Seroepidemiology Survey and Isolation of Swine Influenza Viruses from Subclinical Infections in Israel, 2009-2011

AttachmentSize
davidson.pdf154.98 KB
Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 69 (2)  June 2014 Davidson, I. 62
INTRODUCTION
Infuenza A virus infection of pigs is of great interest and
concern worldwide, particularly since the emergence of the
pandemic virus (A(H1N1)pdm09) in 2009. Te circulating
swine infuenza viruses (SIVs) are extensively surveyed vi-
rologically and seroepidemiologically in many parts of the
world, including North and South America (1), Europe (2),
China (3)
and other Far Eastern countries. Infuenza viruses
have the potential for rapid spread amongst the swine pop-
ulation. Swine are infected through contact with infuenza-
infected humans (4), turkeys (5) and other birds, and are in
a continuous course of genetic change. Airway epithelia of
the upper respiratory tract of pigs express the two types of
cellular sialic acid receptors that preferentially bind avian
(NeuAc -alpha2,3-Gal) and human (NeuAc -alpha2,6-Gal),
infuenza viruses facilitating the formation of inter-species
reassortments. As a result, the emergence of new SIV variants
with potential zoonotic signifcances is a realistic scenario,
and pigs can serve as mixing vessels for the generation of new
human and avian viruses.
Seroepidemiology Survey and Isolation of Swine Infuenza Viruses
from Subclinical Infections in Israel During the Years 2009-2011
Davidson, I.,
1
* Al-Touri, A.,
1
Raibshtein, I.,
1
Hadani, Y.,
2
Bombarov, V.,
3
Yadin, H.,
4
ESNIP 3 consortium
5
and Reid, S.M.
6
1
Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, Israel, 50250.
2
State of Israel, Ministry of Agriculture and Rural development, Field Veterinary Services and Animal Health, Akko.
3
Division of Virology, Kimron Veterinary Institute, Bet Dagan, Israel, 50250.
4
Veterinary Private Consultant, Shefer 129, Israel 20120.
5
ESNIP 3 (European Surveillance Network for Infuenza in Pigs 3) consortium, FP7-INFLUENZA-2010, Grant # 25 9949,
Coordinated Action ESNIP 3.
6
Department of Avian Virology, Animal Health and Veterinary Laboratories Agency-Weybridge, Addlestone, Surrey, KT15
3NB, United Kingdom.
* Corresponding Author: Dr. Irit Davidson, Division of Avian Diseases, Kimron Veterinary Institute, Bet Dagan, Israel 50250. Tel: +972 3 9681602,
Fax: +972 3 9688964. Email: davidsoni@int.gov.il.
ABSTRACT
Te exposure of the swine population in Israel to swine infuenza viruses (SIV) was actively assessed
during the years 2009-2011 by serological and virological assays. Around 90% of 777 sera from 52 herds
and 2 wild boars were positive by ELISA. Te antibody subtype specifcity was determined on 407 sera
from 27 herds by haemagglutination inhibition assay with 4 viruses, A/sw/Flandre/1/98(H3N2), A/sw/
Scotland/410440/94(H1N2), A/sw/Cotes d’Armor/0388/09(H1N1) and A/ck/Israel/1525(H9N2). All
herds had antibodies to SIV H1N2 and H3N2 while only 10 herds had antibodies to SIV H1N1. Te
highest HI titers against SIV H1N2, SIV H3N2 HI titers were of intermediate values, while SIV H1N1
exposure produced the lowest titers. No antibodies to AIV subtype H9N2 were detected. Sub-clinically
infected pigs yielded fve positive samples, of which two were identifed as H3N2 and the pandemic H1N1,
respectively. We suspect that these were acquired by contact of the pigs with infected humans.
