Abstract
Avian influenza and Newcastle disease are the highly contagious and most economically important diseases in poultry industry throughout the world. A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for the rapid and specific discrimination of H5 and H9 subtypes of avian influenza viruses (AIV) and Newcastle disease virus (NDV). Three sets of specific primers were applied in the assay based on the sequences of the hemagglutinin gene of H5-AIV, H9-AIV and fusion protein gene of NDV. 59 clinical samples including the throat washes, oral swabs, and cloacal scrapings were detected by mRT-PCR and single RT-PCR (sRT-PCR), respectively. The results indicated that the sensitivity and specifity of mRT-PCR were in accordance with sRT-PCR. The mRT-PCR developed in this study may therefore provide a new avenue to rapid detection of these important pathogens in one reaction.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Avian influenza (AI) and Newcastle disease (ND) are two of the most devastating avian diseases in the world, which is caused by avian influenza virus (AIV) and Newcastle disease virus (NDV), respectively. AIV is responsible for major disease problem in birds as well as in humans (Alexander 2000; Cameron et al. 2000; Li et al. 2003; Tran et al. 2004; Normile 2005; Ng and To 2007), There are 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes of AIV (Fouchier et al. 2005), but only a few subtypes have been recovered from mammals and humans (Xie et al. 2006). Recently, the repeated incidences of H5N1 subtype of AIV in commercial chickens (Webster 2006) and the high prevalence of H9N2 in chickens throughout Asia, and along with their potential important role in the emergence of avian influenza pandemic (Lin et al. 2000), have accelerated the development of detection of AIV subtypes. It is difficult to discriminate AI and ND according to the clinical signs because both diseases may cause loss of egg production, acute respiratory infection and serosal hemorrhages of the gastrointestinal tract of domestic avian species. (Capua and Alexander 2004; Hubalek 2004; de Leeuw et al. 2005; Sakai et al. 2006). Rapid differential diagnosis of H5-AIV, H9-AIV and NDV is therefore a matter of urgency in order to prevent and control the epidemics.
Although virus isolation in embryonating eggs is a routinely sensitive method for diagnosis of AIV and NDV (Alexander et al. 1999; Alexander 2000), it may be time-consuming and laborious. Other tests such as reverse transcription polymerase chain reaction (RT-PCR) and real time RT-PCR have been previously applied to the detection of AIV and NDV (Bruckner et al. 1996; Fouchier et al. 2000; Wise et al. 2004; Pham et al. 2005; Ng et al. 2006; Das et al. 2006), but the single RT-PCR (sRT-PCR) only recognizes one specific virus in one reaction and expensive equipment and specific technical training requirements limit usefulness of real-time RT-PCR as routine laboratory tests. No multiplex RT-PCR (mRT-PCR) has been reported so far to detect H5-AIV, H9-AIV and NDV. In this study, a specific and sensitive mRT-PCR was developed for simultaneous detection and differentiation of these viruses.
Materials and methods
Viruses and plasmids
The viruses including field isolates of H5-AIV (A/duck/HN/AH01/2007(H5N1)), H9-AIV (A/chicken/GD/GZ01/2007(H9N2)), and reference strain of NDV (Roakin/NDV) from Chinese Veterinary Microorganism Conservation Center were used to standardize the mRT-PCR. All of the strains were identified by sRT-PCR and sequencing.
Recombinant plasmids of pT-H9, pT-H5 and pT-F were constructed by ligation of pGEM-T easy vector (Promega, Shanghai, China) and HA genes of H5-AIV, H9-AIV and fusion (F) protein gene of NDV (Roakin/NDV) and served as standard for determining the specificity of the assays.
