An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS

doi:10.1016/j.vetmic.2009.03.003

Veterinary Microbiology

Volume 138, Issues 1–2, 2 July 2009, Pages 34-40

https://doi.org/10.1016/j.vetmic.2009.03.003 Get rights and content

Abstract

Porcine infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) cause significant morbidity and mortality and currently there are no effective vaccines for disease prevention. An attenuated strain, HuN4-F112, was obtained by passaging the HP-PRRSV HuN4 on Marc-145 cells (112th-passage). PRRSV-free pigs were inoculated intramuscularly with HuN4-F112 (10^2.0, 10^3.0, 10^4.0, 10^5.0 and 10^6.0 TCID₅₀ for groups 1–5, respectively). The groups 3–5 could resist the lethal challenge and did not show any obvious changes in body temperature nor clinical signs throughout the experiment, the pathological lesions were milder and the gained weight at a greater rate (P < 0.05), compared to group 1 and control. Sequence analysis of the HuN4 passages showed a conserved epitope in GP5 protein was mutated (¹⁹⁶QWGRL/P²⁰⁰ → ¹⁹⁶RWGRL/P²⁰⁰), as a result the monoclonal antibody could not recognize the HuN4-F112 any more. These results suggested that the HuN4-F112 could protect piglets from lethal challenge and might be a candidate vaccine against the HP-PRRSV.

Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded, positive RNA virus of the Arteriviridae family that is the causative agent of porcine reproductive and respiratory syndrome (PRRS) that can result in reproductive failure in pregnant sows or in respiratory complications. PRRS was first described in North America in 1987 (Keffaber, 1989, Cavanagh, 1997) and today presents health problems to the swine industry worldwide.

The PRRSV genome contains nine open reading frames (ORF). ORF1a and ORF1b encode viral replicase polyproteins that are immediately translated upon viral entry and then proteolytically processed by virally encoded proteinases into 12 mature nonstructural proteins (NSP1-NSP12) (Snijder and Meulenberg, 1998). ORF2a, ORF2b, and ORFs 3–7 encode the viral structural proteins GP2, E, GP3, GP4, GP5, M, and N, respectively (Benfield et al., 1992, Stadejek et al., 2002).

In May 2006, HP-PRRSV (a highly virulent form of PRRSV) severely affected the pork industry of the Jiangxi province (China). Infections were characterized by high morbidity (characterized by high fever, 50–100%) and mortality (20–100%) (Tian et al., 2007, Li et al., 2007, Tong et al., 2007). Since the initial outbreak, disease spread rapidly to most provinces in China, resulting in more than one million deaths. In previous studies, we identified the HP-PRRSV HuN4 strain from a farm outbreak of ‘pig high fever syndrome’ in 2006 in China and established pathogenicity assays for the characterization of this strain (Tong et al., 2007, Zhou et al., 2008). Because the live-attenuated North-American-type PRRSV vaccines currently in use were limited effective against HP-PRRSV infections, novel, safe and effective formulations are needed to prevent HP-PRRSV infections.

In this study, we describe the development and efficacy evaluation of a live-attenuated vaccine derived from the HP-PRRSV HuN4 strain. The results presented in this report indicated that the live vaccine conferred effective protection against a lethal HP-PRRSV HuN4-F5 challenge and could serve as a vaccine against HP-PRRSV in pigs.

Section snippets

Viral culture

PRRSV HuN4 strain (GenBank accession no. EF635006) was isolated and maintained in our laboratory as described (Tong et al., 2007). The Marc-145 cell line (an African green monkey kidney cell line) was employed for viral propagation and titration. The monoclonal antibody (MAb) 53D8 specific for the conserved ¹⁹⁶QWGRL²⁰⁰ epitope of GP5 was generated and maintained by our research group as described (Zhou, 2005).

Viral titer assessment and passaging

HP-PRRSV HuN4 titers were assessed every 5–10 passages by seeding Marc-145 cells into

Molecular marker in GP5 of HuN4-derivate viruses

RNA extracted from different HuN4 passages (i.e., F1, F20, F40, F65, F84, F100, F112 and F127) identified two amino acids that mutated in the GP5 protein; one at position Q¹⁹⁶ → R¹⁹⁶, and the other at position N³⁴ → D³⁴ (Table 1). In our previous study we identified the GP5 Q¹⁹⁶WGRL/P²⁰⁰ epitope as immunodominant and highly conservative in all North American type PRRSVs examined (Zhou, 2005). As a result, the 53D8 MAb directed against Q¹⁹⁶WGRL/P²⁰⁰ was unable to recognize the mutated R¹⁹⁶WGRL/P²⁰⁰

Discussion

In May 2006 a new HP-PRRSV (i.e., HP-PRRSV HuN4) variant was identified in China that caused severe disease in pigs of all ages and resulted in more than one million deaths. HP-PRRSV sequence analysis revealed two discontinuous deletions in the NSP2 protein. A single amino acid deletion was found at position 482 (L⁴⁸²) of NSP2 and a second deletion of 29 amino acids was located between positions 533–561 (S⁵³³–A⁵⁶¹) (Li et al., 2007, Tong et al., 2007, Zhou et al., 2008). After a comparing

Acknowledgments

The study was supported by grants from the National Basic Research Program (973) of China (No. 2005CB523200), National High-Tech Development Plan (863 Plan) of China (No. 2006AA10A204) and National Scientific Supporting Program (No. 2006BAD06A04/18/03/01).

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¹: These authors contributed equally to this study.

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An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS

Abstract

Introduction

Section snippets

Viral culture

Viral titer assessment and passaging

Molecular marker in GP5 of HuN4-derivate viruses

Discussion

Acknowledgments

Vet. Microbiol.

Vet. Microbiol.

Vet. J.

Vet. Immunol. Immunopathol.

Vaccine

Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332)

J. Vet. Diagn. Invest.

Nidovirales: a new order comprising Coronaviridae and Arteriviridae

Arch. Virol.

Reproductive failure of unknown etiology

Am. Assoc. Swine Pratitioners Newslett.