Porcine reproductive respiratory syndrome virus (PRRSV); replicons
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Publication Number
US-7666585-B2
Publication Date
2010-02-23
Expiration Date
Abstract
Chimeric replicons of North American Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) containing the 5′ sequence of an avirulent strain of PRRSV and a 3′ sequence of a virulent strain of PRRSV are provided. Further provided is a method of producing attenuated PRRSV from the chimeric replicon. Also provided are compositions containing the replicon or attenuated virus. Vaccines and a method of vaccinating pigs against PRRSV are also provided.
Core Innovation
The invention relates to chimeric PRRSV replicons and infectious attenuated chimeric PRRSV virus produced from those replicons. The chimeric construct combines a 5′ sequence derived from an avirulent strain, including ORF1, ORF2, part of ORF3 or combinations thereof, with a 3′ sequence derived from a virulent field isolate that completes the genome. The resulting recovered virus particle is attenuated relative to the virulent strain of PRRSV.
The document also defines chimeric replicons with particular 5′ sequence sources, including ptkPRRS and ptkPRRS-1 and degenerate variants, and the specified linkage between ORF1/ORF2 and part of ORF3 within the 5′ region and the virulent-derived 3′ region. It further defines a genetic marker PSA for differentiating vaccine versus field strains and describes replicon behavior including marker stability.
The chimeric replicons generate attenuated infectious chimeric PRRSV and maintain replication characteristics while limiting disease outcomes. The document reports reduced lung lesion scores demonstrating avirulence in pigs, limited-duration viremia, and immunogenicity after vaccination including PRRSV-specific antibodies and cross-protection against homologous and heterologous virulent challenges.
Claims Coverage
The document provides four independent claims. Across these claims, the inventive features are organized around producing an infectious chimeric PRRSV virus or attenuated chimeric PRRSV by transfecting cells with specified chimeric replicon or nucleic acid inputs, incubating for production of infectious or chimeric particles, and recovering virus, with attenuation as a functional requirement in two independent claims and named chimeric sequences as the defining inputs in the other two.
Chimeric replicon with ptkPRRS-derived 5′ and virulent 3′ with attenuation outcome
A method of producing a chimeric PRRSV virus by transfecting a cell with a replicon comprising a 5′ sequence derived from ptkPRRS (SEQ ID NO: 1) or a degenerate variant of ptkPRRS, including ORF1, ORF2, part of ORF3 or a combination thereof, and a 3′ sequence derived from a virulent strain of PRRSV, incubating under conditions suitable for production of an infectious virus particle, and recovering the virus particle wherein the recovered virus is attenuated relative to the virulent strain of PRRSV.
Chimeric replicon with ptkPRRS-1-derived 5′ and virulent 3′ with attenuation outcome
A method of producing a chimeric PRRSV virus by transfecting a cell with a replicon comprising a 5′ sequence derived from ptkPRRS-1 (SEQ ID NO: 2) or a degenerate variant of ptkPRRS-1, including ORF1, ORF2, part of ORF3 or a combination thereof, and a 3′ sequence derived from a virulent strain of PRRSV, incubating under conditions suitable for production of an infectious virus particle, and recovering the virus particle wherein the recovered virus is attenuated relative to the virulent strain of PRRSV.
Production using chimeric sequence SEQ ID NO: 13 (pPRRSPTK-3)
A method of producing a chimeric PRRSV virus by transfecting a cell with a chimeric sequence comprising SEQ ID NO: 13 (pPRRSPTK-3) or a degenerate variant of SEQ ID NO: 13, incubating the cell under conditions suitable for production of an infectious, chimeric virus particle, and recovering the chimeric virus particle.
Production using chimeric sequence SEQ ID NO: 14 (pPRRSPTK-6)
A method of producing a chimeric PRRSV virus by transfecting a cell with a chimeric sequence comprising SEQ ID NO: 14 (pPRRSPTK-6) or a degenerate variant of SEQ ID NO: 14, incubating the cell under conditions suitable for production of an infectious, chimeric virus particle, and recovering the virus particle.
Overall, the claims cover methods that generate infectious chimeric PRRSV, including attenuated infectious chimeric PRRSV, by using defined chimeric nucleic acid inputs, specifically combining a defined avirulent-derived 5′ region with a virulent-derived 3′ region and requiring attenuation relative to the virulent strain for those replicon-design claims, or by transfecting with named chimeric sequences SEQ ID NO: 13 or SEQ ID NO: 14, or degenerate variants, and recovering the resulting chimeric virus.
Stated Advantages
Recovered virus particle is attenuated relative to the virulent strain of PRRSV.
Reduced lung lesion scores demonstrating avirulence in pigs.
Limited-duration viremia with maintained replication characteristics.
Immunogenicity after vaccination, including PRRSV-specific antibodies and cross-protection against homologous and heterologous virulent challenges.
Documented Applications
Vaccination use cases are described, including vaccination with homologous and heterologous virulent challenges and evaluation via PRRSV-specific antibodies, cross-protection, and lung lesion scores.
Vaccine and composition formulations are described, including inclusion of adjuvant information in the document.
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