Clinical specimens
Clinical specimes include throat washes, oral swabs and cloacal scrapings. Throat washes were collected with 1 ml syringe filled with 0.05M pH7.4 phosphate buffer to rinse the throat of the bird one or two times. 49 specimens comprised of 15 throat washes, 13 oral swabs, and 21 cloacal scrapings were collected from 25 chickens and 24 ducks that were infected naturally after initial appearance of clinical syndrome. In addition, 10 throat washes from specific-pathogen-free (SPF) chickens were used as negative control. The clinical specimens were suspended immediately in viral transport medium, which was made up of 0.05 M phosphate buffered saline (PBS), pH 7.4, containing antibiotics of penicillin (2000 units/ml), streptomycin (2 mg/ml), gentamycin (50 μg/ml) and mycostatin (1000 units/ml) for throat washes and oral swabs, and the antibiotic concentration is five-fold higher for cloacal scraping samples. The details of clinical samples were listed in Table 1.
RNA extraction
The processing of specimens was performed under biosafety level 3 containment facilities. The clinical sample suspensions were centrifuged at 6,500 × g for 1 min and the supernatants were collected. Viral RNAs were extracted from 140 μl of the supernatants by using a QIAamp viral RNA mini kit (Qiagen). After lysis of the specimens, the mixture was applied to a spin column as described by the manufacturer’s protocol. The extracted RNAs were eluted in a total volume of 60 μl of elution buffer and were stored at −70°C until further use.
sRT-PCR and sequencing
Three different sets of primers specific for amplifying conserved region of HA gene of H5-AIV, H9-AIV and F gene of NDV were designed for sRT-PCR and mRT-PCR and listed in Table 2. The sRT-PCR was performed using SuperScript one-step RT-PCR kit (Invitrogen). Primers of H5F/H5R, H9F/H9R of AIV and F1/F2 of NDV were employed in the reaction, respectively. The sRT-PCR was performed in a 50 μl total reaction volume with 50 pmol of each primer according to the manufracturer’s protocol. The thermal profile for sRT-PCR was 94°C for 2 min, followed by 30 cycles of 94°C for 1 min, 55°C for 30 s, and 72°C for 1 min and a final extension cycle at 72°C for 5 min.
All of the 59 clinical samples including negative control were identified by sRT-PCR and sequencing for H5-AIV, H9-AIV and NDV, respectively.
The purified sRT-PCR products for clinical samples were sent out to Takara Biotechnology Co., Ltd, Dalian, China for DNA sequencing with an automated ABI model 373A Stretch DNA sequencer. DNA sequences of the products were then analyzed using the DNAStar to confirm the amplified DNA sequence.
mRT-PCR
The mRT-PCR was performed using SuperScript one-step RT-PCR kit (Invitrogen). Three sets of primers were employed in one reaction. The mRT-PCR was carried out in a reaction volume of 50 μl containing 10 pmol of each primer, 1 μl of RT/Taqase Mix, 25 μl of 2× Reaction Mix and 5 μl of each viral RNA or plasmids of pT-F, pT-H5 and pT-H9. Reverse transcription reaction was carried out at 52°C for 30 min followed by the cycling protocol of an initial denaturing at 94°C for 2 min, then 30 cycles that each consisted of denaturing at 94°C 30 s, annealing at 52°C 30 s, and extension at 72°C for 1 min. The sample was then heated at 72°C for 2 min. The mRT-PCR products were separated in 1% agarose gels, run in 0.5× TAE buffer with 0.5 μg/ml ethidium bromide at 10 V/cm for 25 min, and visualized under UV light. These different mRT-PCR products were subjected to DNA sequencing.
Results
The specificity of mRT-PCR
The mRT-PCR products visualized by gel electrophoresis were 266 bp for H9-AIV, 229 bp for H5-AIV and 136 bp for NDV (Fig. 1). The mRT-PCR was found to be a specific assay for H5-AIV, H9-AIV and NDV, with no amplification of nucleic acid from SPF chicken throat washes. After DNA sequencing, DNAStar software analysis indicated that the mRT-PCR amplified DNA products were similar to sequences of HA of H5-AIV and H9-AIV, and F gene of NDV, respectively.
