Development of a Real-time RT-PCR Method for Detection of Porcine Rubulavirus (PoRV-LPMV)

Overview

Journal J Virol Methods

Specialty Microbiology

Date 2013 Jan 12

PMID 23305816

Citations 4

Authors

Sandra Cuevas-Romero

Anne-Lie Blomstrom

Arcelia Alvarado

Pablo Hernandez-Jauregui

Francisco Rivera-Benitez

Humberto Ramirez-Mendoza

Mikael Berg

Affiliations

Soon will be listed here.

Abstract

In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.

Citing Articles

Immunogenicity of a recombinant hemagglutinin neuraminidase-Porcine rubulavirus produced by Escherichia coli of Porcine rubulavirus gives protective immunity of litter after challenge.

Cuevas-Romero J, Cerriteno-Sanchez J, Lara-Romero R, Vega-Lopez M, Ramirez-Estudillo C, Ramirez-Mendoza H J Vet Med Sci. 2022; 84(12):1595-1604.

PMID: 36273875 PMC: 9791230. DOI: 10.1292/jvms.22-0207.

Development of Novel Recombinant Antigens of Nucleoprotein and Matrix Proteins of Antigenicity and Structural Prediction.

Lara-Romero R, Cerriteno-Sanchez J, Mendoza-Elvira S, Garcia-Cambron J, Castaneda-Montes M, Perez-Aguilar J Viruses. 2022; 14(9).

PMID: 36146753 PMC: 9504402. DOI: 10.3390/v14091946.

Molecular and epidemiological studies of Porcine rubulavirus infection - an overview.

Cuevas-Romero J, Blomstrom A, Berg M Infect Ecol Epidemiol. 2015; 5:29602.

PMID: 26584829 PMC: 4653323. DOI: 10.3402/iee.v5.29602.

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation.

Pisanelli G, Laurent-Rolle M, Manicassamy B, Belicha-Villanueva A, Morrison J, Lozano-Dubernard B Virus Res. 2015; 213:11-22.

PMID: 26546155 PMC: 5538256. DOI: 10.1016/j.virusres.2015.10.027.

References

Stephan H, Gay G, Ramirez T . Encephalomyelitis, reproductive failure and corneal opacity (blue eye) in pigs, associated with a paramyxovirus infection. Vet Rec. 1988; 122(1):6-10. DOI: 10.1136/vr.122.1.6. View

Berg M, Hjertner B, Moreno-Lopez J, Linne T . The P gene of the porcine paramyxovirus LPMV encodes three possible polypeptides P, V and C: the P protein mRNA is edited. J Gen Virol. 1992; 73 ( Pt 5):1195-200. DOI: 10.1099/0022-1317-73-5-1195. View

Allan G, McNeilly F, Walker I, Linne T, Moreno-Lopez J, Hernandez P . A sequential study of experimental porcine paramyxovirus (LPMV) infection of pigs: immunostaining of cryostat sections and virus isolation. J Vet Diagn Invest. 1996; 8(4):405-13. DOI: 10.1177/104063879600800401. View

Nordengrahn A, Svenda M, Moreno-Lopez J, Bergvall A, Hernandez P, McNeilly F . Development of a blocking ELISA for screening antibodies to porcine rubulavirus, La Piedad Michoacan Virus. J Vet Diagn Invest. 1999; 11(4):319-23. DOI: 10.1177/104063879901100404. View

McNeilly F, Walker I, Allan G, Foster J, Linne T, Merza M . A comparative study on the use of virus and antibody detection techniques for the diagnosis of La Piedad Michoacan paramyxovirus (LPMV) infection in pigs. J Vet Diagn Invest. 1997; 9(1):3-9. DOI: 10.1177/104063879700900102. View

Svenda M, Berg M, Moreno-Lopez J, Linne T . Analysis of the large (L) protein gene of the porcine rubulavirus LPMV: identification of possible functional domains. Virus Res. 1997; 48(1):57-70. DOI: 10.1016/s0168-1702(96)01426-8. View

