Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK 12 . To rescue infectious virus, capped RNA was transcribed in vitro from the pOK 12 clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5 end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

An infectious clone of the highly pathogenic porcine reproductive and respiratory syndrome virus: Topology of glycoprotein 3 (GP3) addressing the intrachain disulfide bonds

“…The titer at the peak of BJSY07 replication was slightly higher than that of vrBJSY07. The same situation was also found in other reports, where the cloned virus was qualitatively similar but quantitatively slightly different from parental virus [30].…”

“…In recent years, several PRRSV stains have been used as parental viruses to construct full-length infectious clones, including FL12, VR2332, P129, CH-1a, Lelystad virus, and the hpPRRSV JXwn06 and JX143 [18,23,24,27,28,30]. Although JXwn06 and JX143 as hpPRRSV were used to construct the infectious clones, the construction of a reverse genetic platform based on the hpPRRSV was still needed in a different strategy for easier screening and testing of new antiviral drugs in future studies.…”

“…To rescue infectious viruses vrBJSY07 and vBJSY07, 293T cells were transfected with the in vitrotranscribed RNA. We chose 293T cells to assemble the first passage virions for their high efficiency of RNA transfection; higher than BHK21 cells which had been successfully used in previous reports [22,27,30]. The harvested cell medium containing the rescued virus vrBJSY07 and vBJSY07 particles were then each transferred to PRRSV permissible cells.…”

The 30-Amino-Acid Deletion in the Nsp2 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Emerging in China Is Not Related to Its Virulence

“…Complete sequence determination showed that the virus had the same genomic characteristics as the isolates described previously (23,41,48), namely, a noncontiguous 30-aa deletion in its Nsp2. In recent years, reverse genetic operation methods have been extensively applied to study the genomic functions of PRRSV (11,21,26,28,31,32,34,43). A new study indicated that the rescued virus from an infectious cDNA clone of a highly pathogenic PRRSV variant was highly virulent for pigs (24).…”

Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies

“…Recently the development of reverse genetic systems for PRRSV has been reported from several laboratories, including ours (33,37,48,49). Evidently, mutational studies with infectious clones have led to a better understanding of the mechanisms of transcription and replication of the viral genome of arteriviruses (46).…”