT TCT TGC GTC CCT CAA AT 3173-3192 E5352R 5′ CGA CAG TCT TTC TGC CAT CAA TG 5330-5352 Fragment f E2229F 5′ GCT GCT GTT GTC CTG TGT T 2229-2247 E3397R 5′ CCG TCG AAG GGG GTG GCA TCC 3377-3397 Fragment g E12482F 5′ TCA TTC GAG CTG ACC ATC AA 12482-12501 E14651R 5′ CTT TAT CAT TGC ACC CAG CAA 14631-14651 Fragment h E1GF 5′ GGC GCG CCT AAT ACG ACT CAC TAT AGA TGA TGT GTA GGG TAT 1-16 E2968R 5′ CGC GGG CGC CTG AGT TCG ACA AAT T 2944-2968 Fragment i E14059F 5′ CAA CGA TCC TAC CGC CGC ACA A 14059-14080 018 Poly AR 5′ GGC GAT CGG GCG TCT AGG AAT TCT AGA (T)41 AAT TTC GGT CAC 15036-15047 018 3′R 5′ GGC GAT CGG GCG TCT AGG AAT TC After poly(A) Unique restriction enzyme site construction in ORF7 E14059F 5′ CAA CGA TCC TAC CGC CGC ACA A 14059-14080 YFp503R 5′ GGC CCC AGT GCT GCA ATG ATA C After poly(A) ScalF 5′ AGA AGA AAA AGA AAA GTA CTG CTC CAA TGG G 14569-14599 ScalR 5′ CCC CAT TGG AGC AGT ACT TTT CTT TTT CTT 14571-14600 GFP insertion in Nsp2 region gfpF 5′ GCT CAG ATG GTG AGC AAG GGC GAG GAG C gfpR 5′ GAG TCT GAA GAG GAC TTG TAC AGC TCG TCC A Nsp2F1 5′ TGC TGA CTT TCT TGC TGA TCC ACC TCC T 1895-1922 Nsp2R1 5′ CCT TGC TCA CCA TCT GAG CAC TCC CG 2408-2420 Nsp2F2 5′ GCT GTA CAA GTC CTC TTC AGA CTC CAA GA 2419-2439 Nsp2R2 5′ GCG GAC CCA GCC AGG ATC AGA C 2732-2753The 5′ and 3′ ends of the genome sequences were determined using a GeneRACER kit (Invitrogen) according to the manufacturer's instructions. The fragment representing the 5′ terminus of the viral genome was prepared using RT-PCR with primers E1GF and E2968R (Table 1), which integrate a T7 RNA polymerase site immediately preceding the authentic 5′-terminal nucleotides and an AscI restriction enzyme site. The fragment containing the 3′-end sequence was constructed by reverse transcription of RNA with primer 018 Poly AR, which flanks the 41 poly(A) residues and the XbaI site. The reverse transcription reaction was followed by PCR with primers E14059F and 018 3′R (Table 1).
Assembly of full-length cDNA clone.A low-copy-number plasmid, pACYC177 (GenBank accession number X06402), was modified by replacing the fragment between the BamHI and BglI sites with a stuffer fragment, which was prepared as a synthetic gene containing the restriction enzyme sites, as shown in Fig. 1. Each of the viral fragments was excised from PCR-Blunt II-Topo by use of restriction enzymes and ligated into the pACYC177 plasmid, which was digested with the same restriction enzymes. After each ligation step, the pACYC177 construct was transformed into Escherichia coli DH5α cells and grown overnight at 37°C in the presence of kanamycin. The completely assembled full-length cDNA clone was sequenced.
Insertion of a unique restriction enzyme site into the cloned virus.A ScaI restriction enzyme site was engineered into the ORF7 region of the cloned virus for discriminating between the cloned virus and parental virus. To create the ScaI restriction enzyme site, the silent mutation (G-to-T mutation) at nucleotide 42 of ORF7 (nucleotide 14588 of the SD01-08 genome) was generated using site-directed mutagenesis. Site-directed mutagenesis was achieved by an overlapping extension PCR technique (10, 11) using primer pairs E14059F/Sca1R and Sca1F/YFp503R. The mutated product was confirmed by DNA sequencing analysis.