Berg M, Sundqvist A, Moreno-Lopez J, Linne T . Identification of the porcine paramyxovirus LPMV matrix protein gene: comparative sequence analysis with other paramyxoviruses. J Gen Virol. 1991; 72 ( Pt 5):1045-50. DOI: 10.1099/0022-1317-72-5-1045. View

Cuevas J, Rodriguez-Ropon A, Kennedy S, Moreno-Lopez J, Berg M, Hernandez-Jauregui P . Investigation of T-cell responses and viral mRNA persistence in lymph nodes of pigs infected with porcine rubulavirus. Vet Immunol Immunopathol. 2008; 127(1-2):148-52. DOI: 10.1016/j.vetimm.2008.09.008. View

Duvigneau J, Hartl R, Groiss S, Gemeiner M . Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. J Immunol Methods. 2005; 306(1-2):16-27. DOI: 10.1016/j.jim.2005.06.021. View

10.

Reyes-Leyva J, Espinosa B, Santos G, ZENTENO R, Hernandez J, Vallejo V . Purification and characterization of the hemagglutinin-neuraminidase of Porcine rubulavirus LPMV. Glycoconj J. 2000; 16(9):517-22. DOI: 10.1023/a:1007022021301. View

11.

Hjertner B, Linne T, Moreno-Lopez J . Establishment and characterisation of a porcine rubulavirus (LPMV) persistent infection in porcine kidney cells. Acta Vet Scand. 1997; 38(3):213-24. PMC: 8057016. View

12.

Linne T, Berg M, Bergvall A, Hjertner B, Moreno-Lopez J . The molecular biology of the porcine paramyxovirus LPMV. Vet Microbiol. 1992; 33(1-4):263-73. DOI: 10.1016/0378-1135(92)90054-w. View

13.

Salas-Rojas M, Sanchez-Hernandez C, Romero-Almaraz Md M, Schnell G, Schmid R, Aguilar-Setien A . Prevalence of rabies and LPM paramyxovirus antibody in non-hematophagous bats captured in the Central Pacific coast of Mexico. Trans R Soc Trop Med Hyg. 2004; 98(10):577-84. DOI: 10.1016/j.trstmh.2003.10.019. View

14.

Sundqvist A, Berg M, Hernandez-Jauregui P, Linne T, Moreno-Lopez J . The structural proteins of a porcine paramyxovirus (LPMV). J Gen Virol. 1990; 71 ( Pt 3):609-13. DOI: 10.1099/0022-1317-71-3-609. View

15.

Berg M, Bergvall A, Svenda M, Sundqvist A, Moreno-Lopez J, Linne T . Analysis of the fusion protein gene of the porcine rubulavirus LPMV: comparative analysis of paramyxovirus F proteins. Virus Genes. 1997; 14(1):55-61. DOI: 10.1023/a:1007987407250. View

16.

Escobar-Lopez A, Rivera-Benitez J, Castillo-Juarez H, Ramirez-Mendoza H, Trujillo-Ortega M, Sanchez-Betancourt J . Identification of antigenic variants of the porcine rubulavirus in sera of field swine and their seroprevalence. Transbound Emerg Dis. 2011; 59(5):416-20. DOI: 10.1111/j.1865-1682.2011.01286.x. View

17.

Stephens K, Hutchins R, Dauphin L . Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5'nuclease assay for Ebola virus. Mol Cell Probes. 2010; 24(6):370-5. DOI: 10.1016/j.mcp.2010.08.004. View

18.

Wang L, Hansson E, Yu M, Chua K, Mathe N, Crameri G . Full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the Americas. Arch Virol. 2007; 152(7):1259-71. PMC: 7086891. DOI: 10.1007/s00705-007-0959-4. View

19.

Sanchez-Betancourt J, Trujillo M, Mendoza S, Reyes-Leyva J, Alonso R . Genetic and antigenic changes in porcine rubulavirus. Can J Vet Res. 2012; 76(1):33-7. PMC: 3244285. View

20.

Hjertner B, Wiman A, Svenda M, Berg M, Moreno-Lopez J, Linne T . Multiple factors including subgenomic RNAs and reduced viral protein expression are associated with a persistent infection by porcine rubulavirus (LPMV). Arch Virol. 1998; 143(3):425-39. DOI: 10.1007/s007050050300. View