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Rapid subtyping of H9N2 influenza virus by a triple reverse transcription polymerase chain reaction
Journal of Virological Methods 158 (2009) 58–62
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Rapid subtyping of H9N2 influenza virus by a triple reverse transcription polymerase chain reaction Hao-Tai Chen 1 , Jie Zhang 1 , Li-Na Ma 1 , Yan-Ping Ma, Yao-Zhong Ding, Meng Wang, Xiang-Tao Liu, Yong-Guang Zhang, Yong-Sheng Liu ∗ Key Laboratory of Animal Virology of Ministry of Agriculture, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
a b s t r a c t Article history: Received 26 May 2008 Received in revised form 20 January 2009 Accepted 22 January 2009 Available online 6 February 2009 Keywords: H9N2 influenza virus Subtyping Triple reverse transcription polymerase chain reaction
The aim of this study was to develop a rapid, cost-saving triple reverse transcription polymerase chain reaction (triple RT-PCR) for subtyping H9N2 avian influenza viruses (AIVs). The three primer pairs for amplification of target sequences of nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA) genes, respectively, were designed for subtyping the viruses in the triple RT-PCR. The sensitivity of triple RT-PCR was found to be 102 copies per reaction for each of NP, H9 and N2 gene. The specificity tests indicated that all of NP, HA and NA genes were positive for H9N2, only NP gene was positive for H5N1 and H1N1 AIVs, and the results were negative for the other avian viruses including Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, duck hepatitis virus and avian encephalomyelitis virus. A total of 112 clinical samples were evaluated by the assay and the results showed that the sensitivity and specificity of triple RT-PCR were in accordance with the virus isolation. In conclusion, this method is rapid and cost-effective making it feasible and attractive for large-scale epidemiological investigation of H9N2 influenza virus. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Avian influenza A viruses are a public health threat worldwide because they are usually associated with severe illness and consequently a high risk of death. The viruses are enveloped, singlestranded negative-sense RNAs and classified further into subtypes 16 hemagglutinin (HA) and 9 neuraminidase (NA), respectively (Fouchier et al., 2005). To date, most HA/NA combinations have been identified in the domestic and wild bird reservoir. Among 16 HA subtypes, the H9N2 subtype of avian influenza viruses (AIVs) was detected first in the United States in 1966 (Homme and Easterday, 1970). In North America, there are no reports of H9N2 influenza virus associated disease in chickens, although these viruses can be found in wild ducks and have caused a number of outbreaks in turkeys (Kawaoka et al., 1988; Sharp et al., 1997). Reports from Europe, Asia and South Africa since the late 1990s indicated widespread distribution of H9N2 influenza virus (Alexander, 2003; Cameron et al., 2000; Lee et al., 2000; Naeem et al., 1999; Nili and Asasi, 2002; Perk et al., 2006). In terrestrial poultry of China, H9N2 influenza viruses were prevalent mainly in chickens and ducks (Guan et al., 1999; Li et al., 2003).
∗ Corresponding author. Tel.: +86 931 8342166; fax: +86 931 8340977. E-mail address: [email protected] (Y.-S. Liu). 1 These authors contributed equally to this work. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.01.026
Several molecular techniques, such as reverse transcription polymerase chain reaction (RT-PCR) and real time RT-PCR (RRTPCR), have been developed as rapid tests for AIVs (Ellis and Zambon, 2002; Playford and Dwyer, 2002; Ong et al., 2007). However, the single RT-PCR only recognizes one specific viral gene in one reaction and RRT-PCR requires expensive or specialized equipment and reagents and are not applied easily in many small laboratories. A triple RT-PCR method was established for typing of avian influenza A viruses, and for subtyping simultaneously of H9N2 influenza virus. 2. Materials and methods 2.1. Viral strains and clinical specimens 2.1.1. Viral strains The field isolate of A/duck/GD/GZ01/2007 (H9N2) was used to establish a triple RT-PCR for subtyping H9N2 of AIV. Eight influenza A viruses field isolates and eight reference strains of other avian viruses including Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), duck hepatitis virus (DHV) and avian encephalomyelitis virus (AEV) from the Chinese Veterinary Microorganism Conservation Center were used to evaluate the specificity of the triple RT-PCR assay (Table 1). All of field isolates were identified by RT-PCR and by sequencing (WHO, 2005; Lee et al., 2001).
H.-T. Chen et al. / Journal of Virological Methods 158 (2009) 58–62
59
Table 1 Strains and specificity of triple RT-PCR for H9N2 influenza viruses. Avian pathogen
Strain namea
HA
NA
NP
Influenza viruses
A/duck/GD/GZ01/2001 (H9N2) A/chicken/GD/GZ01/1998 (H9N2) A/duck/GD/GZ02/2007 (H9N2) A/chicken/GD/GZ02/2008 (H9N2) A/duck/HN/AH01/2007 (H5N1) A/chicken/HN/AH01/2007 (H5N1) A/human/GD/GZ01/2005 (H1N1) A/human/GS/LZ02/2005 (H1N1)
+ + + + − − − −
+ + + + − − − −
+ + + + + + + +
Infectious bronchitis viruses
IBV01 IBV02 IBV03
− − −
− − −
− − −
Infectious bursal disease viruses
IBDV01 IBDV02
− −
− −
− −
Newcastle disease virus
R/NDV
−
−
−
Duck hepatitis virus
DHV01
−
−
−
Avian encephalomyelitis virus
AEV01
−
−
−
a b
Resultb
The identity of these virus strains had been determined by RT-PCR and sequencing. Results of triple RT-PCR: (+) positive result; (−) negative result.