GFP insertion.The pSD01-08-GFP clone was constructed by inserting the GFP gene sequence (Clontech) into the Nsp2 region (nucleotides 2420 and 2421) of the viral genome in the plasmid pSD01-08. The GFP gene was amplified from the pEGFP-N1 plasmid (Clontech) with forward primer gfpF and reverse primer gfpR. GFP was inserted by an overlapping extension PCR technique (10, 11) using primer pairs Nsp2F1/Nsp2R1 and Nsp2F2/Nsp2R2. The PCR product was digested with RsrlI and AclI restriction enzymes and ligated into the pSD01-08 plasmid, which was digested with the same restriction enzymes.
In vitro transcription and rescue of PRRSV.The plasmid pSD01-08 or pSD01-08-GFP was linearized with restriction enzyme XbaI. Capped RNA was transcribed with T7 RNA polymerase using an mMessage Machine kit (Ambion) and transfected to BHK-21 cells using DMRIE-C reagent (Invitrogen) according to the manufacturer's instructions. To rescue the virus, cell culture supernatant obtained 48 h posttransfection was serially passaged on MARC-145 cells. Rescue of infectious virus was confirmed by indirect immunofluorescence assay (IFA) as described in our previous publication (26). Monoclonal antibodies (MAbs) used in the IFA were developed in our laboratory, including MAb ES3-4 58-46, which specifically recognizes Nsp2 of SD01-08 (Y. Fang, B. Neiger, T. Hawkins, J. Christopher-Hennings, R. Rowland, and E. Nelson, Proc. Conf. Res. Work. Anim. Dis., abstr. 78, 2004); MAb MR39, which specifically recognizes the N protein of the North American type 2 PRRSV; and MAb SDOW17, which recognizes the N protein of both genotypes of PRRSV (21, 26). For rescue of GFP virus, the expression of GFP was also visualized directly under a fluorescent microscope.
Characterization of in vitro growth properties.Growth kinetics were examined by infecting MARC-145 cells with cloned virus and parental virus at an MOI of 0.1. Infected cells were collected at 0, 6, 12, 24, 36, 48, 60, and 72 h postinfection, and the virus titers were determined by IFA on MARC-145 cells and quantified as fluorescent focus units (FFU) per ml. Plaque morphologies of the cloned virus and parental virus were compared by plaque assay on MARC-145 cells. Confluent cell monolayers were infected with viruses at an MOI of 0.1. After 2 h, cell culture supernatant was removed and an agar overlay was applied. Plaques were detected after 5 days at 37°C and stained by using 0.1% crystal violet.
Animals/challenge groups.Twenty-one 4-week-old, PRRSV-naïve pigs from a certified PRRSV-negative herd were obtained and randomly divided into four groups housed separately in isolation facilities at Boehringer Ingelheim Vetmedica (BI), Ames, IA. After a 4-day acclimation period, pigs from each group (n = 6 for cloned-virus-infected group; n = 5 for the remaining groups) were inoculated intranasally with 1 ml 105 50% tissue culture infective doses (TCID50) of cloned virus (group 1) or parental virus (group 2). The third group of animals was inoculated with the current modified live virus (MLV) Ingelvac PRRSV vaccine. The negative-control (group 4) animals were mock challenged with MARC-145 cell culture supernatant.
Clinical signs and serum and tissue sampling.Pigs were observed daily for clinical signs and body temperatures were taken for the first 7 days after infection. Blood samples were obtained from all pigs on days 0, 7, 14, 21, 28, 35, and 42. Serum samples were stored at −80°C for further tests. Two pigs from each group were euthanized at 21 days postinoculation (dpi) for postmortem analysis of acute infection. The remaining three pigs from each group were euthanized at 42 dpi. Lung lesions of the study animals were evaluated using a previously developed system based on the approximate volume that each lobe contributes to the entire lung: the left and right apical lobes, the left and right cardiac lobes, and the intermediate lobe each contribute 10% of the total lung volume, and the left and right caudal lobes each contribute 25%. These scores were then used to calculate the total lung lesion score based on the relative contributions of each lobe (9).
