A multiplex RT-PCR for detection of type A influenza virus and differentiation of avian H5, H7, and H9 hemagglutinin subtypes

• Zhixun Xie^{a, ,} ,
• Yao-shan Pang^a,
• Jiabo Liu^a,
• Xianwen Deng^a,
• Xiaofei Tang^a,
• Jianhua Sun^a,
• Mazhar I. Khan^{b, 1, ,}

• ^a Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning, Guangxi 530001, People's Republic of China
• ^b Department of Pathobiology and Veterinary Science, University of Connecticut, 61 North Eagleville Road, Storrs, CT 06269-3089, USA

• Received 24 August 2005. Accepted 17 January 2006. Available online 20 March 2006.

• http://dx.doi.org/10.1016/j.mcp.2006.01.003, How to Cite or Link Using DOI

• Permissions & Reprints

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Keywords

• Multiplex;
• Polymerase chain reaction;
• Avian influenza virus;
• H5 subtype;
• H7 subtype;
• H9 subtype;
• Hemagglutinin

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1. Introduction

Influenza is a zoonotic disease, infecting a wide variety of warm-blooded animals, including birds and mammals. Influenza viruses are classified into types A, B, and C. Influenza A viruses are responsible for major disease problem in birds, as well as in humans [1], [2], [3], [4], [5], [6], [7], [8], [9] and [10]. Infections among domestic or confined birds have been associated with a variety of disease syndromes ranging from sub-clinical to mild upper respiratory disease, to loss of egg production, to acute generalized fatal disease. In domestic avian species, influenza viruses have caused considerable economic losses [1] and [2].

Influenza A are enveloped, negative-sense RNA viruses. Influenza A viruses are further classified into subtypes on the basis of the antigenic properties of their two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). To date, 15 HA and 9 NA subtypes have been identified. All influenza A virus subtypes have been found in aquatic and domestic birds, but only a few subtypes have been recovered from mammals and humans.

Among 15 HA subtypes, only H5 and H7 are highly virulent in poultry [1]. Historically, highly pathogenic avian influenza viruses (AIV) of poultry only belonged to the H5 and H7 hemagglutinin (HA) subtypes. Therefore, because there is a greater risk for these subtypes to become highly pathogenic, it is important to identify them specifically in surveillance programs [10] and [16]. Recently, H9 subtypes have been seen to cause infections in poultry [3] and [6]. Separate from the H5N1 influenza virus, another subtype of influenza virus, H9N2, has become panzootic in the last decade and has been isolated from different types of terrestrial poultry worldwide [3], [6] and [9].

Diagnosis of influenza A virus infection is routinely done by the isolation and identification of the virus. Serotyping is required to differentiate the subtypes of the AI viruses and is laborious and time-consuming. Furthermore, other tests required to determine the HA cleavage site sequence must be done to determine its potential virulence [12] and [13]. Although single band PCR has been used to detect and differentiate subtypes, it only recognizes one specific subtype at a time [14]. Standard RT-PCR has been previously applied to the detection of avian influenza virus [10], [15] and [16] and each of the 15 HA subtypes [14]. In addition, real time-RT-PCR assays for influenza virus have been developed for the detection of influenza virus types A and B [17] and differentiation of two subtypes H5, and H7 [11]. Equipment costs and specific technical training requirements limit usefulness of these assays as routine laboratory tests. In our study, we have developed a specific and sensitive multiplex RT-PCR that can simultaneously detect and differentiate the three most important subtypes of avian influenza viruses.

2. Materials and methods

2.1. Avian pathogens and culture conditions

The avian pathogens used in this study are listed in Table 1. All avian influenza virus (AIV) subtypes, Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) were propagated in the allantoic cavity of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs, whereas infectious laryngotracheitis virus (ILT) was propagated on the chorioallantoic membrane in 10-day-old SPF embryonated chicken eggs as described [18]; the allantoic fluids from embryonated eggs infected with AI, NDV, ILT and IBV were harvested after 36 h of incubation at 37 °C [18]. Mycoplasma gallisepticum (MG) was propagated in Frey's broth and incubated at 37 °C as described [19].