Table 2 Comparison of virus isolation, mRT-PCR and triple RT-PCR for detection of H9N2 influenza viruses in 112 clinical samples. Specimens
No. of samples
c
Oral swabs of chicken Oral swabs of ducks Cloacal scrapings of chicken Cloacal scrapings of ducks Total a b c
No. of positive samples for assay Virus isolationa
mRT-PCRb
Triple RT-PCR
51 40 11 10
35 35 7 8
35 35 7 8
35 35 7 8
112
85
85
85
Virus isolation culture-positive samples were all confirmed to be H9N2 influenza viruses by sequencing. mRT-PCR assay were conducted according to the methods described by Ong et al. (2007). Ten out of 51 oral swabs were from SPF chickens working as the negative controls in the experiments.
2.1.2. Clinical specimens A total of 112 clinical specimens were subjected to the triple RT-PCR. Among them, 102 specimens including 81 oral swabs and 21 cloacal scrapings were collected from the chicken and
duck farms with avian influenza outbreak. In addition, 10 oral swab samples from SPF chickens were used as negative controls (Table 2). The clinical specimens were prepared immediately as 10% suspension in viral transport medium, which was made
Fig. 1. (A–C) The mismatch between the primers of triple RT-PCR for subtyping H9N2 AIV and sequences of H9, N2 and NP gene of different avian influenza viruses, respectively.
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H.-T. Chen et al. / Journal of Virological Methods 158 (2009) 58–62
up of 0.05 M phosphate buffered saline (PBS), pH 7.4, containing penicillin (10,000 units/ml), streptomycin (10 mg/ml), gentamycin (250 g/ml) and mycostatin (5000 units/ml).
2.2. Laboratory methods 2.2.1. Primers design The primers were selected from the highly conserved regions of target genes specific for the nucleoprotein (NP), HA and NA of H9N2 influenza viruses, respectively, by the sequence data available in the GenBank. The sequence data were generated using the sequence analysis of the influenza database at http://www.flu.lanl.gov. The three primer sets were analyzed with the OLIGO 6.0 primer design software to ensure that they would be used together in a triple format. The sequence information and mismatch of the primers used to amplify each target gene with different AIVs are shown in Fig. 1.
Fig. 2. The electrophoresis analysis of the specificity of the triple RT-PCR results. Lane M, markers (2000, 1000, 750, 500, 250, 100 bp); lane 1, host-derived RNA; lane 2, DHV/2008/01; lane 3, AEV/2008/01; lane 4, R/NDV; lane 5, IBV/2008/01; lane 6, IBV/2008/02; lane 7, IBV/2008/03; lane 8, IBDV/2008/01; lane 9, IBDV/2008/02; lane 10, A/duck/HN/AH01/2007 (H5N1); lane 11, A/chicken/HN/AH01/2007 (H5N1); lane 12, A/human/GD/GZ01/2005 (H1N1); lane 13, A/human/GS/LZ02/2005 (H1N1); lane 14, A/duck/GD/GZ01/2007 (H9N2); lane 15, A/chicken/GD/GZ01/2008 (H9N2); lane 16, A/duck/GD/GZ02/2007 (H9N2); lane 17, A/chicken/GD/GZ02/2008 (H9N2).
3. Results 3.1. Triple RT-PCR for subtyping AIVs H9N2
2.2.2. RNA isolation All experiments with viruses were performed in a biosafety level 3 laboratory. Viral genomic RNA was extracted from the supernatant of the allantoic fluids or suspension solutions of clinical samples by using a QIAamp RNA extraction kit (QIAGEN), according to the protocol suggested by the manufacturer. The RNA was eluted from the QIAspin columns in a final volume of 50 l of elution buffer and immediately stored at −70 ◦ C until used.
The specificity of triple RT-PCR indicated that all of NP, HA and NA genes were positive for H9N2, only NP gene was positive for H5N1 and H1N1 AIVs, and the results were negative for the other avian viruses (Table 1). The expected triple RT-PCR products were 328 bp for H5N1 and H1N1 AIVs, 488, 238 and 328 bp for subtyping H9N2, and the other avian viruses and host-derived RNA did not give any visual band (Fig. 2). 3.2. Sensitivity of the triple RT-PCR system
2.2.3. Triple RT-PCR for typing and subtyping simultaneously The triple RT-PCR was carried out in a reaction mixture (50 l volume) containing 1× RT-PCR reaction buffer (Promega), 0.325 mM each of four dNTPs (Promega), 2.5 mM MgCl2 (Promega), 6 U AMV reverse transcriptase (Promega), 24 U RNase inhibitor (Genview), 4 U Taq polymerase (Promega), 0.4 M of each primer for H9F and H9R, 0.6 M of each primer for N2F and N2R, 0.8 M of each primer for NPF, and NPR, 3 l of RNA template. The triple RT-PCR conditions were 50 ◦ C for 35 min, 95 ◦ C for 1 min, 30 cycles of 94 ◦ C for 30 s, 55 ◦ C for 40 s and 68 ◦ C for 1 min, followed by a final 68 ◦ C for 2 min. The triple RT-PCR products were subjected to electrophoresis on a 1.5% agarose gel.
