Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays

Abstract

Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

Introduction

Newcastle disease (ND) and avian influenza (AI) are two of the most devastating avian diseases in the world. Both diseases cause acute respiratory infection and lead to mortality in poultry flocks (Sakai et al., 2006). Newcastle disease virus (NDV) and avian influenza virus (AIV) are associated with transmission from wild to domestic birds, and can lead to human infections such as conjunctivitis or influenza-like syndrome (Artois et al., 2002, Capua and Alexander, 2004b). Wild birds may function as a reservoir for both viruses, playing a role as potential vectors with few or no clinical signs (Alexander, 2000, Pfitzer et al., 2000, Hubalek, 2004). NDVs have been isolated from many free-living avian species, including Pelecaniformes, Falconiformes, Strigiformes and Anatiformes (Kaleta and Baldauf, 1988, Wobeser et al., 1993, Farley et al., 2001, Schettler et al., 2003). The Anatiformes has also provided the highest rate of AIV isolations (Kaleta et al., 2005).

The pathogenicity of NDV is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Mutation will change the virulence from non-virulent (lentogenic) to intermediate (mesogenic) or highly virulent (velogenic) strains (de Leeuw et al., 2003, de Leeuw et al., 2005). There are 16 (H1–H16) haemagglutinin (HA) subtypes of AIV. The H16, a novel haemagglutinin subtype, has recently been found from black-headed gulls (Fouchier et al., 2005). The highly pathogenic avian influenza (HPAI) viruses have been restricted to H5 and H7, although not all viruses of these subtypes cause HPAI. Others cause a milder respiratory disease, designated low pathogenicity avian influenza (LPAI) viruses (Alexander, 2000). The virulence of AIV depends on the cleavage site of the haemagglutinin precursor protein (HA0). HPAI H5 and H7 can arise from the HA gene mutation of LPAI H5 and H7 (Capua and Alexander, 2004a). The virulent determination of NDV is required, because control measures for avirulent viruses are very different from those for virulent viruses (de Leeuw et al., 2003). The AIV subtyping, likewise, is imperative and most countries have recently implemented a stamping-out policy on H5 and H7 outbreaks whether it is LPAI or HPAI (Capua and Alexander, 2004a).

Outbreaks of ND are regular and frequent throughout Africa, Asia and parts of Central and South America. It appears to be a sporadic epizootic disease despite vaccination programs (Chen and Wang, 2002, Capua and Alexander, 2004b). In recent years, many outbreaks of both HPAI and LPAI have been reported in Asia and Europe (Capua and Alexander, 2004a). In addition, NDV and AIV H9N2 and H7N3 were isolated in various combinations in poultry flocks in Pakistan in 2001. Interestingly, a H7N3 virus showing close genetic similarity to the Pakistan virus was isolated from a peregrine falcon (Falco peregrinus) in the United Arab Emirates prior to the outbreak (Manvell et al., 2000, Swayne and Suarez, 2001). All of these indicate the necessity for detecting and typing NDV and AIV in both wild and domestic birds in order to quickly prevent and control the epidemics. Both NDV and AIV may cause serosal haemorrhages of the gastrointestinal tract and be difficult to discriminate. Therefore, differential diagnosis is imperative.

Many rapid serosurveys of both NDV and AIV have been done in wild and domestic birds in recent years (Pfitzer et al., 2000, Artois et al., 2002, Peterson et al., 2002a, Peterson et al., 2002b, Shengoing et al., 2002, Hua et al., 2005, Sakai et al., 2006). However, the antibodies against NDV and AIV were examined as two separate procedures, with no pathogenicity and subtype information obtained in these investigations except for performing further molecular manipulations. Some molecular approaches have been applied to NDV detection and pathotyping, e.g. reverse transcription polymerase chain reaction (RT-PCR) followed by restriction endonuclease analysis (Creelan et al., 2002), real-time PCR (Aldous et al., 2001) and real-time reverse-transcription PCR (RRT-PCR) (Wise et al., 2004). A number of molecular methods for the detection of AIV and subtyping of H5 and H7 have also been developed, e.g. RT followed by enzyme-linked immunosorbent assay (Dybkaer et al., 2004), multiplex RRT-PCR combined with haemagglutinin inhibition test (Spackman et al., 2003b), and RRT-PCR targeting matrix and haemagglutinin genes with separate procedures (Spackman et al., 2003a). Nucleic acid sequence-based amplification (NASBA) was employed to detect AIV H5 or H7 (Collins et al., 2002, Collins et al., 2003, Lau et al., 2004). Using microarrays to type and subtype human influenza viruses has been recently reported (Li et al., 2001, Sengupta et al., 2003, Kessler et al., 2004, Townsend et al., 2006); however, none focused on the detection of avian viruses. No integrated manipulations have been reported so far to detect NDV and AIV simultaneously, although these two viruses are important wild bird-carried zoonoses and the intervention of differential diagnosis is needed in many cases.

We have developed a rapid approach to differentiate NDV and AIV using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined simultaneously. This method, thus, may provide a new avenue to rapid detection, differentiation and typing of multiple pathogens. It could also be used to screen for potential carriers in both wild and domestic birds.