Myristoylation of the small envelope protein of porcine reproductive and respiratory syndrome virus is non-essential for virus infectivity but promotes its growth

ABSTRACT The objective of this research was to optimize sampling parameters for increased recovery and detection of airborne porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV). Collection media containing antifoams, activated carbons, protectants, and ethylene glycol were evaluated for direct effects on factors impacting the detection of PRRSV and SIV, including virus infectivity, viability of continuous cell lines used for the isolation of these viruses, and performance of reverse transcriptase PCR assays. The results showed that specific compounds influenced the likelihood of detecting PRRSV and SIV in collection medium. A subsequent study evaluated the effects of collection medium, impinger model, and sampling time on the recovery of aerosolized PRRSV using a method for making direct comparisons of up to six treatments simultaneously. The results demonstrated that various components in air-sampling systems, including collection medium, impinger model, and sampling time, independently influenced the recovery and detection of PRRSV and/or SIV. Interestingly, it was demonstrated that a 20% solution of ethylene glycol collected the greatest quantity of aerosolized PRRSV, which suggests the possibility of sampling at temperatures below freezing. Based on the results of these experiments, it is recommended that air-sampling systems be optimized for the target pathogen(s) and that recovery/detection results should be interpreted in the context of the actual performance of the system.

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N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus ReplicationIn Vivo

Journal of Virology ◽

10.1128/jvi.07067-11 ◽

2012 ◽

Vol 86 (18) ◽

pp. 9941-9951 ◽

Cited By ~ 40

Author(s):

Zuzhang Wei ◽

Tao Lin ◽

Lichang Sun ◽

Yanhua Li ◽

Xiaoming Wang ◽

...

Keyword(s):

Antibody Response ◽

Cultured Cells ◽

Neutralizing Antibody ◽

Respiratory Syndrome Virus ◽

Virus Infectivity ◽

Serum Samples ◽

Virus Life Cycle ◽

Glycosylation Sites ◽

High Level

It has been proposed that the N-linked glycan addition at certain sites in GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) is important for production of infectious viruses and viral infectivity. However, such specific N-linked glycosylation sites do not exist in some field PRRSV isolates. This implies that the existence of GP5-associated glycanper seis not vital to the virus life cycle. In this study, we found that mutation of individual glycosylation sites at N30, N35, N44, and N51 in GP5 did not affect virus infectivity in cultured cells. However, the mutants carrying multiple mutations at N-linked glycosylation sites in GP5 had significantly reduced virus yields compared with the wild-type (wt) virus. As a result, no viremia and antibody response were detected in piglets that were injected with a mutant without all N-linked glycans in GP5. These results suggest that the N-linked glycosylation of GP5 is critically important for virus replicationin vivo. The study also showed that removal of N44-linked glycan from GP5 increased the sensitivity of mutant virus to convalescent-phase serum samples but did not elicit a high-level neutralizing antibody response to wt PRRSV. The results obtained from the present study have made significant contributions to better understanding the importance of glycosylation of GP5 in the biology of PRRSV.

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Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity

Journal of General Virology ◽

10.1099/vir.0.81160-0 ◽

2005 ◽

Vol 86 (11) ◽

pp. 3091-3096 ◽

Cited By ~ 23

Author(s):

Changhee Lee ◽

Dongwan Yoo

Keyword(s):

North American ◽

Infectious Clone ◽

Progeny Virus ◽

Respiratory Syndrome Virus ◽

Cysteine Residues ◽

Virus Infectivity ◽

Reading Frame ◽

N Protein ◽

Cytopathic Effects ◽

E Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 73 aa, termed E protein. The function of E protein is currently unknown. The E protein possesses two cysteines at positions 49 and 54 that are highly conserved among North American isolates. In the present study, it was shown that E protein did not homodimerize with itself nor did it heterodimerize with the nucleocapsid (N) protein. However, E protein was interactive non-covalently with itself or with the N protein as shown by pull-down assays. The significance of the E protein cysteine residues on virus replication was determined using an infectious clone. Each cysteine was substituted by serine and the mutations were introduced into a full-length clone of PRRSV. When transfected into Marc-145 cells, all cysteine mutant clones induced PRRSV-specific cytopathic effects and produced infectious progeny virus. The data indicate that cysteine residues in the E protein are not essential for replication of North American genotype PRRSV.

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Analysis of immunogenicity of minor envelope protein GP3 of porcine reproductive and respiratory syndrome virus in mice

Virus Genes ◽

10.1007/s11262-006-0067-7 ◽

2007 ◽

Cited By ~ 2

Author(s):

Wenming Jiang ◽

Ping Jiang ◽

Yufeng Li ◽

Xianwei Wang ◽

Yijun Du

Keyword(s):

Envelope Protein ◽

Respiratory Syndrome Virus

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

Journal of Virology ◽

10.1128/jvi.80.8.3994-4004.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3994-4004 ◽

Cited By ~ 183

Author(s):

Israrul H. Ansari ◽

Byungjoon Kwon ◽

Fernando A. Osorio ◽

Asit K. Pattnaik

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Protective Efficacy ◽

Neutralizing Antibody ◽

Respiratory Syndrome Virus ◽

Virus Infectivity ◽

Neutralization Epitope ◽

Enhanced Sensitivity

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.

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