2.2.4. Sensitivity and specificity of triple RT-PCR The preparation of RNA transcripts of NP, H9 and N2 and calculation of copies of different templates for triple RT-PCR were conducted with the strain of A/duck/GD/GZ01/2007 (H9N2) according to the methods described by Ong et al. (2007). The sensitivity of the triple RT-PCR were evaluated with different copies of templates ranging from 1 to 106 with 10-fold dilutions and compared with the mRT-PCR assay described by Ong et al. (2007). The assay of each RNA dilution was conducted in triplicates. The specificity of the three primer sets was examined by triple RTPCR using templates extracted from AIV, host-derived RNA and other avian viruses, including NDV, IBV, IBDV, DHV and AEV described above. All of the clinical samples were investigated with virus isolation, the triple RT-PCR and the mRT-PCR, respectively.
2.2.5. DNA sequencing The amplification products of all the stains and clinical samples were sequenced with an automated ABI model 373 Stretch DNA sequencer. DNAStar software was applied to align the sequences and BLAST searching of GenBank was used to assess homology with the known NP, HA and NA gene.
The sensitivity of the triple RT-PCR using the three sets of primers was determined by testing a serial dilution of RNA standards of the NP, H9 and N2 gene. The sensitivity of the triple RT-PCR for each gene was found to be 102 copies/tube and the mRT-PCR was 103 copies/tube (Table 3). 3.3. Evaluation of the triple RT-PCR assay using clinical samples To evaluate the clinical sensitivity of the triple RT-PCR method, a total of 112 samples were tested by the mRT-PCR, the triple RTPCR, and virus isolation. The results indicated that the sensitivity and specificity of triple RT-PCR were in accordance with mRT-PCR and virus isolation. Among 85 specimens that were culture positive, all the specimens were also positive by the triple RT-PCR and
Table 3 Comparison of detection sensitivities of triplex RT-PCR and mRT-PCR. Method
RNA copy numbers (copies/tube)
NP
HA
NA
mRT-PCR
1 10 102 103 104 105 106
− − − + + + +
− − − + + + +
− − − + + + +
Triplex RT-PCR
1 10 102 103 104 105 106
− − + + + + +
− − ± + + + +
− − ± + + + +
a
Result of triplex RT-PCR with separate dilutions (106 –100 ) of RNA standardsa
(+) clearly visible; (±) visible but not clear; (−) not visible.
H.-T. Chen et al. / Journal of Virological Methods 158 (2009) 58–62
the mRT-PCR (Table 2). All the positive samples produced the three expected specific bands, which was confirmed to be AIVs H9N2 by sequencing. 4. Discussion Influenza A viruses H9N2 are present worldwide in poultry populations and derive from two major influenza virus gene pools, the Eurasian and the North American (Guo et al., 2000; Webster et al., 1992). In terrestrial poultry of Southern China, two H9N2 influenza virus lineages have become established since the mid1990s (Guan et al., 1999). Previous studies have shown that H9N2 influenza viruses are prevalent in chickens, ducks, and other minor poultry species in Asia and have demonstrated the ability to infect human beings (Li et al., 2003; Nicholson et al., 2003). Currently, human infections with wild-type strains of these viruses could occur in the USA in poultry and turkey farm workers and in travelers returning from countries where H9N2 influenza viruses are prevalent in birds. Laboratory-acquired infections could also occur in vaccine researchers (Chen et al., 2003). The prerequisite for controlling the disease is rapid and accurate identification of this virus. Successful amplification by a triple RT-PCR method relies on the specificities of designed primers. The study aimed at the establishment of a rapid method for the screening or detection of not only the NP genes, the relatively highly conserved gene in all type A viruses (Portela and Digard, 2002), but also the H9 and N2 gene in a single reaction. The primer pairs of the NP, H9 and N2, designed for the triple RT-PCR assay, were examined separately to ascertain their use for multiplex amplification under similar conditions. All primers were designed to ensure that the final reaction products could be differentiated easily on the basis of their size in a 1.5% agarose gel analysis. As a result, the triple RT-PCR method produced three bands of different sizes for H9N2 AIVs. The H9 and N2 primers are highly specific when detecting the H9N2 influenza viruses and the addition of NP primers aimed at determining viruses whose subtypes are neither H9 nor N2. Furthermore, validation of the specificity of the triple RT-PCR for H9N2 AIVs revealed that there was no cross-reactivity with other avian viruses including NDV, IBV, IBDV, DHV, AEV and host-derived RNA. In general, the relative sensitivity of the triple RT-PCR was lower than that of the single RT-PCR (Huang et al., 2004), but the similar sensitivity and specificity of the triple RT-PCR for clinical samples were obtained compared with that of the mRT-PCR. These findings suggest that the system may be specific in addition to high sensitivity. The present method provides a screening technique for differentiating H9N2 AIVs infection from the other respiratory diseases. In the experiment described above, oral swabs and cloacal scrapings are the samples of choice during the early stage of infection, which may have a higher predictive