2.2. Extraction of RNA and DNA

Allantoic fluids from embryonated eggs infected with AI, NDV, IBV and ILT were first clarified by centrifugation at 500g for 15 min. The supernatants were transferred to new tubes and then centrifuged at 45,000g for 30 min at 4 °C. The supernatants were discarded and the pellets treated with 25 μl Rnase-free TE buffer (10 mM Tris–HCl and 1 mM ethylendiaminetetraacetic acid (EDTA)). The RNA extraction from AI, NDV, and IBV was carried out according to the Trizol LS manufacturer's protocol (Trizol, Invitrogen, Carlsbad, CA, USA). DNA from ILT and MG was extracted using the phenol:chloroform:isoamyl alcohol (25:24:1 v/v) (Amersham Life Science, Cleaveland, OH, USA) method as described by Pang et al. [20]. The concentrations of the RNA or DNA were determined by spectrophotometry using the UV2501PC (Shimadzu Corporation, Tokyo, Japan) and stored at −20 °C. An aliquot of 250 μl each was used for RNA extraction from the AI virus isolates listed in Table 1 according to the Trizol LS manufacturer's protocol (Trizol, Invitrogen, Carlsbad, CA, USA). Four-week-old SPF chicken (Jinan City, Shandong, China) was euthanized and 30 mg tissue samples from lung, liver, and small intestine were minced in TE buffer and RNA extraction was carried out according to the Trizol LS manufacturer's protocol for tissue samples (Trizol, Invitrogen, Carlsbad, CA, USA).

2.3. Primers designs and selection

Four sets of primers that specifically amplify type A influenza virus and simultaneously detect and differentiate H5, H7 and H9 subtypes of hemagglutinins are listed in Table 2. The primers for type A avian influenza were designed after reviewing the matrix gene sequences of over 10 different subtypes of AIV from chickens, ducks, geese, swine and humans; whereas primers for H5 subtypes were designed from hemagglutinin genes of avian origin. Primers for H7 and H9 subtypes were used from published data [14]. All four sets of oligonucleotide primers were synthesized at the Takara Shuzo Co., Ltd (Dalian, Shandong, China). The primers were aliquoted to a final concentration of 100 pmol/μl and stored at −20 °C.

2.4. Optimization of Reverse transcription and multiplex PCR reaction

The mRT-PCR consists of a two-step procedure, which includes reverse transcription (RT) and PCR amplification. An RT-PCR kit (Takara Shuzo Co., Ltd, Dalian, Shandong, China) was used for the reverse transcription reaction. RT was performed in 20 μl volumes, in which the reaction mixture contained 2 μl RNA in different concentrations and 5 mM MgCl₂, in PCR buffer Buffer (500 mM KCl, 100 mM Tris–HCl, pH 8.3), 1 mM of each dinucleoside triphosphate (dNTP), 2 units RNase inhibitor, 0.25 units avian myeloblastosis virus (AMV) reverse transcriptase, 1.25 pmol upstream primers of XZ145-2, XZ H5-1, XZ H7-1 and XZ H9-1. DEPC treated water was added to bring the final volume to 20 μl. RT was performed in a thermal cycler (Model 9600, Perkin Elmer Cetus, Norwalk, CT) for one cycle at 42 °C for 25 min, 99 °C for 3 min and 4 °C for 5 min.

The multiplex PCR was performed in a 50 μl volume using a PCR kit (Takara Shuzo Co., Ltd, Dalian, Shandong, China). The reaction contained 5 mM MgCl₂, 1×PCR Buffer, 10 mM of each dNTP, 0.5 pmol of each down stream primer XZ146, XZ H5-5, XZ H7-2 and XZ H9-2, and 1 unit TaKaRa LA Taq™ (Takara Shuzo Co., Ltd, Dalian, Shandong, China). This mixture was added to the RT reaction tubes. Sterile deionized water was added to the mixture to bring the total volume to 50 μl. The mPCR was carried out in the same thermal cycler used for RT. The cycling protocol consisted of an initial denaturing at 94 °C for 5 min, then 35 cycles that each consisted of denaturing at 94 °C for 45 s, annealing at 55 °C for 45 s, and extension at 72 °C for 105 s. The sample was then heated at 72 °C for 10 min for a final extension. A negative control did not contain template cDNA and consisted of PCR master mix, all four sets of primers and deionized water.