Keywords: Swine Infuenza Virus, Active Surveillance, Haemagglutination Inhibition, Virus
Isolation, Real Time Reverse Transcription RRT-PCR, ESNIP 3.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 69 (2)  June 2014 63 Swine Infuenza Survey in Israel
Te main enzootic SIV subtypes are H1N1, H3N2
and H1N2, except pandemic H1N1 virus that evaded the
swine population since 2009, as reviewed by Zell et al. (6)
European SIV lineages difer genetically from classical
SIVs and from the triple (avian, human and swine) North
American and Asian SIV reassortants. Avian-like H1N1
SIVs emerged in 1979 and spread to Europe. From further
reassortment with the human seasonal H3N2, the human-
like swine H3N2 SIV emerged in 1984. During 1994, the
human-like swine H1N2 also emerged after reassortment
of human-like swine H3N2 with human seasonal H1N1.
Tese reassortants, comprising genes of avian and human
origin, prevailed in European pigs and replaced the clas-
sical swine H1N1 viruses. Among the various SIV reas-
sortant viruses, the most eminent SIV is A(H1N1)pdm09,
which emerged in swine and caused the 2009 pandemic,
and then re-infected the swine population. Emergence of A
(H1N1)pdm09 resulted from the reassortment of two SIVs,
the North American triple reassortant, H1N1 or H1N2 and
the European avian-like H1N1 (2).
Active monitoring of swine infuenza is conducted on
apparently healthy, subclinically-infected pigs, side-by-side
with passive monitoring, performed on clinically-afected
pigs with apparent morbidity. Lately, subclinical infections
became a topic of increased awareness as the movement and
exhibition of pigs at agricultural fairs in the US presents a
risk of SIV dissemination
(7).
Te present study reports for the frst time evidence of
subclinical SIV infections in Israel in apparently healthy an-
imals, as in the previous serological survey, no evidence of
SIV infection of Israeli pigs was demonstrated (8). We now
illustrate an active serosurveillance of the swine population
at slaughter and attempts to isolate virus from subclinically
infected pigs. In so doing, the SIV subtype that prevailed in
the most eastern European border of swine herds before en-
tering the Far East was revealed.
MATERIALS AND METHODS
Israeli swine population
Te yearly swine production, coordinated by the Israeli
Veterinary Services, comprises around 200,000 pigs. Most
farms are concentrated in four regions, three of them are lo-
cated in the Northern part of the country and the fourth is
located in the Southern part. Te countries bordering Israel
do not focus on swine breeding; therefore, the swine popu-
lation in Israel is relatively isolated and refects the epide-
miological situation in the most easterly European region,
before the Far East.
ELISA
Te presence of SIV nucleocapsid antibodies was determined
in sera collected during the years 2009-2010 by ELISA us-
ing the ID Screen Infuenza A Antibody Competition kit
(IDVet Innovative Diagnostics, Fluaca ver 0509 GB), and on
sera collected during 2011 using the LSIVET Suis Infuenza
(LSI version SIVL 002 VA – 070409). Both kits performed
similarly.
Serum samples tested by the haemagglutination
inhibition assay
Sera from 27 herds were obtained at slaughter from appar-
ently healthy animals. Each was treated with receptor de-
stroying enzyme (RDE) to eliminate competitive inhibitors
of the haemagglutination inhibition (HI) test, according to
the method of Hofing et al.
(9), as recommended by the OIE
(10), and then inactivated at 56°C for 30 minutes. Briefy, 50
µl of sera were incubated at 37°C for 1 hr with 100 Units/ml
of RDE in calcium saline solution.
Reference sera and viruses
In the context of the ESNIP 3 Consortium, reference sera
and viruses were distributed to all consortium members by
Dr. Gaëlle Simon (Anses, Laboratory National de Référence
Pestes Porcines, Ploufragan, France). Te reference viruses
and their respective hyperimmune sera were:
1. A/sw/Flandre/1/98 H3N2
2. A/sw/Cotes d’Armor/0388/09 H1N1
3. A/sw/California/04/09 pH1N1
4. A/sw/Scotland/410440/94 H1N2
5. A/sw/Finistre/2899/82 H1N1
In addition, an avian infuenza virus, subtype H9N2, A/
ck/Israel/1525/H9N2
(11) was added to the HI panel.