value of detecting H9N2 AIVs infection during disease surveillance screening. The early detection of H9N2 AIVs suggests that the triple RT-PCR method may be useful for disease control. This method is expected to detect H9N2 influenza viruses from preclinical specimens. The amplification outcome with this triple RT-PCR method could be explained in the case that the negative control is reasonable. The results may be interpreted as demonstrated in Table 4 according to the bands shown by agarose gel electrophoresis. DNA band of 328 bp corresponding to the NP was the prerequisite for determination of AIV by the triple RT-PCR. The high sensitivity and specificity observed with the use of triple RT-PCR described above suggest that the method could be of potential value for rapid detection of H9N2 AIVs in clinics. The specificity and sensitivity of the triple RT-PCR should be validated further and evaluated on a larger number of clinical specimens
61
Table 4 Interpretation of the amplification results for detecting clinical specimens with the triplex RT-PCR assay. Amplification product (bp)
Result
328 328 and 488 238 and 328 238, 328 and 488
Influenza A virus H9 influenza virus, not N2 N2 influenza virus, not H9 H9N2 influenza virus
and compared with that of the conventional virus isolation. This work is in progress and the preliminary results show the potential application of this system for rapid detection of the H9N2 influenza viruses. Acknowledgements This work was supported in part by grants from the National Key Technologies R&D Program of China (no. 2006BAD06A03 and 2008BADB4B05). This study also was supported by the National Natural Science Foundation of China (nos. 30671563 and 30700597) and National Scientific Program for Academy and Institute (no. BRF080305). References Alexander, D.J., 2003. Report on avian influenza in the eastern hemisphere during 1997–2002. Avian Dis. 47, 792–797. Cameron, K.R., Gregory, V., Banks, J., Brown, I.H., Alexander, D.J., Hay, A.J., Lin, Y.P., 2000. H9N2 subtype influenza A viruses in poultry in Pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong. Virology 278, 36–41. Chen, H., Subbarao, K., Swayne, D., Chen, Q., Lu, X., Katz, J., Cox, N., Matsuoka, Y., 2003. Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate. Vaccine 21, 1974–1979. Ellis, J.S., Zambon, M.C., 2002. Molecular diagnosis of influenza. Rev. Med. Virol. 12, 375–389. Fouchier, R.A.M., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D.M.E., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from blackheaded gulls. J. Virol. 79, 2814–2822. Guan, Y., Shortridge, K.F., Krauss, S., Webster, R.G., 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong. Proc. Natl. Acad. Sci. U.S.A. 16, 9363–9367. Guo, Y.J., Krauss, S., Senne, D.A., Mo, I.P., Lo, K.S., Xiong, X.P., Norwood, M., Shortridge, K.F., Webster, R.G., Guan, Y., 2000. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology 267, 279–288. Homme, P.J., Easterday, B.C., 1970. Avian influenza virus infections. I. Characteristics of influenza A-turkey-Wisconsin-1966 virus. Avian Dis. 14, 66–74. Huang, C.J., Hung, J.J., Wu, C.Y., Chien, M.S., 2004. Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Vet. Microbiol. 101, 209–214. Kawaoka, Y., Chambers, T.M., Sladen, W.L., Webster, R.G., 1988. Is the gene pool of influenza viruses in shorebirds and gulls different from that in wild ducks? Virology 163, 247–250. Lee, C.W., Song, C.S., Lee, Y.J., Mo, I.P., Garcia, M., Suarez, D.L., Kim, S.J., 2000. Sequence analysis of the hemagglutinin gene of H9N2 Korean avian influenza viruses and assessment of the pathogenic potential of isolate MS96. Avian Dis. 44, 527–535. Lee, M.S., Chang, P.C., Shien, J.H., Cheng, M.C., Shieh, H.K., 2001. Identification and subtyping of avian influenza viruses by reverse transcription-PCR. J. Virol. Meth. 97, 13–22. Li, K.S., Xu, K.M., Peiris, J.S., Poon, L.L., Yu, K.Z., Yuen, K.Y., Shortridge, K.F., Webster, R.G., Guan, Y., 2003. Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans? J. Virol. 77, 6988–6994. Naeem, K., Ullah, A., Manvell, R.J., Alexander, D.J., 1999. Avian influenza A subtype H9N2 in poultry in Pakistan. Vet. Rec. 145, 560. Nicholson, K.G., Wood, J.M., Zambon, M., 2003. Influenza. Lancet 362, 1733–1745. Nili, H., Asasi, K., 2002. Natural cases and an experimental study of H9N2 avian influenza in commercial broiler chickens of Iran. Avian Pathol. 31, 247–252. Ong, W.T., Omar, A.R., Ideris, A., Hassan, S.S., 2007. Development of a multiplex realtime PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A. J. Virol. Meth. 144, 57–64. Perk, S., Panshin, A., Shihmanter, E., Gissin, I., Pokamunski, S., Pirak, M., Lipkind, M., 2006. Ecology and molecular epidemiology of H9N2 avian influenza viruses isolated in Israel during 2000–2004 epizootic. Dev. Biol. 124, 201–209. Playford, E.G., Dwyer, D.E., 2002. Laboratory diagnosis of influenza virus infection. Pathology 34, 115–125.
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Portela, A., Digard, P., 2002. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J. Gen. Virol. 83, 723–734. Sharp, G.B., Kawaoka, Y., Jones, D.J., Bean, W.J., Pryor, S.P., Hinshaw, V., Webster, R.G., 1997. Coinfection of wild ducks by influenza A viruses: distribution patterns and biological significance. J. Virol. 71, 6128–6135.
Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152–179. World Health Organization (WHO), 2005. Recommended Laboratory Tests to Identify Avian Influenza A virus in Specimens from Humans. Available at www.who.int/ csr/disease/avian influenza/country/cases table 2006 06 06/en/index.html.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Rapid subtyping of H9N2 influenza virus by a triple reverse transcription polymerase chain reaction Hao-Tai Chen 1 , Jie Zhang 1 , Li-Na Ma 1 , Yan-Ping Ma, Yao-Zhong Ding, Meng Wang, Xiang-Tao Liu, Yong-Guang Zhang, Yong-Sheng Liu ∗ Key Laboratory of Animal Virology of Ministry of Agriculture, Key Laboratory of Veterinary Public Health of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
a b s t r a c t Article history: Received 26 May 2008 Received in revised form 20 January 2009 Accepted 22 January 2009 Available online 6 February 2009 Keywords: H9N2 influenza virus Subtyping Triple reverse transcription polymerase chain reaction
The aim of this study was to develop a rapid, cost-saving triple reverse transcription polymerase chain reaction (triple RT-PCR) for subtyping H9N2 avian influenza viruses (AIVs). The three primer pairs for amplification of target sequences of nucleoprotein (NP), hemagglutinin (HA) and neuraminidase (NA) genes, respectively, were designed for subtyping the viruses in the triple RT-PCR. The sensitivity of triple RT-PCR was found to be 102 copies per reaction for each of NP, H9 and N2 gene. The specificity tests indicated that all of NP, HA and NA genes were positive for H9N2, only NP gene was positive for H5N1 and H1N1 AIVs, and the results were negative for the other avian viruses including Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, duck hepatitis virus and avian encephalomyelitis virus. A total of 112 clinical samples were evaluated by the assay and the results showed that the sensitivity and specificity of triple RT-PCR were in accordance with the virus isolation. In conclusion, this method is rapid and cost-effective making it feasible and attractive for large-scale epidemiological investigation of H9N2 influenza virus. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Avian influenza A viruses are a public health threat worldwide because they are usually associated with severe illness and consequently a high risk of death. The viruses are enveloped, singlestranded negative-sense RNAs and classified further into subtypes 16 hemagglutinin (HA) and 9 neuraminidase (NA), respectively (Fouchier et al., 2005). To date, most HA/NA combinations have been identified in the domestic and wild bird reservoir. Among 16 HA subtypes, the H9N2 subtype of avian influenza viruses (AIVs) was detected first in the United States in 1966 (Homme and Easterday, 1970). In North America, there are no reports of H9N2 influenza virus associated disease in chickens, although these viruses can be found in wild ducks and have caused a number of outbreaks in turkeys (Kawaoka et al., 1988; Sharp et al., 1997). Reports from Europe, Asia and South Africa since the late 1990s indicated widespread distribution of H9N2 influenza virus (Alexander, 2003; Cameron et al., 2000; Lee et al., 2000; Naeem et al., 1999; Nili and Asasi, 2002; Perk et al., 2006). In terrestrial poultry of China, H9N2 influenza viruses were prevalent mainly in chickens and ducks (Guan et al., 1999; Li et al., 2003).
∗ Corresponding author. Tel.: +86 931 8342166; fax: +86 931 8340977. E-mail address: [email protected] (Y.-S. Liu). 1 These authors contributed equally to this work. 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.01.026
Several molecular techniques, such as reverse transcription polymerase chain reaction (RT-PCR) and real time RT-PCR (RRTPCR), have been developed as rapid tests for AIVs (Ellis and Zambon, 2002; Playford and Dwyer, 2002; Ong et al., 2007). However, the single RT-PCR only recognizes one specific viral gene in one reaction and RRT-PCR requires expensive or specialized equipment and reagents and are not applied easily in many small laboratories. A triple RT-PCR method was established for typing of avian influenza A viruses, and for subtyping simultaneously of H9N2 influenza virus. 2. Materials and methods 2.1. Viral strains and clinical specimens 2.1.1. Viral strains The field isolate of A/duck/GD/GZ01/2007 (H9N2) was used to establish a triple RT-PCR for subtyping H9N2 of AIV. Eight influenza A viruses field isolates and eight reference strains of other avian viruses including Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), duck hepatitis virus (DHV) and avian encephalomyelitis virus (AEV) from the Chinese Veterinary Microorganism Conservation Center were used to evaluate the specificity of the triple RT-PCR assay (Table 1). All of field isolates were identified by RT-PCR and by sequencing (WHO, 2005; Lee et al., 2001).