2.5. Detection of amplified nucleic acid products

Agarose gel electrophoresis was used to detect mRT-PCR nucleic acid products. A volume of 10 μl of amplified PCR nucleic acid products was subjected to electrophoresis at 80 V in horizontal gels containing 1% agarose with Tris–borate buffer (45 mM Tris–borate, 1 mM EDTA) as described [21]. The Gels were stained with ethidium bromide (0.5 mg/ml or 0.5 μg/ml^−l), and exposed to UV light to visualize the amplified nucleic acid products, and photographed using a Bio-vision Post-electrophoresis instrument (Vilber Lourmat, Paris, France).

2.6. Multiplex-RT-PCR sensitivity and specificity

Specificity of the mRT-PCR was determined by examining the ability of the test to detect type A influenza viruses and simultaneously differentiate H5, H7, and H9 subtypes. The mRT-PCR was tested using other avian pathogens that produce similar clinical signs or that can be present in mixed infections with AI subtypes. These pathogens and AIV subtypes are listed in Table 1. Random samples from the amplified mRT-PCR DNA bands were isolated from the gel and purified using a DNA Glass-milk purification kit (BioVed, Beijing, China). The purified DNA products were sent out to Takara Shuzo Co., Ltd, Dalian, Shandong, China for DNA sequencing. DNA sequences of the amplified products were than analyzed using the DNAStar software to confirm the amplified DNA sequence in the products. To determine the ability of the multiplex PCR assay to detect type A influenza virus and differentiate three subtypes in the same reaction, we used a mixture of RNA concentrations ranging from 200 ng to 10 fg RNA in various combinations of all subtypes of avian influenza virus. Sensitivity of the mRT-PCR was determined by making 10-fold serial dilutions of a mixture containing 10 ng of the RNA templates of each of the three viruses of AIV.

3. Results

Multiplex RT-PCR was developed to detect group A avian influenza viruses and simultaneously differentiate three hemagglutinin subtypes H5, H7, and H9 in a single reaction through 35 cycles of PCR. The mRT-PCR products were 244 bp for type A avian influenza viruses (group specific), 860 bp for H5, 634 bp for H7, 488 bp for H9. These products were visualized by electrophoresis (Fig. 1 and Fig. 2). This mRT-PCR was found to be a specific assay for type A avian influenza and hemagglutinin subtypes H5, H7, and H9, with no amplification of nucleic acid from NDV-F48, ILTV, IBV-M41, MG-S6 and SPF chicken tissues. The mRTPCR specifically detected the H5, H7, and H9 hemagglutinin subtypes along with their group specific type A specification. Thirty-seven avian influenza type A, including nine H5 subtypes, two H7 subtypes, and five H9 subtypes (listed in Table 1) were detected by the mPCR with the amplification of 244 bp DNA products. Limit of detection by visualization of PCR-amplified DNA products was 100 pg for the subtypes hemagglutinins, and 10 pg for type A avian influenza viruses (Fig. 2). Throughout the development of mRT-PCR, various modifications were made to the annealing temperature, extension time, cycle quantity, and primer concentrations in order to obtain the optimal conditions for this mRT-PCR. No spurious PCR amplification reactions among all influenza subtypes and other pathogens were noticed with various amounts of DNA and RNA mixtures. All the negative controls including RNA samples from the SPF chickens tissues were negative. DNAStar software analysis indicated that the mRT-PCR amplified DNA products were similar to the group A, subtypes H5, H7, and H9 genes sequences of avian influenza viruses.

4. Discussion and conclusions

The mRT-PCR developed here was able to detect type A influenza virus hemagglutin subtypes from H1 to H13 as tested (Table 1) and simultaneously detected and differentiated the very important hemagglutinin subtypes H5, H7 and H9 in one single reaction (Fig. 1 and Fig. 2). These hemagglutin subtypes recently have been a cause of human infections [2], [4], [5], [6] and [7]. Therefore, a mRT-PCR which can rapidly identify type A influenza as well as subtypes H5, H7 and H9, will be very important for the control of disease transmission in poultry and in humans. Use of this assay will also help reduce the economic losses in poultry associated with an AIV outbreak. This mRT-PCR is sensitive, specific, cost effective and it may be useful in diagnosis, screening and surveillance of poultry, including live bird market population. This mRT-PCR has the added benefits of being time saving, and using fewer reagents. Studies will be carried out to further test the specificity and sensitivity of this mRT-PCR on avian influenza subtypes from the various diagnostic and research laboratories as well as on clinical samples originated in the USA and South East China.