A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region

Abstract

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.

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.

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.

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.

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.

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.

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.

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.