Quantification of viral load.For the detection of viral RNA and determination of viral load, serum samples from 0, 7, 14, 21, 28, 35, and 42 dpi were examined using a real-time, quantitative PCR (Tetracore VetAlert PRRS [32]) which is routinely performed at the South Dakota Animal Disease Research and Diagnostic Laboratory (SDSU-ADRDL).
Determination of humoral immunity.All serum samples were evaluated for anti-PRRSV antibodies by use of an IDEXX HerdChek PRRS 2XR enzyme-linked immunosorbent assay (ELISA) and a virus neutralization assay (VN). These tests are also routinely performed at SDSU-ADRDL under strict quality assurance guidelines.
Nucleotide sequence accession numbers.The genome sequence of the full-length cDNA clone was deposited in GenBank under accession number DQ489311.
RESULTS Construction of a full-length cDNA clone of a North American type 1 PRRSV and determination of its infectivity.A full-length genomic cDNA clone of a North American type 1 PRRSV, pSD01-08, was constructed using the strategy shown in Fig. 1. This construct contains a bacteriophage T7 RNA polymerase promoter at the 5′ terminus of the viral genome, one additional guanosine residue introduced between the T7 promoter and the first nucleotide of the viral genome, the 15,047-nucleotide full-length genome of SD01-08, and a poly(A) tail of 41 residues incorporated at the 3′ end of the genome. Compared to the genome sequence of the parental virus, the DNA sequence of pSD01-08 contained six nucleotide differences (Table 2). Four of these differences were silent mutations. The mutation at nucleotide 14588 was introduced to create a unique ScaI restriction enzyme site into ORF7 for differentiating the cloned virus from the parental virus. Two of the nucleotide mutations resulted in amino acid changes, a C-to-T substitution at nucleotide 9492 (amino acid P to L) located at Nsp10 and a T-to-C substitution at nucleotide 11261 (amino acid Y to H) located at Nsp11.
TABLE 2.Nucleotide differences between the parental SD01-08 isolate and the full-length cDNA clone
Nucleotide position within SD01-08 genome Nucleotide in parental virus Nucleotide in cDNA clone Amino acid change Gene position 1331 T C Silent Nsp1β 6158 T C Silent Nsp5 8191 A G Silent Nsp9 9492 C T P to L Nsp10 11261 T C Y to H Nsp11 14588 G T Silent ORF7The plasmid pSD01-08 was linearized by restriction enzyme XbaI and used for in vitro transcription by T7 RNA polymerase to synthesize capped RNAs. The in vitro-transcribed capped RNA was transfected into BHK-21 cells. At 48 h posttransfection, cells were examined for the expression of N protein by fluorescent antibody staining with MAb SDOW17 (Fig. 2A). Results showed that about 5% of the transfected cells expressed the N protein (Fig. 2A). Supernatants from the transfected cells were passaged onto MARC-145 cells. After 72 h, MARC-145 cells were stained using the SD01-08-specific, anti-Nsp2 MAb ES3-4 58-46 (Fig. 2B) and anti-N MAb SDOW17 (Fig. 2C). A North American type 2 PRRSV-specific, anti-N MAb, MR39 (Fig. 2D), was incorporated as a negative control. The results showed that both Nsp2 and N proteins were detected in MARC-145 cells inoculated with supernatant from the pSD01-08-transfected BHK-21 cells. Upon further passage of the supernatant onto fresh MARC-145 cells (passage 2 on MARC-145 cells), cytopathic effects were observed within 48 to 72 h postinfection (hpi). Titration of virus from passage 2 on MARC-145 cells showed an average titer of 3.6 × 107 FFU/ml. These results indicate that viable and infectious North American type 1 PRRSV was rescued from the cells transfected with in vitro-transcribed RNA.
FIG. 2.Rescue and passage of cloned North American type 1 virus SD01-08. (A) BHK-21C cells were transfected with in vitro-transcribed RNA from the full-length cDNA clone. (B, C, and D) MARC-145 cells were infected with cloned virus rescued from BHK. Cells were fixed and stained with PRRSV-specific MAbs at 48 h posttransfection (or postinfection): (A) anti-N MAb SDOW17, (B) anti-Nsp2 MAb ES3-4 58-46 (type 1 PRRSV specific), (C) anti-N MAb SDOW17, and (D) anti-N MAb MR39 (type 2 PRRSV specific). (E and F) PAMs were infected with parental virus (E) and cloned virus (F) and stained with anti-N MAb SDOW17.
In vitro characterization of cloned virus.The parental virus and cloned virus (passage 2 on MARC-145 cells) were titrated on PAMs. Immunofluorescent staining using anti-N MAb showed that both viruses replicated in PAMs (Fig. 2E and F) and produced similar virus yields (2.1 × 104 to 2.8 × 104 FFU/ml) at 72 hpi.
To further compare the growth properties of the cloned and parental viruses, MARC-145 cells were infected with each of the viruses at an MOI of 0.1 and harvested at 6, 12, 24, 36, 48, 60, and 72 hpi. Growth curve results showed that the cloned virus possessed growth kinetics similar to that of the parental virus (Fig. 3). Titers peaked at 48 hpi for both viruses. The peak titer of the cloned virus was 1.39 × 107 FFU/ml, versus 2.34 × 107 FFU/ml for the parental virus. Plaque morphology of these viruses was also determined, and the plaque size produced by the cloned virus was similar to that of the parental virus (data not shown). These results indicate that the cloned virus possesses in vitro properties similar to those of the parental wild-type virus.
FIG. 3.Growth kinetics of cloned virus, parental virus, and GFP-expressing virus. MARC-145 cells were infected with each virus at an MOI of 0.1. At 6, 12, 24, 36, 48, 60, and 72 hpi, cells were harvested and the virus titers were determined by IFA on MARC-145 cells.
To differentiate the cloned virus from the parental virus, we engineered a ScaI restriction enzyme site at nucleotide 42 of ORF7. As shown in Fig. 4, a 1,054-bp RT-PCR fragment derived from amplifying nucleotides 13875 to 14928 was cleaved by ScaI in the cloned virus. In contrast, the RT-PCR fragment derived from the parental virus was not cleaved by ScaI.
FIG. 4.Differentiation between cloned virus and parental virus SD01-08. A ScaI restriction enzyme site was introduced in the full-length cDNA clone for distinguishing cloned virus from parental virus.
Pathogenic and immunological properties of cloned virus derived from pSD01-08 in a pig model.We performed an in vivo study of the replication properties of virus derived from the infectious clone by using a nursery pig model. Pigs, divided into four groups, were infected with cloned virus, parental virus, or the MLV Ingelvac PRRSV vaccine or were mock infected. All pigs that received viruses became infected; this was evident by positive RT-PCR results for the presence of viral RNA in serum and by serology. Virus in serum peaked at about 14 dpi (Fig. 5A; Table 3). At 14 dpi, five of six pigs in the cloned-virus group, four of five pigs in the parental-virus group, and all five pigs in the vaccine group had seroconverted (Fig. 5B; Table 3). Four of the six pigs in the cloned-virus-challenged group had detectable neutralizing antibody titers at 21 dpi, while one of the five pigs in the parental-virus-challenged group developed neutralizing antibodies by 21 dpi. Two of the pigs from the MLV vaccine-challenged group developed detectable neutralizing antibody titers at 42 dpi (Table 3). All mock-infected pigs remained RT-PCR and PRRSV antibody negative throughout the study period. No significant clinical signs were observed in any of the infected pigs. Only mild pathological lung lesions characteristic of PRRSV, such as minor interstitial pneumonia, were observed in three of six pigs from the cloned-virus group, five of five pigs from the parental-virus group, and two of five pigs from the vaccine group. The rest of the pigs did not show gross lung lesions (Table 4). Interestingly, comparing the pathological lesions among the pigs from different groups, the lesion scores appear slightly higher in pigs infected with parental virus.