H.-T. Chen et al. / Journal of Virological Methods 158 (2009) 58–62
59
Table 1 Strains and specificity of triple RT-PCR for H9N2 influenza viruses. Avian pathogen
Strain namea
HA
NA
NP
Influenza viruses
A/duck/GD/GZ01/2001 (H9N2) A/chicken/GD/GZ01/1998 (H9N2) A/duck/GD/GZ02/2007 (H9N2) A/chicken/GD/GZ02/2008 (H9N2) A/duck/HN/AH01/2007 (H5N1) A/chicken/HN/AH01/2007 (H5N1) A/human/GD/GZ01/2005 (H1N1) A/human/GS/LZ02/2005 (H1N1)
+ + + + − − − −
+ + + + − − − −
+ + + + + + + +
Infectious bronchitis viruses
IBV01 IBV02 IBV03
− − −
− − −
− − −
Infectious bursal disease viruses
IBDV01 IBDV02
− −
− −
− −
Newcastle disease virus
R/NDV
−
−
−
Duck hepatitis virus
DHV01
−
−
−
Avian encephalomyelitis virus
AEV01
−
−
−
a b
Resultb
The identity of these virus strains had been determined by RT-PCR and sequencing. Results of triple RT-PCR: (+) positive result; (−) negative result.
Table 2 Comparison of virus isolation, mRT-PCR and triple RT-PCR for detection of H9N2 influenza viruses in 112 clinical samples. Specimens
No. of samples
c
Oral swabs of chicken Oral swabs of ducks Cloacal scrapings of chicken Cloacal scrapings of ducks Total a b c
No. of positive samples for assay Virus isolationa
mRT-PCRb
Triple RT-PCR
51 40 11 10
35 35 7 8
35 35 7 8
35 35 7 8
112
85
85
85
Virus isolation culture-positive samples were all confirmed to be H9N2 influenza viruses by sequencing. mRT-PCR assay were conducted according to the methods described by Ong et al. (2007). Ten out of 51 oral swabs were from SPF chickens working as the negative controls in the experiments.
2.1.2. Clinical specimens A total of 112 clinical specimens were subjected to the triple RT-PCR. Among them, 102 specimens including 81 oral swabs and 21 cloacal scrapings were collected from the chicken and
duck farms with avian influenza outbreak. In addition, 10 oral swab samples from SPF chickens were used as negative controls (Table 2). The clinical specimens were prepared immediately as 10% suspension in viral transport medium, which was made
Fig. 1. (A–C) The mismatch between the primers of triple RT-PCR for subtyping H9N2 AIV and sequences of H9, N2 and NP gene of different avian influenza viruses, respectively.
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up of 0.05 M phosphate buffered saline (PBS), pH 7.4, containing penicillin (10,000 units/ml), streptomycin (10 mg/ml), gentamycin (250 g/ml) and mycostatin (5000 units/ml).
2.2. Laboratory methods 2.2.1. Primers design The primers were selected from the highly conserved regions of target genes specific for the nucleoprotein (NP), HA and NA of H9N2 influenza viruses, respectively, by the sequence data available in the GenBank. The sequence data were generated using the sequence analysis of the influenza database at http://www.flu.lanl.gov. The three primer sets were analyzed with the OLIGO 6.0 primer design software to ensure that they would be used together in a triple format. The sequence information and mismatch of the primers used to amplify each target gene with different AIVs are shown in Fig. 1.
Fig. 2. The electrophoresis analysis of the specificity of the triple RT-PCR results. Lane M, markers (2000, 1000, 750, 500, 250, 100 bp); lane 1, host-derived RNA; lane 2, DHV/2008/01; lane 3, AEV/2008/01; lane 4, R/NDV; lane 5, IBV/2008/01; lane 6, IBV/2008/02; lane 7, IBV/2008/03; lane 8, IBDV/2008/01; lane 9, IBDV/2008/02; lane 10, A/duck/HN/AH01/2007 (H5N1); lane 11, A/chicken/HN/AH01/2007 (H5N1); lane 12, A/human/GD/GZ01/2005 (H1N1); lane 13, A/human/GS/LZ02/2005 (H1N1); lane 14, A/duck/GD/GZ01/2007 (H9N2); lane 15, A/chicken/GD/GZ01/2008 (H9N2); lane 16, A/duck/GD/GZ02/2007 (H9N2); lane 17, A/chicken/GD/GZ02/2008 (H9N2).
3. Results 3.1. Triple RT-PCR for subtyping AIVs H9N2
2.2.2. RNA isolation All experiments with viruses were performed in a biosafety level 3 laboratory. Viral genomic RNA was extracted from the supernatant of the allantoic fluids or suspension solutions of clinical samples by using a QIAamp RNA extraction kit (QIAGEN), according to the protocol suggested by the manufacturer. The RNA was eluted from the QIAspin columns in a final volume of 50 l of elution buffer and immediately stored at −70 ◦ C until used.
The specificity of triple RT-PCR indicated that all of NP, HA and NA genes were positive for H9N2, only NP gene was positive for H5N1 and H1N1 AIVs, and the results were negative for the other avian viruses (Table 1). The expected triple RT-PCR products were 328 bp for H5N1 and H1N1 AIVs, 488, 238 and 328 bp for subtyping H9N2, and the other avian viruses and host-derived RNA did not give any visual band (Fig. 2). 3.2. Sensitivity of the triple RT-PCR system
2.2.3. Triple RT-PCR for typing and subtyping simultaneously The triple RT-PCR was carried out in a reaction mixture (50 l volume) containing 1× RT-PCR reaction buffer (Promega), 0.325 mM each of four dNTPs (Promega), 2.5 mM MgCl2 (Promega), 6 U AMV reverse transcriptase (Promega), 24 U RNase inhibitor (Genview), 4 U Taq polymerase (Promega), 0.4 M of each primer for H9F and H9R, 0.6 M of each primer for N2F and N2R, 0.8 M of each primer for NPF, and NPR, 3 l of RNA template. The triple RT-PCR conditions were 50 ◦ C for 35 min, 95 ◦ C for 1 min, 30 cycles of 94 ◦ C for 30 s, 55 ◦ C for 40 s and 68 ◦ C for 1 min, followed by a final 68 ◦ C for 2 min. The triple RT-PCR products were subjected to electrophoresis on a 1.5% agarose gel.