Haemagglutination inhibition assay
Te HI assay (10) was used for serological identifcation of
SIV antibodies using the reference viruses and their hyper-
immune sera. As the initial serum dilution was 1:10, the HI
detection limit was 1:20. Te mean HI titer of the herd was
calculated on the basis of the titer dilution antilog.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 69 (2)  June 2014 Davidson, I. 64
SIV sampling and virus isolation
Five apparently healthy swine holdings were actively sam-
pled. From each holding fve diferent groups of 7-10 week-
old piglets, housed separately, were sampled. From each
group fve animals were sampled for virus identifcation and
isolation in embryonated specifc pathogen free fowls’ eggs
using both dry and transport medium (Copan, Ltd., Brescia,
Italy) soaked nasal swabs; a total of 125 swabs, each. Te fve
animals from each group were pooled, obtaining 25 pools.
Separate pools of dry and “wet” swabs in transport media
were used to purify RNA directly, and to amplify the virus-
es in embryonated eggs, respectively. Te RNA purifcation
was carried out using the QIAamp Viral RNA Mini KIT
(QIAGEN, GmbH, Germany) according to the manufac-
turer’s instructions. Te swabs in transport media were in-
oculated into allantoic cavities of 10-day-old embryonated
eggs (SPAFAS, U.S.A.) which were incubated at 37° C for
6 days. Te embryos were observed for 5 days after inocula-
tion; those dead within the frst day were discarded. Allantoic
fuids from eggs in which embryos were dead from the sec-
ond day post-inoculation, or from eggs with surviving em-
bryos up to 5 days, were used to demonstrate the presence
of SIV by the haemagglutination activity (HA) assay (10)
and by molecular amplifcation of RNA purifed from the
allantoic fuid.
SIV detection by real-time reverse transcription and
conventional PCR amplifcation
RNA samples were amplifed by real-time reverse tran-
scription PCR (RRT-PCR) for the SIV Matrix gene us-
ing the “perfect match” method described by Slomka et al.
(12), as well as for the presence of A(H1N1)pdm09 (14-
16), both by the TaqMan chemistry (Ambion Ag-Path,
Life Technologies, USA). Conventional amplifcation of the
RNA samples was performed using the multiplex RT-PCR
assay (Qiagen OneStep RT-PCR kit, QIAGEN) for difer-
entiating European SIV subtypes H1N1, H1N2 and H3N2
as described by Chiapponi et al., 2012 (13). Te presence
of A(H1N1)pdm09 was determined by RRT-PCR using
TaqMan chemistry, with the RRT-PCR assay (Ambion Ag-
Path master mix (Life Technologies, USA)) (14-16).
RESULTS AND DISCUSSION
Subtype specifcity of reference viruses and their
homologous and heterologous hyperimmune sera
Table 1 shows the assessment of the homologous and heter-
ologous HI assays using reference prototype SIVs and hyper-
immune sera raised against these viruses. It was apparent that
the homologous HI reactivity of the four reference viruses,
A/sw/Flandre/1/98 (H3N2), A/sw/Cotes d’Armor/0388/09
(H1N1), A/sw/California/04/09 A(H1N1)pdm09 and A/
sw/Scotland/410440/94 (H1N2) was very robust, while the
ffth virus, A/sw/Finistre/2899/82 (H1N1) was weaker, and
the virus titer did not allow it to be included in the HI check-
erboard matrix.
Active monitoring of antibodies to SIV in swine sera
and determination of their subtype specifcity
An initial serological survey was performed by ELISA to
demonstrate the presence of antibodies to the SIV nucleo-
capsid antigens. Table 2 shows that 90% of the 777 swine sera
tested, which were sampled between the years 2009-2011,
were positive for SIV antibodies, as well as the sera from two
wild boars. Tis therefore strongly suggests that the swine
population in Israel was extensively exposed to SIV.