FIG. 5.In vivo characterization of cloned virus. Twenty-one 4-week-old, PRRSV-naïve pigs from a certified PRRSV-negative herd were obtained and randomly divided into four groups. Pigs from each group were inoculated intranasally with 1 ml 105 TCID50 of cloned virus (group 1, n = 6) or parental virus (group 2, n = 5). The third group of animals (n = 5) was inoculated with the current MLV Ingelvac PRRSV. The negative-control (group 4, n = 5) animals were mock challenged with MARC-145 cell culture supernatant. (A) Viral load in serum samples from different groups of pigs, quantified by real-time PCR. (B) Serum antibody responses of challenged groups of pigs, measured by IDEXX ELISA. S/P, sample-to-positive ratio.
TABLE 3.Summary of serological and PCR results from sera of inoculated pigs at different days postinfection
Day postinfection No. of pigs with positive result/total no. of pigs in groupa Parental virus Cloned virus MLV vaccine PCR ELISA VN PCR ELISA VN PCR ELISA VN 0 0/5 0/5 0/5 0/6 0/6 0/6 0/5 0/5 0/5 7 4/5 1/5 0/5 5/6 0/6 0/6 5/5 0/5 0/5 14 4/5 4/5 0/5 6/6 5/6 0/6 5/5 5/5 0/5 21b 5/5 5/5 1/5 6/6 5/6 4/6 5/5 5/5 0/5 28 2/3 3/3 2/3 4/4 4/4 2/4 3/3 3/3 0/3 35 1/3 3/3 2/3 2/4 4/4 2/4 3/3 3/3 0/3 42 0/3 3/3 3/3 1/4 4/4 2/4 2/3 3/3 2/3 TABLE 4.Percentages of lung with gross pneumonia lesions in infected pigs
Pig no. Gross lung lesion score (%)a Cloned virus Parental virus MLV vaccine Mock control 1 0 0.6 0.7 0 2 1.0 0.5 0 0 3 0.9 10.5 0 0 4 0 2.1 0.3 0 5 0 3.25 0 0 6 1.5 — — — Introduction of green fluorescent protein into the Nsp2 region of the infectious clone.We explored the potential of using the infectious clone for foreign-gene expression. Previous studies showed that Nsp2 is an excellent candidate site for foreign-gene insertion. The C-terminal region of Nsp2 for both type 1 and type 2 contains hypervariable domains, including amino acid insertions and deletions (7, 8, 27). One of the major differences between SD01-08 and LV, the prototypic member of European type 1 viruses, is the presence of a 17-amino-acid deletion in Nsp2, which is located between amino acids 734 and 750 in ORF1 of LV. We inserted a GFP into this unique deletion site of Nsp2 (at amino acids 733 and 734 of SD01-08 ORF1a [see Fig. 7]). The construct, pSD01-08-GFP, was in vitro transcribed and transfected into BHK-21 cells. Live cells were examined directly under a fluorescence microscope at 48 h posttransfection. The cell culture supernatant from transfected BHK cells was passaged onto MARC-145 cells, resulting in the appearance of GFP-expressing cells, which could be clearly visualized as early as 6 h after infection (Fig. 6A). To confirm the expression of the GFP-Nsp2 fusion protein, at 48 h postinfection, cells were fixed and stained with an Nsp2-specific MAb, ES3-4 58-46. ES3-4 58-46 was generated by immunizing mice with synthetic peptide made from ES3 epitope sequence, which is located immediately upstream of the GFP insertion site (Fig. 7). A red fluorescent Cy3-conjugated goat anti-mouse immunoglobulin G was used as the secondary antibody. Confocal microscopy showed the perinuclear localizations of both GFP and Nsp2, which were similar to Nsp2 localization of the parental virus (Fig. 6B and C). To determine if the expression of GFP affected virus replication, the growth characteristics of the GFP virus were compared to those of the parental wild-type and cloned viruses. The replication cycle of the pSD01-08-GFP virus was similar to those of the other viruses, including peak viral titers at 48 hpi; however, the peak titer for the GFP virus infection was reduced approximately 10-fold (Fig. 3).
FIG. 7.Schematic diagram of the pSD01-08-GFP construct. The pSD01-08-GFP clone was constructed by inserting the GFP gene sequence into the Nsp2 deletion region, between amino acids 733 and 734 of ORF1a of SD01-08 (arrow). Boxes show the B-cell epitope sites (ES) identified by Oleksiewicz et al. (25). The N-terminal 1 to 159 amino acids of GFP deleted by the virus are underlined. The two amino acids inserted by the virus upstream of GFP amino acid 160 are shown in bold type and parentheses.