2.2.4. Sensitivity and specificity of triple RT-PCR The preparation of RNA transcripts of NP, H9 and N2 and calculation of copies of different templates for triple RT-PCR were conducted with the strain of A/duck/GD/GZ01/2007 (H9N2) according to the methods described by Ong et al. (2007). The sensitivity of the triple RT-PCR were evaluated with different copies of templates ranging from 1 to 106 with 10-fold dilutions and compared with the mRT-PCR assay described by Ong et al. (2007). The assay of each RNA dilution was conducted in triplicates. The specificity of the three primer sets was examined by triple RTPCR using templates extracted from AIV, host-derived RNA and other avian viruses, including NDV, IBV, IBDV, DHV and AEV described above. All of the clinical samples were investigated with virus isolation, the triple RT-PCR and the mRT-PCR, respectively.
2.2.5. DNA sequencing The amplification products of all the stains and clinical samples were sequenced with an automated ABI model 373 Stretch DNA sequencer. DNAStar software was applied to align the sequences and BLAST searching of GenBank was used to assess homology with the known NP, HA and NA gene.
The sensitivity of the triple RT-PCR using the three sets of primers was determined by testing a serial dilution of RNA standards of the NP, H9 and N2 gene. The sensitivity of the triple RT-PCR for each gene was found to be 102 copies/tube and the mRT-PCR was 103 copies/tube (Table 3). 3.3. Evaluation of the triple RT-PCR assay using clinical samples To evaluate the clinical sensitivity of the triple RT-PCR method, a total of 112 samples were tested by the mRT-PCR, the triple RTPCR, and virus isolation. The results indicated that the sensitivity and specificity of triple RT-PCR were in accordance with mRT-PCR and virus isolation. Among 85 specimens that were culture positive, all the specimens were also positive by the triple RT-PCR and
Table 3 Comparison of detection sensitivities of triplex RT-PCR and mRT-PCR. Method
RNA copy numbers (copies/tube)
NP
HA
NA
mRT-PCR
1 10 102 103 104 105 106
− − − + + + +
− − − + + + +
− − − + + + +
Triplex RT-PCR
1 10 102 103 104 105 106
− − + + + + +
− − ± + + + +
− − ± + + + +
a
Result of triplex RT-PCR with separate dilutions (106 –100 ) of RNA standardsa
(+) clearly visible; (±) visible but not clear; (−) not visible.
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the mRT-PCR (Table 2). All the positive samples produced the three expected specific bands, which was confirmed to be AIVs H9N2 by sequencing. 4. Discussion Influenza A viruses H9N2 are present worldwide in poultry populations and derive from two major influenza virus gene pools, the Eurasian and the North American (Guo et al., 2000; Webster et al., 1992). In terrestrial poultry of Southern China, two H9N2 influenza virus lineages have become established since the mid1990s (Guan et al., 1999). Previous studies have shown that H9N2 influenza viruses are prevalent in chickens, ducks, and other minor poultry species in Asia and have demonstrated the ability to infect human beings (Li et al., 2003; Nicholson et al., 2003). Currently, human infections with wild-type strains of these viruses could occur in the USA in poultry and turkey farm workers and in travelers returning from countries where H9N2 influenza viruses are prevalent in birds. Laboratory-acquired infections could also occur in vaccine researchers (Chen et al., 2003). The prerequisite for controlling the disease is rapid and accurate identification of this virus. Successful amplification by a triple RT-PCR method relies on the specificities of designed primers. The study aimed at the establishment of a rapid method for the screening or detection of not only the NP genes, the relatively highly conserved gene in all type A viruses (Portela and Digard, 2002), but also the H9 and N2 gene in a single reaction. The primer pairs of the NP, H9 and N2, designed for the triple RT-PCR assay, were examined separately to ascertain their use for multiplex amplification under similar conditions. All primers were designed to ensure that the final reaction products could be differentiated easily on the basis of their size in a 1.5% agarose gel analysis. As a result, the triple RT-PCR method produced three bands of different sizes for H9N2 AIVs. The H9 and N2 primers are highly specific when detecting the H9N2 influenza viruses and the addition of NP primers aimed at determining viruses whose subtypes are neither H9 nor N2. Furthermore, validation of the specificity of the triple RT-PCR for H9N2 AIVs revealed that there was no cross-reactivity with other avian viruses including NDV, IBV, IBDV, DHV, AEV and host-derived RNA. In general, the relative sensitivity of the triple RT-PCR was lower than that of the single RT-PCR (Huang et al., 2004), but the similar sensitivity and specificity of the triple RT-PCR for clinical samples were obtained compared with that of the mRT-PCR. These findings suggest that the system may be specific in addition to high sensitivity. The present method provides a screening technique for differentiating H9N2 AIVs infection from the other respiratory diseases. In the experiment described