Next, the SIV subtype specifcity was determined by
HI on a total of 407 sera from 27 herds and on serum
from one wild boar. Te HI was performed with three
SIV prototypes; namely, A/sw/Flandre/1/98 (H3N2),
Table 1: Subtype specifcity of reference hyper-immune sera and viruses by the HI assay
Hyperimmune serum H3N2
Flandre
H1N2
Scotland
pH1N1
California
H1N1
Cotes d’Armor
A/sw/Flandre/1/98 (H3N2) 1:5120 1:160 Negative Negative
A/sw/Cotes d’Armor/0388/09 (H1N1) 1:20 1:80 1:160 1:2560
A/sw/California/04/09 (pH1N1) 1:20 1:80 1:1280 1:40
A/sw/Scotland/410440/94 (H1N2) 1:160 1:5120 1:160 Negative
A/sw/Finistre/2899/82 (H1N1) 1:20 1:80 1:320 1:320
Negative serum Negative Negative Negative Negative
Table 2: Survey of antibodies to SIV nucleocapsid antigens by ELISA
Year
No. of swine
herds
Total sera
tested
No. total positive
sera (%)
Mean (%)
positive sera per
herd ± s.d.
2009
1 (wild boar) 1 1 100
19 241 151 (63) 91.5±18.6
2010
1 (wild boar) 1 1 100
21 346 306 (88) 84.6±30.4
2011 12 190 164 (86.3) 90.8±18.3
Research Articles
Israel Journal of Veterinary Medicine  Vol. 69 (2)  June 2014 65 Swine Infuenza Survey in Israel
A/sw/Scotland/410440/94 (H1N2) and A/sw/Cotes
d’Armor/0388/09 (H1N1). As pigs are receptive to infec-
tion with avian infuenza viruses, and as the AIV subtype33
H9N2 is endemic in Israel, an AIV H9N2 virus (A/ck/
Israel/1525/H9N2) was included in the subtype specifcity
examination of the swine sera.
Table 3 presents the mean antilog titers of the HI assay
performed with the four viruses. Firstly, it is evident that the
swine sera did not possess any antibodies to AIV H9N2,
indicating that although AIV H9N2 is endemic in Israel
within the poultry population (11), and its biological ability
to infect swine (17-19), it has not infected the pig popula-
tion, at least not the animals sampled in the present study. All
swine sera had antibodies to SIV H1N2 and H3N2, while
SIV H1N1 HI antibodies were detected only in 10 herds.
Te highest HI titers were against SIV H1N2, SIV H3N2
HI with titers of intermediate values, while SIV H1N1 ex-
posure initiated antibody with the lowest titers. Te wild boar
sera contained a high antibody HI titer to SIV H1N1. No
antibodies to H9N2 virus were detected.
Virus isolation and identifcation from subclinically
infected, apparently healthy pigs
Table 4 shows the results of the PCR assays that were per-
formed on seven samples obtained from the subclinically
infected pigs. Five apparently healthy swine holdings were
selected for investigation. From each holding, fve groups
of 7-10 week-old piglets were sampled. Five animals were
swabbed from each of the 25 groups. Pools of fve swabs, rep-
resenting the fve animals from each group, were examined
both directly and after being passaged twice in embryonated
fowls’ eggs for virus replication.
RNA was purifed from dry swabs and from allantoic
fuids after virus amplifcation in embryonated eggs. In fve
cases, SIV was detected by RRT-PCR, three of them were
positive in both RNAs, while the swab RNA from one case
had a low C
T
value, representing a low virus load, in the other
case, the virus could be detected only after the egg inocula-
tion had been performed.
