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Further Characterization of PCV2 Isolate

AVAILABLE TESTS

Detection of PCV2 Nucleic Acids

Detection of Anti-PCV2 Antibodies

Detection of PCV2 Virus or Viral Antigen

Available Tests

 

 PCV2 homepage

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

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Restriction Fragment Length Polymorphism (RFLP)

RFLP uses enzyme digestion of viral nucleic acid (partial or whole) which results in a specific cutting pattern that is visualized on a gel. If there are differences between viruses at the site of enzyme cutting, different patterns can be observed. This fingerprinting technique has been commonly used for DNA viruses. An ORF2 based PCR-RFLP assay described in 2000 using HinfI, HinP1I, KpnI, MseI, and RsaI enzymes is able to distinguish among PCV2 isolates (PCV2A, B, C, D, and E) (Hamel et al., 2000).

Restrictions fragment length polymorphism (RFLP):
From: Hamel et al., Can J Vet Res. 2000.

 

A PCR-RFLP assay using NcoI enzyme that differentiates between PCV1 and PCV2 was described in 2000 (Fenaux et al., 2000).

Restrictions fragment length polymorphism (RFLP):
From: Fenaux et al., J Clin Microb. 2000

 

An ORF2 based PCR-RFLP assay using Sau3AI, BanII, NspI, XbaI, and CfrI enzymes has been described recently and is able to distinguish 9 different PCV2 genotypes (Wen et al., 2005). PCV2 RFLP analysis showed that there was a significant change from RFLP type 422 to type 321 in 2005 in Ontario, Canada (Delay et al., 2005).

 

Trends in RFLP types in Canada from 2000-2005.
From: AHL Newsletter, 9:3, 2005 

 

Sequencing

PCV2 sequencing:
Entire genome =1,769 bp 
 
 
PCV2 sequencing: Open reading frame 2 (ORF2) = 688 bp (red area); encodes for the capsid protein 
Dendogram based on entire PCV2 sequencing and comparsion of PCV2 sequences available from the GenBank. From: T. Opriessnig, Thesis, Austria, 2002.

With sequence analysis it is possible to characterize the genetic information and compare isolates to each other (Choi et al., 2002; De Boisséson et al., 2004; Fenaux et al., 2000; Grierson et al., 2004; Kim and Lyoo, 2002; Mankertz et al., 2000). To further investigate possible differences among PCV2 isolates it is possible to sequence the entire PCV2 genome or to sequence only ORF2.

At this point in time, sequencing results may be a useful epidemiological tool but the knowledge to determine virulence based on sequence information is not yet available.

Based on ORF2 sequencing, our isolates are less than 95% homologous to recent eastern Canadian isolates. Recent Canadian and related European strains have been summarized as PCV2b which can be distinguished from PCV2a which includes older North American and European isolates by open reading frame 2 (ORF2) sequencing. There is limited field evidence that PCV2B strains are associated with increased mortality in affected herds.

References:

Choi J, Stevenson GW, Kiupel M, Harrach B, Anothayanontha L, Kanitz CL, Mittal SK: Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome. Can J Vet Res. 66:217-224, 2002

De Boisséson C, Béven V, Bigarré L, Thiéry R, Rose N, Eveno E, Madec F, Jestin A: Molecular characterization of porcine circovirus type 2 isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs. J Gen Virol. 85:293-304, 2004

Delay J, McEwen B, Carman S, van Dreuel T, Fairles J: Porcine circovirus type 2-associated disease is increasing. AHL Newsletter. 9:22, 2005

Fenaux M, Halbur PG, Gill M, Toth TE, Meng XJ: Genetic characterization of type 2 porcine circovirus (PCV-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1 and PCV-2. J Clin Microbiol. 38:2494-2503, 2000

Grierson SS, King DP, Sandvik T, Hicks D, Spencer Y, Drew TW, Banks M: Detection and genetic typing of type 2 porcine circovirus in archived pig tissues from the UK. Arch Virol. 149:1171-1183, 2004

Hamel AL, Lin LL, Sachvie C, Grudeski E, Nayar GP: PCR detection and characterization of type-2 porcine circovirus. Can J Vet Res. 64:44-52, 2000

Kim JH, Lyoo YS: Genetic characterization of porcine circovirus-2 field isolates from PMWS pigs. J Vet Sci. 3:31-39, 2002

Mankertz A, Domingo M, Folch JM, LeCann P, Jestin A, Segalés J, Chmielewicz B, Plana-Durán J, Soike D: Characterisation of PCV-2 isolates from Spain, Germany and France. Virus Res. 66:65-77, 2000

Wen L, Guo X, Yang H: Genotyping of porcine circovirus type 2 from a variety of clinical conditions in China. Vet Microbiol. 110:141-146, 2005