Abstract Book of EAVLD2012 - eavld congress 2012

S1 - P - 07
ANALYTICAL EVALUATION OF A SWINE INFLUENZA VIRUS PCR ON AVIAN SAMPLES
J. C. Pedersen 1 , C. Boss 2 , A. Burrell 3 , C. O’Connell 3
1
National Veterinary Services Laboratory (NVSL), Ames, Iowa. USA
2
Life Technologies,Darmstadt, Germany
3
Life Technologies, Austin, USA
Introduction
Swine influenza is a highly contagious viral infection of pigs that
has a significant economic impact on affected herds. The disease
is caused by Swine Influenza Virus (SIV) which is a group of
specified viruses that are members of the family
Orthomyxoviridae and placed in the genus influenzavirus A.
Pigs play a unique role in the ecology of Influenza Type A, in that
they have receptors in their respiratory tract that will bind swine,
human, and avian influenza viruses. Consequently, pigs have
been called the ‘mixing vessels’ for the development of new
influenza viruses when swine, avian, and/or human influenza
viruses undergo genetic reassortment in pigs. Subtypes of SIV
that are most frequently identified in pigs include classical and
avian H1N1, human (hu) H1N1 and H1N2, reassortant (r) H3N2,
and rH1N2.
In theory, a PCR based test which detects RNA coding for the
matrix and nucleoprotein genes of SIV, an Influenzavirus A,
should be able to detect similar viral RNA sequences in avian
samples. The USDA licensed Swine Influenza Virus RNA Test Kit
(VetMAX ® -Gold SIV Detection Kit) was used to test RNA
extracted from a panel of clinical Avian Influenza Virus samples
representing all 16 HA subtypes in order to assess the
performance of the test in species other than swine.
Materials & methods
A panel of 34 cultured isolates (stock virus) representing all 16
HA Influenzavirus A subtypes was assembled at the NVSL. RNA
was extracted from the samples using the MagMAX ® Viral RNA
Isolation Kit following manufactures’ instructions. Undiluted RNA
extracted from the stock viruses was tested using the VetMAX ® -
Gold SIV Detection Kit real time PCR from Life Technologies
following the product insert instructions. The test detects RNA
representing two different targets on the SIV matrix gene as well
as an additional target on the nucleoprotein gene.
Results
Strong amplification signals were obtained (Ct range: 13.2-20.9)
for each of the panel members tested. The quality standards
including positive and negative extraction conotrols, positive kit
control and no template controls performed as expected.
Discussion & conclusions
This preliminary study gives an indication that further validation
studies are warranted to support additional claims for use of this
product for the detection of Influenzavirus A in avian samples.
On a global level, the use diagnostic assays for the detection of
certain reportable diseases is becoming more highly regulated in
order to ensure that clinical decisions are being made from high
quality laboratory results. In light of this, the advantages listed
above must be understood along with the limitations of these
reported data.
Limitations: The VetMAX ® -Gold SIV Detection Kit is a USDA
licensed product for the detection of Swine Influenza Virus (SIV)
RNA isolated from nasal swab samples. Off-label uses of the
product in the United States are not supported by the
manufacturer and any additional claims for performance of the kit
must be supported by rigorous studies which must be submitted
and approved by the USDA before any additional claims can be
made in the US. Usage claims in other countries outside the US
are subject to the regulatory requirement(s) within each country
of use.
References
OIE Terrestrial Manual 2010, Chapter 2.8.8. Swine Influenza, accessed 30
Mar 2012
SIV, PCR, Avian Samples