Evaluation of mRT-PCR with clinical samples
The applicability of mRT-PCR assay for detection of H5-AIV, H9-AIV and NDV in the diagnosis was validated by evaluating the assay system with the 59 clincal samples as mentioned above. Among 49 positive clinical samples, 37 birds were infected individually and 12 birds were co-infected. The assay indicated that 7 samples were positive for NDV, 19 samples were positive for H9-AIV, 11 samples were positive for H5-AIV, 7 samples were coinfected with both H5-AIV and NDV, 2 samples were coinfected with both H9-AIV and NDV, and 3 samples were coinfected with both H5-AIV and H9-AIV (Table 1). In addition, all the negative controls from the SPF chickens throat washes were shown to be free of these pathogens. All of these clinical samples were also tested separately by sRT-PCR. The results indicated that the sensitivity and specificity of mRT-PCR were in accordance with sRT-PCR. The mRT-PCR gel electrophoresis analysis of partial clinical samples was shown in Fig. 2.
Discussion
Upon typical conditions of intensive poultry production, several clinically similar viral diseases can occur which require laboratory differential diagnosis. Avian influenza is the most important viral zoonosis from avian sources (Antal et al. 2007; Tsuda et al. 2007; Wakamatsu et al. 2007) and Newcastle disease is also an economically important disease in poultry industries (Steel et al. 2008). Since 2002 the incidence of H5N1 in domestic poultry has increased substantially, while H9N2 has become endemic in Europe and Asia. Recent studies have shown the presence of multiple subtypes of AIV from samples collected from chickens and waterfowls (Panigrahy et al. 2002; Hanson et al. 2005; Nguyen et al. 2005). The mRT-PCR assay which can rapidly identify H5-AIV, H9-AIV and NDV will be therefore very important for the control of disease transmission from avian species to humans and reduction of the economic losses in poultry industries (Shu et al. 2006; Corbanie et al. 2007; Han et al. 2008).
In this study, mRT-PCR assay was demonstrated to be capable of rapid detection and differentiation of three important avian RNA viruses in clinical specimens. Precise primer design and the appropriate ration of each primer pair are crucial for a successful amplification in mRT-PCR (Bej et al. 1990). In general, the relative sensitivity of mRT-PCR was lower than that of sRT-PCR (Huang et al. 2004), but the similar sensitivity and specificity of mRT-PCR for clinical samples was obtained compared with that of sRT-PCR in the study. This may result from the optimization of primer designation or limited samples. Further studies have to be evaluated with a large-scale investigation in order to advance our understanding in this respect.
The throat washes, oral swabs, and cloacal scrapings are the samples of choice during the early stages of infection, which may have a higher predictive value of detecting H5-AIV, H9-AIV and NDV infection during disease surveillance screening. Importantly, the early detection suggests two potential uses in disease control for the assay: as a surveillance tool in areas free of the disease and as a screening assay for monitoring a disease outbreak.
In conclusion, the mRT-PCR method for rapid detection of H5-AIV, H9-AIV and NDV provides a more convenient and reliable means for routine diagnosis of bird disease and may be employed to screen for potential carriers in birds.
