Newcastle disease and Avian Influenza are considered to be the most dangerous fowl diseases which may cause huge economic losses. Newcastle disease is caused by the enveloped, and single-stranded RNA virus (NDV, APMV-1; belonging to Paramyxoviridae family), which can be further divided into sixteen different genotypes grouped into five pathotypes according to their pathogenicity. It has been reported that low pathogenic virus can greatly increase its pathogenicity even during a single passage. Additionally, due to the widespread use of live vaccines, a mixture of two or more different viruses in one sample can be detected. Hence, there is a great need for establishment of fast, inexpensive, sensitive, and relatively simple diagnostic method for multistrain and quasispecies detection of NDV infection. In this paper we describe a diagnostic method based on RT-PCR followed by a modified version of single-stranded conformational polymorphism analysis using short DNA fragments of gene encoding viral F protein. The method allows for rapid diagnosis of genetic variant emerging from previously stable population which may prevent the spread of the pathogenic viral variant.
Newcastle disease is an avian viral disease that endemically occurs in Asia, Africa, and Central and South America [
NDV genome contains 15186, 15192, or 15198 nucleotides depending on genotype and encodes six proteins in the following order: NP (nucleocapsid protein), P (phosphate polymerase cofactor protein), M (membrane associated matrix protein), F (fusion surface glycoprotein), HN (hemagglutinin-neuraminidase surface glycoprotein), and L (large RNA dependent RNA-polymerase) [
Newcastle disease is widely spread throughout the globe and is considered to be one of the two most dangerous poultry diseases (second is the avian influenza) that cause huge economic losses in poultry production. The loss of US$161 million by the US Government in 2002 after the outbreak of Newcastle disease in California is one of the most recent cases of the disease.
The name of the disease is derived from the British city “Newcastle-upon-Tyne,” near which it was diagnosed in 1926 for the first time. Almost simultaneously, in the same year, the disease was discovered on Java Island in Indonesia [
The main reservoirs of the APMV-1 are waterfowl and migratory birds [
Although vaccines based on low pathogenic variants of NDV protect against clinical signs and mortality, they do not prevent infection with highly pathogenic strains, which may be the cause for uncontrolled spread of the virus [
Molecular diagnostic methods that employ different variations of RT-PCR and real-time PCR techniques allow for sensitive detection and pathotyping of APMV-1 [
One of the methods which may help in differentiation as well as in quasi- and multistrain NDV detection is one-step RT-PCR followed by single-stranded conformational polymorphism. Single-stranded conformational polymorphism (SSCP) analysis was first described by Orita et al. [
Possible ssDNA conformers of 123 nt fusion protein fragments of La Sota (a), Roakin (b), Italy (c), lentogenic 98-1154 (d), mesogenic 99-0868-2 (e), and velogenic 99-0655 (f) NDV strains. Structures were drawn in Geneious R6 PRO software using ssDNA energy model at 20°C.
The purpose of this study was to develop a method for inexpensive, quick, and sensitive identification of NDV variants with a point mutation in gene F cleavage site that can lead to increase of the pathogenicity. Ideally, the method should allow for detection of minor quasispecies at relatively low cost in routine diagnostic laboratories.
Full size or fragments of genomes from three reference strains (vaccine La Sota, vaccine Roakin, and Italy) and three Australian strains (isolated during 1998–2002 outbreak in New South Wales) were tested in this study and are listed in Table
List of NDV strains with different types of templates for PCR or RT-PCR and highlighted point mutations affecting their virulence.
