key: cord-0777835-66c2tg5r
authors: Nidzworski, Dawid; Smietanka, Krzysztof; Minta, Zenon; Szewczyk, Bogusław
title: Detection of avian influenza virus and newcastle disease virus by duplex one step RT PCR
date: 2013-03-16
journal: Cent Eur J Biol
DOI: 10.2478/s11535-013-0164-7
sha: 86b25f6daeeb952b927c53a104fd46e0a91573a2
doc_id: 777835
cord_uid: 66c2tg5r

Newcastle disease Virus (NDV), a member of the Paramyxoviridae family, and Influenza virus, from the Orthomyxoviridae family, are two main avian pathogens that cause serious economic problems in poultry farming. NDV strains are classified into three major pathotypes: velogenic, mesogenic, and lentogenic. Avian influenza viruses (AIV) are also divided into: low pathogenic (LPAI) and highly pathogenic (HPAI) strains. Both viruses are enveloped, single stranded, negative-sense RNA viruses which give similar symptoms ranging from sub-clinical infections to severe disease, including loss in egg production, acute respiratory syndrome, and high mortality, depending on their level of pathogenicity. This similarity hinders diagnosis when based solely on clinical and post mortem examination. Most of the currently available molecular detection methods are also pathogenspecific, so that more than one RT-PCR is then required to confirm or exclude the presence of both pathogens. To overcome this disadvantage, we have applied a One Step Duplex RT-PCR method to distinguish between those two pathogens. The main objective of the project was to develop a universal, fast, and inexpensive method which could be used in any veterinary laboratory.

Avian influenza virus (AIV) is a member of the Orthomyxoviridea family. It has a spherical, enveloped capsid composed of a matrix protein. The virus contains linear, segmented, single-stranded RNA of negative polarity, covered by nucleoprotein. Eight segments encode twelve proteins. On the surface of the virion, there are ion channels and two main glycoproteins, hemagglutinin and neuraminidase, which are responsible for the diversity of this virus. Highly pathogenic avian influenza (HPAI) strain cause severe symptoms in the respiratory tract, as well as in the digestive and nervous systems which lead to a high mortality rate of up to 100%. This group includes, in particular, some H5 and H7 influenza strains. Other strains cause milder symptoms: apathy, decrease in laying capacity, and mild respiratory tract disorders, and are known as LPAI (low pathogenic avian influenza) [1, 2] . The differences in virulence level have a molecular basis, namely changes in the cleavage site of HA0. In HPAI strains, in the cleavage site, there is a region rich with basic amino acids. This motif can be cleaved by the majority of enzymes present in various cells of the host organism [3] .

Newcastle disease virus (NDV) belongs to Mononegavirales order, Paramyxoviridae family, and Avulavirus genus. The viral particle is composed of a matrix protein, surface glycoproteins (hemagglutininneuraminidase and a fusion protein), and has a lipid double layer on its surface. The genome of NDV is linear, non-segmented, single-stranded RNA encoding 8 proteins [4] . NDV strains are classified into one of the three major pathotypes: velogenic, mesogenic, and lentogenic [5] , according to pathogenicity in vivo. Similarly to influenza virus, NDV pathogenicity has a molecular background. The product of F gene of highly pathogenic strains (velogenic) is recognized by a common, subtilisin-like protease, while the products of genes of low pathogenic strains (mesogenic, lentogenic) are recognized only by trypsin-like proteases present mainly in the respiratory tract [6, 7] .

Newcastle disease virus and avian influenza virus cause similar symptoms in infected birds, which makes quick diagnosis difficult. The symptoms are observed in the digestive tract, respiratory tract, and nervous system. The incubation period normally lasts 2 to 6 days, and this timeframe depends on the level of virulence of the strain and the pre-infection condition of the bird. The most frequent clinical symptoms are apathy, loss of appetite, diarrhoea, secretion from nostrils and beak, breathing difficulties, strong convulsions, neck torsion, drooping wings, paralysis. In internal organs, hemorrhagic lesions occur. Also, a significant decrease in laying capacity, and laying eggs without shells, can be observed [1, 8, 9] .

Traditional methods of isolation and identification of viruses, such as propagation in chicken embryos or cell culture followed by identification in hemagglutinationinhibition test (HI) are time-consuming and laborious. Rapid-developing molecular biology tools are an alternative to traditional diagnostic methods and allow fast and definite identification of a particular pathogen in a sample.

The previously developed tests are focus mostly on identification of only a few types of influenza serotypes (H3, H5, H7, and H9) and NDV. The primers used in those tests are based on fragments of hemagglutinin gene (for detection of influenza virus) and fusion (F) protein (for detection of NDV) [10] [11] [12] [13] . These genes are the most variable fragments of AIV and NDV genome respectively, therefore applying such methods may lead to some false negative results.

Naturally, there are a few more publications related to detection of NDV and AIV [14] [15] [16] [17] [18] but the main disadvantage of the tests presented there is the need to use two-step methods, which are more complicated, expensive and time-consuming.

In this study, we present a new duplex One step RT-PCR assay for the simultaneous detection and differentiation of avian influenza virus and Newcastle disease virus. Using primers designed to conserve fragments of the genes of both viruses assures the universality of this method. One step RT-PCR method reduces the time and cost of analysis.

Eighteen NDV strains, fourteen influenza and twelve other (negative control strains) viruses listed in Table 1 were isolated from allantoic fluids of SPF embryonated eggs using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). All strains with the exception of A/ostrich/Denmark-Q/72420/96 and A/African starling/ England/983/79 (source: AHVLA Weybridge, UK), were from the collection of the National Veterinary Research Institute (Pulawy, Poland). APMV strains were purchased from x-OvO Limited (Dunfermline, United Kingdom). Reovirus (Reo S1133) and infectious bronchitis virus (IB K+ 4/91) were components of Nobilis REO 1133 and Nobilis IB 4-91 vaccines (Intervet/Schering-Plough Animal Health, Holland).

In silico analyses were performed with Geneious software (http://www.geneious.com/) using sequences available in GenBank (http://www.ncbi.nlm.nih. gov/genbank/). Two sets of primers based on the conserved fragments of the M gens of NDV and IV were used in this study: Primers: IV-F-VII/25 (5'-AGATGAGTCTTCTAACCGAGGTCG-3') and IV-R-VII/100 (5'-TGCAAAAACATCTTCAAGTCTCTG-3') amplifies a 100 bp long fragment of the matrix protein gene of AIV [19] , while primers: NDV-F-4011 (5'-GTCCCAAATACCGGAGACCT-3') and NDV-R-4142 (5'-TTGTTTGCCACAACCCTACAG-3') are specific to the conserved fragment of matrix protein gene of NDV, and the amplification product is 152 bp long. BLAST sequence analyses were also performed to confirm the specificity of the desigend primers.

The reaction was carried out according to the Transcriptor One-Step RT-PCR Kit protocol (Roche Diagnostics, Mannheim, Germany). The reaction mixture (50 µl) contained: viral RNA (5 µl), both sets of primers (0.3 µM each), buffer (1x), Transcriptor Enzyme Mix, and water. The conditions of the reaction were established through optimization and are presented in Table 2 . The results were visualized in 2.5% agarose gel.

The detection limit was determined using a known number of copies of M genes fragments of both viruses. In vitro transcription of RNA was performed using TranscriptAid T7 High Yield Transcription Kit (Fermentas, Thermo Fisher Scientific, Vilnius, Lithuania), according to protocol instructions. The concentration of plasmid RNA was determined by spectrophotometry. Copy number was calculated and the RNA samples were serially, 10-fold diluted to serve as standards for determination of the sensitivity of One

Step RT-PCR assay. The specificity of the newly developed assay was investigated using the RNA extracted from NDV and AIV strains, as well as from 12 other avian viruses presented in Table 1 .

In order to check the new method and to compare it to the previously described tests, an experiment with co-infected chickens was performed. Four-week-old SPF chickens were infected with both viruses: NDV -LaSota and AIV -H7N1 using a dose of 10 6,0 EID 50 and 10 7,5 EID 50 , respectively, intraocularly and intranasally. Swabs from the cloaca and trachea were collected on the fourth and fifth day of the experiment and examined by the new method and by two previously described tests [12, 19] . Animal experiments in this study were approved by the Local Animal Welfare Committee (22/2006 ).

Step RT-PCR assay; specificity of the assay Eighteen strains of Newcastle Disease Virus (including ten lentogenic, two mesogenic, and six velogenic isolates) and twelve avian influenza virus strains were tested. The expected PCR products of 152 bp and 100 bp of length, respectively, were amplified in all NDV/AIV samples. The new One

Step RT-PCR assay was shown to detect and differentiate both viruses even in co-infection ( Figure 1 , Table 1 ). The specificity of the new assay was investigated by analysing 12 heterologous avian pathogens (Table 1) . No amplification products were detected when templates originating from those twelve other, unrelated viruses were used, while all the templates from NDV and AIV strains gave distinctive products.

The detection limit of this assay was assessed by analysing serial dilutions of in vitro transcribed RNA of M genes of NDVs and AIVs. The detection limits were 10 3 copies of RNA per reaction, even in co-infection. Additional analysis in the presence of contamination with other viruses' RNA did not change the detection limits. Moreover, samples from chickens infected with two viruses: NDV (LaSota) and AIV (H7N1), were also analysed by the new method as well as two previously described methods. The results of the analysis show the possibility to use this new assay as an alternative method for detection and differentiation of both viruses ( Table 3) . The new test also detects viruses in samples with low concentrations of pathogens (Ct value higher than 30) [19] ;B-Ctvaluesobtained fromreal-timePCRmethodofdetectionbasedonKimetal. [12] ;OS-OneStepRT-PCRMethod. 

In this study, we present a new duplex One

Step RT-PCR assay for the detection and differentiation of avian influenza and Newcastle disease viruses. The test is relatively quick and very easy to perform. It is based on the analysis of conserved fragments of genes coding M proteins of both viruses. Both genes in both viruses show the highest level of conservation, and thus can serve as a universal tool for diagnostics and differentiation od NDVs and AIVs. Owing to the substantial difference in product lengths, the risk of a false assessment of the sample is minimized. At the same time, it allows detection of both viruses in co-infection. In previously described tests for differentiation of these two viruses, the authors selected hemagglutinin gene for influenza virus and fusion protein gene for NDV. Other test are more universal but need a two-step procedure. However, these genes are very variable, and the tests based on their detection may fail in some cases. Moreover, those tests focus on the detection of particular subtypes of AIV (H3, H5, H7, and/or H9). Though they are very useful, they do not allow detection of other subtypes of influenza virus that pose a danger to wild and domestic birds [11, 13, 16, 18, 20, 21] .

The advantage of the newly developed test is the ability to detect a broad spectrum of isolates, as the primers bind to the conserved fragment of M genes. This method is naturally less sensitive when compared to real-time PCR assays, however it may be a good alternative for laboratories without RTqPCR equipment. Moreover, the one step approach used in this method reduces the time and cost of analysis.

This assay may be the first step of the complete analysis of infected birds. The international regulations require a more precise characterization of the detected virus based on the virulence markers located in HA (AIV) or F (NDV) genes [22, 23] . Therefore, the method can serve as a useful preliminary tool, but all positive samples should be submitted to a more specific molecular test, e.g. HA-and F-specific, for determination of the pathogenicity of NDV and AIV and its subtypes [13, [22] [23] [24] .

Birds infected by Newcastle disease or influenza virus show very similar symptoms which are hard to distinguish by veterinarians even during necropsy. For this reason, the new One Step RT-PCR assay for direct detection and differentiation of NDV and IV was developed. The conserved fragments of M genes used to design primers and probes make this method widely applicable. The assay can be applied by veterinary diagnosticians for the preliminary differentiation of NDV and influenza virus.

The ability to detect both these major, avian pathogens, even in co-infections, through a very easy procedure, makes this method very attractive for application in veterinary laboratories.

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Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus

Role of fusion protein cleavage site in the virulence of Newcastle disease virus

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Standardization of a duplex RT-PCR for the detection of Influenza A and Newcastle disease viruses in migratory birds

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Multiplex polymerase chain reaction for the detection and differentiation of avian influenza viruses and other poultry respiratory pathogens

Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes

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Detection and differentiation of virulent and avirulent strains of Newcastle Disease Virus by real-time PCR

The authors wish to thank their colleagues from NVRI Pulawy, Poland: Monika Olszewska, Elzbieta Juszczuk and Michal Jozwiak for technical support in experiments.This research work was supported by the European Union in the framework of the European Social Fund; Pomorskie Voivodeship "InnoDoktorant -Scholarships for PhD students, II edition".