Abbreviations
- AI:
-
avian influenza
- AIV:
-
avian influenza virus
- F:
-
fusion protein
- HA:
-
hemagglutinin
- mRT-PCR:
-
multiplex RT-PCR
- ND:
-
Newcastle disease
- NDV:
-
Newcastle disease virus
- RT-PCR:
-
reverse transcription polymerase chain reaction
- sRT-PCR:
-
single RT-PCR
References
Alexander, D.J., 2000. A review of avian influenza in different bird species. Veterinary Microbiology, 74, 3–13
Alexander, D.J., Banks, J., Collins, M.S., Manvell, R.J., Frost, K.M., Speidel, E.C. and Aldous, E.W., 1999. Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997. Veterinary Record, 145, 417–421
Antal, M., Farkas, T., German, P., Belak, S. and Kiss, I., 2007. Real-time reverse transcription-polymerase chain reaction detection of Newcastle disease virus using light upon extension fluorogenic primers. Journal of Veterinary Diagnostic Investigation, 19, 400–404
Bej, A.K., Mahbubani, M.H., Miller, R., Dicesard, T.L., Haff, L. and Atlas, R.M., 1990. Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water. Molecular and Cell Probes, 4, 353–365
Bruckner, L., Stäuber, N., Brechtbühl, K. and Hofmann, M.A., 1996. Detection of extraneous agents in vaccines using the polymerase chain reaction of Newcastle disease virus in poultry biologicals. Develepments in Biological Standard, 86, 175–182
Cameron, K.R., Gregory, V., Banks, J., Brown, I.H., Alexander, D.J. and Hay, A.J., 2000. H9N2 subtype influenza viruses in poultry in Pakistan are closely related to the H9N2 viruses responsible for human infection in Hong Kong. Virology, 278, 36–41
Capua, I. and Alexander, D.J., 2004. Avian influenza: recent developments. Avian Pathology, 33, 393–404
Corbanie, E.A., Remon, J.P., Van Reeth, K., Landman, W.J., van Eck, J.H. and Vervaet, C., 2007. Spray drying of an attenuated live Newcastle disease vaccine virus intended for respiratory mass vaccination of poultry. Vaccine, 25, 8306–8317
Das, A., Spackman, E., Senne, D., Pedersen, J. and Suarez, D.L., 2006. Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents. Journal of Clinical Microbiology, 44, 3065–3073
de Leeuw, O.S., Koch, G., Hartog, L., Ravenshorst, N. and Peeters, B.P.H., 2005. Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein. Journal of General Virology, 86, 1759–1769
Fouchier, R.A.M., Bestebroer, T.M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G.F., and Osterhaus, A.D., 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. Journal of Clinical Microbiology, 38, 4096–4101
Fouchier, R.A.M., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B. and Osterhaus, A.D.M.E., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology, 79, 2814–2822
Han, G.Z., He, C.Q., Ding, N.Z., and Ma, L.Y., 2008. Identification of a natural multi-recombinant of Newcastle disease virus. Virology, 371, 54–60
Hanson, B.A., Swayne D.E., Senne D.A., Lobpries D.S., Hurst J. and Stallknecht D.E., 2005. Avian influenza viruses and paramyxoviruses in wintering and resident ducks in Texas. Journal of Wildlife Diseases, 41, 624–628
Huang, C.J., Hung, J.J., Wu, C.Y. and Chien, M.S., 2004. Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Veterinary Microbiology, 101, 209–214
Hubalek, Z., 2004. An annotated checklist of pathogenic microorganisms associated with migratory birds. Journal of Wildlife Diseases, 40, 639–659
Li, K.S., Xu, K.M., Peiris, J.S., Poon, L.L., Yu, K.Z., Yuen, K.Y., 2003. Characterization of H9 subtype influenza viruses from the ducks of Southern China: a candidate for the next influenza pandemic in humans? Journal of Virology, 77, 6988–6994
Lin, Y.P., Shaw, M., Gregory, V., Cameron, K., Lim, W. and Klimov, A., 2000. Avian-to-human transmission of H9N2 subtype influenza viruses: relationship between H9N2 and H5N1 human isolates. Proceedings of the National Academy of Sciences of the United States of America, 97, 9654–9658