Te multiplex conventional PCR was used to identify
the SIV subtype. Isolate A/sw/Israel/3/2011 had a high C
T
value for the M-gene by RRT-PCR, was positive for N1,
but not for H1 or H3, therefore, the isolate was suspected to
be A(H1N1)pdm09 (Figure 1). By applying the RRT-PCR
Table 4: Virus identifcation from sub-clinically infected, apparently
healthy pigs using RT-PCR
SIV samples
Real-time RT-PCR (C
T
value)
against
M- gene using RNA purifed from
SIV subtype
specifcity
identifcation
Nose Swab Allantoic fuid
A/sw/Israel/1/2011 32-36
A
Negative Negative
A/sw/Israel/2/2011 Negative Negative Negative
A/sw/Israel/3/2011 27-30 22.0 pH1N1
B
A/sw/Israel/4/2011 27-30 30-34 Negative
A/sw/Israel/5/2011 27-30 34 Negative
A/sw/Israel/6/2011 Negative Negative Negative
A/sw/Israel/7/2011 Negative 17-19 H3N2
C
A – Range of C
T
values obtained from several assay repeats performed at
KVI and the AHVLA.
B - Multiplex cPCR for H1N1, H1N2 and H3N2 and real-time RT-PCR
for pandemic H1N1
C - Multiplex cPCR for H1N1, H1N2 and H3N2
Table 3: Te subtype specifcity of SIV antibodies by HI assay
Sampling date Herd No. sera AIV H9N2 H3N2 H1N2 H1N1
28.01.2010 1 33 Negative 905 5120 Negative
05.08.2010 2 7 Negative 905 1810 Negative
11.01.2011 3 2 Negative 183 2228 Negative
11.01.2011 4 2 Negative 92 3151 Negative
12.01.2011 5 3 Negative 226 970 Negative
25.01.2011 6 2 Negative 184 1114 Negative
06.02.2011 7 3 Negative 1280 1575 Negative
15.02.2011 8 6 Negative 485 788 Negative
16.03.2011 9 20 Negative 32 2560 49
16.03.2011 10 20 Negative 69 905 12
21.03.2011 Wild boar Negative Negative 3151 6303
21.03.2011 11 20 Negative 160 3151 139
22.03.2011 12 20 Negative 519 1351 40
22.03.2011 13 20 Negative 211 1700 17
23.03.2011 14 10 Negative 905 735 14
23.03.2011 15 10 Negative 5120 2743 Negative
23.03.2011 16 10 Negative 452 1470 14
23.03.2011 17 10 Negative 686 3620 Negative
24.03.2011 18 20 Negative 905 5881 Negative
01.08.2011 19 20 Negative 485 2560 Negative
01.08.2011 20 20 Negative 1371 2940 40
01.08.2011 21 20 Negative 459 1940 Negative
04.08.2011 22 20 Negative 1575 2560 Negative
04.08.2011 23 20 Negative 367 1810 Negative
04.08.2011 24 20 Negative 442 2079 Negative
04.08.2011 25 20 Negative 970 3377 40
28.08.2011 26 20 Negative 905 1810 Negative
24.11.2011 27 29 Negative 1280 6303 Negative
Research Articles
Israel Journal of Veterinary Medicine  Vol. 69 (2)  June 2014 Davidson, I. 66
against A(H1N1)pdm09, A/Sw/Israel/3/2011 was indeed
identifed as the pandemic virus (14-16).
Samples A/sw/Israel/4/2011 and A/sw/Israel/5/2011 re-
acted weakly with the RRT-PCR for the M-gene and by
HA, and could not be subtyped. Sample A/Sw/Israel/7/2011
was positive by the M-gene RRT-PCR and was identifed as
SIV isolate subtype H3N2 by conventional PCR (Figure 1).
CONCLUSION
Te present study provides evidence for the exposure of the
Israeli swine population to all European SIV subtypes H1N2,
H3N2 and H1N1 and also to A(H1N1)pdm09, based on our
results from seroprevalence, on the one hand, and virus iso-
lation, on the other. Te virological evidence described here
confrms the presence of SIV in subclinical infections, be-
cause of the asymptomatical clinical appearance and the low
virus loads. Wild boars are also known to disseminate SIV,
as evidenced now by the presence of antibodies to H1N1 in
one wild boar. Tis study demonstrates for the frst time the
exposure to, and isolation of SIV from subclinically-infected
pigs in Israel, emphasizing the veterinary and public health
importance of SIV infections in domestic and in wild pigs.