above, oral swabs and cloacal scrapings are the samples of choice during the early stage of infection, which may have a higher predictive value of detecting H9N2 AIVs infection during disease surveillance screening. The early detection of H9N2 AIVs suggests that the triple RT-PCR method may be useful for disease control. This method is expected to detect H9N2 influenza viruses from preclinical specimens. The amplification outcome with this triple RT-PCR method could be explained in the case that the negative control is reasonable. The results may be interpreted as demonstrated in Table 4 according to the bands shown by agarose gel electrophoresis. DNA band of 328 bp corresponding to the NP was the prerequisite for determination of AIV by the triple RT-PCR. The high sensitivity and specificity observed with the use of triple RT-PCR described above suggest that the method could be of potential value for rapid detection of H9N2 AIVs in clinics. The specificity and sensitivity of the triple RT-PCR should be validated further and evaluated on a larger number of clinical specimens
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Table 4 Interpretation of the amplification results for detecting clinical specimens with the triplex RT-PCR assay. Amplification product (bp)
Result
328 328 and 488 238 and 328 238, 328 and 488
Influenza A virus H9 influenza virus, not N2 N2 influenza virus, not H9 H9N2 influenza virus
and compared with that of the conventional virus isolation. This work is in progress and the preliminary results show the potential application of this system for rapid detection of the H9N2 influenza viruses. Acknowledgements This work was supported in part by grants from the National Key Technologies R&D Program of China (no. 2006BAD06A03 and 2008BADB4B05). This study also was supported by the National Natural Science Foundation of China (nos. 30671563 and 30700597) and National Scientific Program for Academy and Institute (no. BRF080305). References Alexander, D.J., 2003. Report on avian influenza in the eastern hemisphere during 1997–2002. Avian Dis. 47, 792–797. Cameron, K.R., Gregory, V., Banks, J., Brown, I.H., Alexander, D.J., Hay, A.J., Lin, Y.P., 2000. H9N2 subtype influenza A viruses in poultry in Pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong. Virology 278, 36–41. Chen, H., Subbarao, K., Swayne, D., Chen, Q., Lu, X., Katz, J., Cox, N., Matsuoka, Y., 2003. Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate. Vaccine 21, 1974–1979. Ellis, J.S., Zambon, M.C., 2002. Molecular diagnosis of influenza. Rev. Med. Virol. 12, 375–389. Fouchier, R.A.M., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D.M.E., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from blackheaded gulls. J. Virol. 79, 2814–2822. Guan, Y., Shortridge, K.F., Krauss, S., Webster, R.G., 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong. Proc. Natl. Acad. Sci. U.S.A. 16, 9363–9367. Guo, Y.J., Krauss, S., Senne, D.A., Mo, I.P., Lo, K.S., Xiong, X.P., Norwood, M., Shortridge, K.F., Webster, R.G., Guan, Y., 2000. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology 267, 279–288. Homme, P.J., Easterday, B.C., 1970. Avian influenza virus infections. I. Characteristics of influenza A-turkey-Wisconsin-1966 virus. Avian Dis. 14, 66–74. Huang, C.J., Hung, J.J., Wu, C.Y., Chien, M.S., 2004. Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Vet. Microbiol. 101, 209–214. Kawaoka, Y., Chambers, T.M., Sladen, W.L., Webster, R.G., 1988. Is the gene pool of influenza viruses in shorebirds and gulls different from that in wild ducks? Virology 163, 247–250. Lee, C.W., Song, C.S., Lee, Y.J., Mo, I.P., Garcia, M., Suarez, D.L., Kim, S.J., 2000. Sequence analysis of the hemagglutinin gene of H9N2 Korean avian influenza viruses and assessment of the pathogenic potential of isolate MS96. Avian Dis. 44, 527–535. Lee, M.S., Chang, P.C., Shien, J.H., Cheng, M.C., Shieh, H.K., 2001. Identification and subtyping of avian influenza viruses by reverse transcription-PCR. J. Virol. Meth. 97, 13–22. Li, K.S., Xu, K.M., Peiris, J.S., Poon, L.L., Yu, K.Z., Yuen, K.Y., Shortridge, K.F., Webster, R.G., Guan, Y., 2003. Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans? J. Virol. 77, 6988–6994. Naeem, K., Ullah, A., Manvell, R.J., Alexander, D.J., 1999. Avian influenza A subtype H9N2 in poultry in Pakistan. Vet. Rec. 145, 560. Nicholson, K.G., Wood, J.M., Zambon, M., 2003. Influenza. Lancet 362, 1733–1745. Nili, H., Asasi, K., 2002. Natural cases and an experimental study of H9N2 avian influenza in commercial broiler chickens of Iran. Avian Pathol. 31, 247–252. Ong, W.T., Omar, A.R., Ideris, A., Hassan, S.S., 2007. Development of a multiplex realtime PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A. J. Virol. Meth. 144, 57–64. Perk, S., Panshin, A., Shihmanter, E., Gissin, I., Pokamunski, S., Pirak, M., Lipkind, M., 2006. Ecology and molecular epidemiology of H9N2 avian influenza viruses isolated in Israel during 2000–2004 epizootic. Dev. Biol. 124, 201–209. Playford, E.G., Dwyer, D.E., 2002. Laboratory diagnosis of influenza virus infection. Pathology 34, 115–125.
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