FIG. 6.Fluorescence microscopy of GFP-expressing PRRSV. BHK-21C cells were transfected with in vitro-transcribed RNA. Forty-eight hours after transfection, cell culture supernatant was passaged on MARC-145 cells. (A) GFP expression in MARC-145 cells after 48 h of infection with the GFP virus. (B) MARC-145 cells were fixed and stained with anti-Nsp2 MAb ES3-4 58-46 and a Cy3-conjugated goat anti-mouse MAb (red). (C) Merged picture of images from panels A and B seen under confocal microscopy.
To investigate the stability of GFP expression over multiple rounds of virus replication, the GFP virus was serially passaged eight times on MARC-145 cells. By the seventh passage, there appeared a subpopulation of non-GFP-expressing virus, which was counted as 15% of the total virus population. The loss of GFP was also analyzed by RT-PCR. Total cellular RNA was isolated from cells infected with the seventh passage of the GFP virus, and RNA was used as a template in an RT-PCR with primers that amplified the GFP insertion region. The RT-PCR product was cloned and sequenced. The results revealed that the N-terminal amino acids 1 to 159 of GFP were deleted (Fig. 7). More interestingly, while the amino acids 1 to 159 were deleted, two amino acids, methionine (M) and glutamic acid (E), were inserted by the virus before the GFP amino acid 160 (Fig. 7). Therefore, the selection of viral genome encoding the deletion in the GFP gene accounted for the decline in the percentage of infected cells expressing GFP. Taken together, these results indicate that the Nsp2 region can tolerate the introduction of a foreign gene. However, inserting a foreign gene reduces the level of viral replication. Selection may have occurred to generate an in-frame deletion so that the polyprotein was translated faithfully, which eventually resulted in variants that no longer encode a functional foreign gene.
DISCUSSIONThe recent and widespread distribution of the unique group of North American type 1 PRRSV isolates represents a new emerging disease problem in U.S. swine herds (7, 26). In order to develop effective vaccines and control strategies, it is essential to understand the fundamental biology of this group of viruses. In the present study, we established a reverse-genetics system for a representative strain of this group. We demonstrated that capped RNA transcripts from the full-length cDNA clone were replication competent when transfected into BHK-21 cells and were infectious when passaged onto MARC-145 cells and PAMs. Experimental infection of pigs with the cloned virus further confirmed that the cDNA clone was infectious in vivo.
SD01-08 (GenBank accession number DQ489311) shares 94.1% identity with Lelystad virus at the nucleotide level. An important distinction between SD01-08 and LV is the growth properties in PAMs and monkey kidney cells. Results reported by Meulenberg et al. (15) showed that wild-type and cloned LV viruses grew well in PAMs but to low levels in the MA-104-derived cell line CL2621. Parental and cloned SD01-08 viruses grew equally well on PAMs and MARC-145 cells, another MA-104-derived cell line. The titer of SD01-08 cloned viruses peaked at 48 hpi, while LV cloned viruses grow to lower titers and had not peaked even at 96 hpi. Therefore, our SD01-08 infectious clone is the first type 1 infectious clone shown to replicate well in the continuous cell line, which provides a significant advantage for future reverse-genetics studies. Another difference between LV and SD01-08 is level of virulence. In PAMs, the LV cloned virus reached high titers, at 107.1 to 107.9 TCID50/ml, and peaked around 32 hpi. Our SD01-08 cloned virus reached the same titer as its parental virus in PAMs, but their titers were both lower than that of LV, reaching only about 104 TCID50/ml, and peaked later, around 72 hpi. This result suggests that the SD01-08 cloned virus may be less virulent than the LV cloned virus. This conclusion is supported by field observations and our experimental animal challenge study. SD01-08 did not cause significant clinical signs, and only mild pathological lesions were observed in the experimental infected pigs. In contrast, LV was reported to cause significant respiratory problems in pigs and abortions in sows (34). Due to the distinct characteristics of the pathogenesis and cell tropism of these two type 1 PRRSV infectious clones, future studies could be performed to determine the genomic regions responsible for virulence and cell tropism by using chimeric constructs between these two infectious clones.