As implicated previously (4), the two SIV isolates might be
acquired by contact of the pigs with infected humans.
ACKNOWLEDGEMENTS
Te study was supported fnancially by the ESNIP 3 consor-
tium, FP7-INFLUENZA-2010, Grant # 25 9949, Coordinated
Action ESNIP 3. We acknowledge the support of Drs. Ella
Mendelson and Michal Mandelboim, National Center for
Infuenza and Respiratory Viruses, Central Virology Laboratory
Sheba Medical Center, Tel-Hashomer, Ramat Gan, Israel, for the
molecular detection of the pandemic H1N1. Te technical assis-
tance of Vadim Pirogov, Israel Veterinary Services, Akko, and of
Anna Gorochov, Shmuel Abramson and Anita Kovtonenko, the
Division of Virology, Kimron Veterinary Institute, Bet Dagan,
Israel is acknowledged.
REFERENCES
1. Nelson, M.I., Detmer, S.E., Wentworth, D.E., Tan, Y., Schwartz-
bard, A., Halpin, R.A., Stockwell, T.B., Lin, X., Vincent, A.L.,
Gramer, M.R. and Holmes, E.C.: Genomic reassortment of in-
fuenza A virus in North American swine, 1998-2011. J. Gen. Vi-
rol.: 93:2584-2589, 2012.
2. Brown, I.H.: History and Epidemiology of Swine Infuenza in
Europe. Curr. Top. Microbiol. Immunol. 370:133-46, 2013.
3. Su, S., Qi, W., Chen, J., Zhu, W., Huang, Z., Xie, J. and Zhang
G.: Seroepidemiological evidence of avian infuenza a virus trans-
mission to pigs in southern china. Clin. Microbiol. 51:601-602,
2013.
4. Nelson, M.I., Gramer, M.R., Vincent, A.L. and Holmes, E.C.:
Global transmission of infuenza viruses from humans to swine.
J. Gen. Virol. 93:2195-2203, 2012.
5. Yassine, H.M., Lee, C.W. and Saif, Y.M.: Interspecies transmis-
sion of Infuenza A viruses between swine and poultry. Curr. Top.
Microbiol. Immunol. 370:227-40, 2013.
6. Zell, R., Scholtissek, C. and Ludwig, S.: Genetics, Evolution, and
the Zoonotic Capacity of European Swine Infuenza Viruses.
Curr Top Microbiol Immunol. 370:29-55, 2013.
7. Bowman, A.S., Nolting, J.M., Nelson, S.W. and Slemons, R.D.:
Subclinical infuenza virus A infections in pigs exhibited at ag-
ricultural fairs, Ohio, USA, 2009-2011. Emerg. Infect. Dis.
18:1945-1950, 2012.
8. Pozzi, S.P. Aborali, G., Cordioli, P. and Rosner, A.: Investigation
of swine infuenza subtypes H1N1, H3N2, H1N2 in pig popula-
tion in Israel (2002-2009). Isr. J. Vet. Med: 65:11-14, 2010.
9. Hofing, K., Klenk, H-D. and Herrler, G.: Inactivation of inhib-
itors by the receptor-destroying enzyme of infuenza C virus. J.
Gen. Virol.; 78:567-570, 1997.
10. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals.
World Organization for Anima Health. NB: Version adopted
by the World Assembly of Delegates of the OIE in May 2010.
Chapter 2. 8. 8. Swine Infuenza.
Figure 1: SIV subtype identifcation of the seven samples by
conventional multiplex PCR based on Chiapponi et al., 2012 (13).
