Multiplex RT-PCR assay for differentiating European swine influenza virus subtypes H1N1, H1N2 and H3N2.
In Europe, three major swine influenza viral (SIV) subtypes (H1N1, H1N2 and H3N2) have been isolated in pigs. Developing a test that is able to detect and identify the subtype of the circulating strain rapidly during an outbreak of respiratory disease in the pig population is of essential importance. This study describes two multiplex RT-PCRs which distinguish the haemagglutinin (HA) gene and the neuraminidase (NA) gene of the three major subtypes of SIV circulating in Europe. The HA PCR was able to identify the lineage (avian or human) of the HA of H1 subtypes. The analytical sensitivity of the test, considered to be unique, was assessed using three reference viruses. The detection limit corresponded to 1×10(-1) TCID(50)/200μl for avian-like H1N1, 1×10(0) TCID(50)/200μl for human-like H1N2 and 1×10(1) TCID(50)/200μl for H3N2 SIV. The multiplex RT-PCR was first carried out on a collection of 70 isolated viruses showing 100% specificity and then on clinical samples, from which viruses had previously been isolated, resulting in an 89% positive specificity of the viral subtype. Finally, the test was able to identify the viral subtype correctly in 56% of influenza A positive samples, from which SIV had not been isolated previously. It was also possible to identify mixed viral infections and the circulation of a reassortant strain before performing genomic studies.
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna Bruno Ubertini, Sezione Diagnostica di Parma, via dei Mercati 13/A, 43126 Parma, Italy. chiara.chiapponi@izsler.it
Chiara
C
Chiapponi
Ana
A
Moreno
Ilaria
I
Barbieri
Marianna
M
Merenda
Emanuela
E
Foni
D000818:Animals; D005060:Europe; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D057221:Influenza A Virus, H1N2 Subtype / Q000235:genetics / Q000302:isolation & purification*; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009439:Neuraminidase / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000662:veterinary* / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000821:virology; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase