A highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cDNA clone retains the in vivo virulence and transmissibility properties of the parental virus

Abstract

The nucleotide sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Transfection of MARC-145 cells with capped in vitro transcripts derived from a full-length cDNA clone of the viral genome resulted in infectious PRRSV with growth characteristics similar to that of the parental virus. Primer extension analysis revealed that during replication, the viral polymerase corrected the two nonviral guanosine residues present at the 5' terminus of the transfected transcripts. Animal studies showed that the cloned virus induced hyperthermia, persistent viremia, and antibody response, similar to that observed with the parental virus. Contact transmission occurred rapidly within 3 days of introduction of naïve pigs into the group of clone virus-inoculated pigs. These results suggest that the cloned virus retains the in vivo virulence and contagion properties of the parental virus, thus, providing the background for reverse genetics manipulation in systematic examination of attenuation and virulence phenotypes.

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

“…The full-length plasmids were digested with AclI and linearized DNA was used as the template to generate capped RNA transcripts using the mMESSAGE mMACHINE Ultra T7 kit as (48,49). The reaction mixture was treated with DNase I to digest the DNA template and extracted with phenol and chloroform and finally precipitated with isopropanol.…”

“…After confirmation, the virus stock was grown and frozen at Ϫ80°C in small aliquots for further studies. In all the experiments, FL-12, containing the wt PRRSV genome, and FL-12pol Ϫ , containing the polymerasedefective PRRSV genome (48,49), were used as controls.…”

“…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).…”

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

“…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.…”

“…Marc-145 cells were grown in 24-well micro-plates with slides inside, as previously described [28], and were infected with 100 L, 10 2 TCID 50 /mL BJSY07, vBJSY07, and vrBJSY07, respectively. Uninfected Marc-145 cells were used as a control.…”