M: molecular size marker, 1 kb lambda ladder (Invitrogen, ltd.);
1-7 suspected SIV samples, (+) SIV positive control, (-) Negative
control. Panel A.: Identifcation of H3 human-like SIV subtype. Te
positive control was RNA purifed from the prototype SIV A/sw/
Flandre/1/98 H3N2; Panel B.: Multiplex identifcation of N1 avian-
like and N2 human-like SIV subtypes. Te positive control was a mix
of RNA purifed from prototypes A/sw/Scotland/410440/94 (H1N2)
and A/sw/Finistre/2899/82 (H1N1). Te same positive control, as in
panel B was used for the multiplex identifcation of H1 avian-like
and H1-human-like SIV subtypes with negative results (not shown).
Research Articles
Israel Journal of Veterinary Medicine  Vol. 69 (2)  June 2014 67 Swine Infuenza Survey in Israel
11. Davidson, I., Shkoda, I., Golender, N., Perk, S., Lapin, K., Khin-
ich, Y. and Panshin, A.: Genetic characterization of HA gene of
low pathogenic H9N2 infuenza viruses isolated in Israel during
2006-2012 periods. Virus Genes. 46:255-263. 2013
12. Slomka, M.J., Densham, A.L., Coward, V.J., Essen, S., Brookes,
S.M., Irvine, R.M., Spackman, E., Ridgeon, J., Gardner, R., Han-
na, A., Suarez, D.L. and Brown, I,H.: Real time reverse transcrip-
tion (RRT)-polymerase chain reaction (PCR) methods for de-
tection of pandemic (H1N1) 2009 infuenza virus and Europe-
an swine infuenza A virus infections in pigs. Infuenza and other
respiratory viruses. 4:277-293, 2010.
13. Chiapponi, C., Moreno, A., Barbieri, I., Merenda, M. and Foni,
E.: Multiplex RT-PCR assay for diferentiating European swine
infuenza virus subtypes H1N1, H1N2 and H3N2. J. Virol.
Meth. 2012; 184: 117-120.
14. CDC. 2009. CDC protocol of realtime RTPCR for infuenza A
(H1N1). http://www.who.int/csr/resources/publications/swine-
fu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf.
15. Panning, M., Eickmann, M., Landt, O., Monazahian, M., Ols-
chläger, S., Baumgarte, S., Reischl, U., Wenzel, J.J., Niller, H.H.,
Günther, S., Hollmann, B., Huzly, D., Drexler, J.F., Helmer, A.,
Becker, S., Matz, B., Eis-Hübinger, A. and Drosten, C.: Detec-
tion of infuenza A(H1N1)v virus by real-time RT-PCR. Euro
Surveill. 14; pii 19329, 2009.
16. Hindiyeh, M.: Evaluation of a multiplex real-time reverse tran-
scriptase PCR assay for detection and diferentiation of infuenza
viruses A and B during the 2001-2002 infuenza season in Israel.
J. Clin. Microbiol., 43:589-95, 2005.
17. Cong, Y.L., Pu, J., Liu, Q.F., Wang, S., Zhang, G.Z., Zhang,
X.L., Fan, W.X., Brown, E.G. and Liu, J.H.: Antigenic and ge-
netic characterization of H9N2 swine infuenza viruses in China.
J. Gen. Virol. 88:2035-2041, 2007.
18. Yu, H., Hua, R.H., Wei, T.C., Zhou, Y.J., Tian, Z.J., Li, G.X.,
Liu, T.Q. and Tong G.Z.: Isolation and genetic characterization
of avian origin H9N2 infuenza viruses prom pigs in China. Vet.
Microbiol., 131: 82-92, 2008.
19. Yu, H., Zhou, Y.J., Li, G.X., Ma, J.H., Yan, L.P., Wang, B., Yang,
F.R., Huang, M. and Tong, G.Z.: Genetic diversity of H9N2 in-
fuenza viruses from pigs in China: a potential threat to human
health. Vet. Microbiol. 149: 254-261, 2011.
Research Articles

Published under a Creative Commons License By attribution, non-commercial