Ng, W.F. and To, K.F., 2007. Pathology of human H5N1 infection: new findings. Lancet, 370, 1106–1108
Ng, L.F., Barr, I., Nguyen, T., Noor, S.M., Tan, R.S., Agathe, L.V., Gupta, S., Khalil, H., To, T.L., Hassan, S.S. and Ren, E.C., 2006. Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay. BioMed Central Infectious Diseases, 6, 40
Nguyen, D.C., Uyeki, T.M., Jadhao, S., Maines, T., Shaw, M. and Matsuoka, Y., 2005. Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001. Journal of Virology, 79, 4201–4212
Normile, D., 2005. Avian flu: First human case in Cambodia highlights surveillance shortcomings. Science, 307, 1027
Panigrahy, B., Senne, D.A. and Pedersen, J.C., 2002. Avian influenza virus subtypes inside and outside the live bird markets, 1993–2000: a spatial and temporal relationship. Avian Disease, 46, 298–307
Pham, H.M., Konnai, S., Usui, T., Chang, K.S., Murata, S., Mase, M., Ohashi, K., and Onuma, M., 2005. Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis. Archives of virology, 150, 2429–2438
Sakai, K., Yada, K., Sakabe, G., Tani, O., Miyaji, K., Nakamura, M. and Takehara, K., 2006. Serological and virological studies of Newcastle disease and avian influenza in slaughter-age ostriches (Struthio camelus) in Japan. Journal of veterinary medical science, 68, 491–494
Shu, Y.L., Yu, H.J., and Li, D.X., 2006. Lethal avian influenza A (H5N1) infection in a pregnant woman in Anhui Province, China. New England Journal of Medicine, 354, 1421–1422
Steel, J., Burmakina, S.V., Thomas, C., Spackman, E., García-Sastre, A., Swayne, D.E., Palese, P.A., 2008. Combination in-ovo vaccine for avian influenza virus and Newcastle disease virus. Vaccine, 26, 522–531
Tran, T.H., Nguyen, T.L., Nguyen, T.D., Luong, T.S., Pham, P.M., and Nguyen, V.C., 2004. Avian influenza A (H5N1) in 10 patients in Vietnam. New England Journal Medicine, 350, 1179–1188
Tsuda, Y., Sakoda, Y., Sakabe, S., Mochizuki, T., Namba, Y. and Kida, H., 2007. Development of an immunochromatographic kit for rapid diagnosis of H5 avian influenza virus infection. Microbiology and Immunology, 51, 903–907
Wakamatsu, N., King, D.J., Seal, B.S. and Brown, C.C., 2007. Detection of Newcastle disease virus RNA by reverse transcription-polymerase chain reaction using formalin-fixed, paraffin-embedded tissue and comparison with immunohistochemistry and in situ hybridization. Journal of Veterinary Diagnostic Investigation, 1, 396–400
Webster, R.G., 2006. H5N1 outbreaks and enzootic influenza. Emerging Infectious Diseases, 12, 3–8
Wise, M.G., Suarez, D.L., Seal, B.S., Pedersen, J.C., Senne, D.A., King, D.J., Kapczynski, D.R. and Spackman, E., 2004. Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples. Journal of Clinical Microbiology, 42, 329–33
Xie, Z., Pang, Y.S., Liu, J., Deng, X., Tang, X. and Sun, J., 2006. A multiplex RT-PCR for detection of type A influenza virus and differentiation of avian H5, H7, and H9 hemagglutinin subtypes. Molecular and Cellular Probes, 20, 245–249
Acknowledgements
This work was supported in part by grants from the National Key Technologies R&D Program of China (No.2006BAD06A03). This study was also supported by the National Natural Science Foundation of China (No.30671563 and No.30700597).
Author information
Authors and Affiliations
Corresponding author
Additional information
Hao-tai Chen and Jie Zhang contributed equally to this work.
Rights and permissions
About this article
Cite this article
Chen, Ht., Zhang, J., Sun, Dh. et al. Rapid discrimination of H5 and H9 subtypes of avian influenza viruses and Newcastle disease virus by multiplex RT-PCR. Vet Res Commun 32, 491–498 (2008). https://doi.org/10.1007/s11259-008-9052-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11259-008-9052-z