In comparison to the parental virus, the cDNA clone contains two amino acid mutations in the nonstructural protein region (nucleotide 9492 at Nsp10 and nucleotide 11261 at Nsp11). These mutations may reflect a quasispecies in the virus stock or may have been introduced by RT-PCR and/or cloning procedures. The cloned virus showed in vitro growth properties similar to those of the parental virus. However, our animal experiment showed that the percentage of pathological lesions caused by the cloned virus is slightly lower than that of the parental virus. Preliminary studies on the expression of immune marker genes from pig peripheral blood mononuclear cells harvested at 35 dpi showed that cloned virus activated more immune marker genes than did parental virus (data not shown). Whether this in vivo difference that we observed was due to these two amino acid changes is a subject of investigation in our laboratory. In addition, due to the inherent genetic variation among individual pigs, our results need to be confirmed in repeated experiments with a larger number of pigs over multiple days postinfection.
One of the major applications of the infectious clone is as a viral backbone for constructing genetically engineered vaccines. Current PRRSV vaccines in the United States target mainly the North American type 2 isolates. The recent emergence of the North American type 1 PRRSV requires that vaccines be effective for both genotypes of PRRSV. An essential requirement for any live-virus vaccine is that it be low virulence, inducing no or, at most, very mild disease manifestations. Our cloned virus is likely to fulfill this requirement. As we discussed before, the parental virus SD01-08 was isolated from a group of pigs showing no clinical signs. Pathogenesis studies in our laboratory further confirmed that SD01-08 possesses low-virulence properties at the acute phase of the disease, which suggested that the pSD01-08 infectious clone may be a potential low-virulence strain and suitable for future vaccine construction. Additional in vivo studies are required for more complete evaluation of the pathogenic properties of this cloned virus; in particular, we need to study the pathogenesis of this virus in the pregnant-sow model.
Besides its potential for vaccine construction, one of the important applications of the infectious clone is the study of the basic viral biology in vitro. GFP commonly serves as a reporter gene to facilitate such studies, since it can be used to monitor virus entry and replication in living cells. For hepatitis C virus, GFP was inserted into Nsp5A, which allowed direct visualization of functional hepatitis C virus replication complexes for studying the assembly and disassembly of functional HCV replicases in living cells (19). In this study, we engineered GFP into the unique 17-amino-acid deletion region of Nsp2. The cloned virus with the GFP insertion was still able to replicate in cell culture. This result demonstrated the flexibility of this viral protein and of the PRRSV RNA replication machinery. Currently, little is known about the mechanism of PRRSV replication. Individual nonstructural protein proteolytic processing products and their functions relating to genome replication and viral assembly have been deduced from the study of equine arteritis virus and have not been determined specifically for PRRSV. This pSD01-08 infectious clone with GFP expression in the Nsp2 region is an attractive system for dynamic study of the function of nonstructural proteins in the formation and turnover of PRRSV replication complexes in vitro in living cells.
In conclusion, we successfully constructed a full-length cDNA infectious clone of a North American type 1 PRRSV and explored its potential as a viral backbone for foreign-gene expression. We believe that the availability of this infectious clone will have significant contributions for the future study of basic viral biology and development of the next generation of PRRSV vaccines.
AcknowledgmentsWe thank Asit K. Pattnaik from the University of Nebraska—Lincoln for confirming the infectivity of our infectious clone, Ruben Donis from the Centers for Disease Control and Prevention, and Kay Faaberg from the University of Minnesota for good suggestions on infectious clone construction. Special thanks to Travis Hawkins, BreAnn Neiger, Jessica Mann, and Nellie Benson for excellent technical assistance.
Funding for this work was derived from USDA-NRI seed grant no. 2005-35204-16112, Boehringer Ingelheim Vetmedica, Inc., the South Dakota Center for Infectious Disease Research and Vaccinology 2010 program, and the South Dakota Animal Disease Research and Diagnostic Laboratory.
Footnotes ▿Published ahead of print on 13 September 2006.
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