Strain | Templates* | GenBank acc. number | Nucleotide motif** | ICPI | Pathotype |
---|---|---|---|---|---|
La Sota | nR, aR, nD | AF077761 | GGGAGACAGGGGCGCCTT |
0.4 [ |
Lentogenic |
Roakin | nR, aR, nD | AY289000 | AGGAGACAGAAACGCTTT | 1.45 [ |
Mesogenic |
Italy | nR, aR, nD | AY562989 | AGGAGGAAGAAACGCTTT | 1.55 [ |
Velogenic |
98-1154 | aR, aD | AY935491 | AGGAGACAG |
0.47 [ |
Lentogenic |
99-0868-2 | aR, aD | AY935496 | AGGAGACAG |
1.38 [ |
Mesogenic |
99-0655 | aR, aD | AY935494 | AGGAGACAG |
1.61 [ |
Velogenic |
nR—allantoic fluid from SPF embryonated flock eggs (Lohman, Germany), previously inoculated with La Sota or Roakin NDV strains, was harvested and subjected to RNA extraction using RNeasy Mini Kit according to the manufacturer protocol (Qiagen, USA). RNA from oral and cloacal swabs from chickens infected with La Sota or Roakin NDV strains was purified with the same procedure.
nD—one set of universal (for all Class II APMV-1) degenerate PCR primers for amplification of 123 nt fusion glycoprotein gene fragment was designed. The DNA sequences for this set of primers were as follows: NDV-F (position 4790–4809 in La Sota genome): 5′-GCATACAACAGRACAYTGAC-3′ and NDV-R (position 4893–4912 in La Sota genome): 5′-GCCDATAATGGCRCCTATAA-3′. Synthesis of the first strand and amplification of the DNA were carried out during one-step RT-PCR (AccessQuick RT-PCR System, Promega, USA) procedure. The reaction mixture contained 1
aD—three 145 bp DNA oligonucleotides with sequences exactly the same as those in Australian strains of NDV (position 4790–4934 in La Sota genome) presented in Table
aR—all purified and checked for correct sequence plasmids were used as template for transcription reaction by TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, USA) as instructed by the manufacturer. RNA was checked for lack of DNA contamination by RT-PCR as described above without addition of AMV Reverse Transcriptase.
Templates nR and aR were subjected to RT-PCR and plasmids (nD and aD) to PCR (no initial 45°C 40 min. step or AMV Reverse Transcriptase). For MSSCP analysis, 3
Conditions of MSSCP electrophoresis.
Step | Vxh | Temp. (°C) |
---|---|---|
1 | 600 | 9 |
2 | 550 | 13 |
3 | 500 | 17 |
4 | 450 | 20 |
5 | 400 | 23 |
During the experiments, different types of templates for polymerase amplification reactions were tested (Table
Until now no data have been presented whether the preamplification procedures (like using only small fragment of transcribed RNA instead of full genomic RNA for RT-PCR) affect secondary ssDNA structures and their quantity, which subsequently may alter MSSCP band pattern. To check this, MSSCP analysis for RT-PCR products derived from different templates was conducted. The result is shown in Figure
Multitemperature single-stranded conformational polymorphism (MSSCP) electrophoresis of 123 nt fusion protein fragments of two vaccine Newcastle disease virus strains obtained from RT-PCR (lanes 2–5 and 8–11) and PCR (lanes 6 and 7). Types of templates (nR, aR, and nD) were described in Section
To test the ability to detect two different NDV variants in a single sample, a series of mixed template PCRs was prepared. After reaction, MSSCP analysis was performed and ssDNA band patterns were visualized as presented in Figures
MSSCP electrophoresis of 123 nt NDV fusion protein fragments RT-PCR and PCR products. Lanes 2–4 are RT-PCR products (genomic RNA—nR template; L—La Sota strain, R—Roakin strain, and I—Italy strain) where lanes 5–19 are PCR products (plasmids with cloned fusion protein gen fragment—nD and aD templates). The PCR strains sample composition, plasmid ratio, and color code explanation can be found in the table below the figure. MSSCP was performed in 11% polyacrylamide gel without any additives as described in Table
MSSCP electrophoresis of 123 nt NDV fusion protein fragments RT-PCR products (nR and aR templates). Labeling: 1. Australian lentogenic strain 98-1154, 2. Australian mesogenic strain 99-0868-2, 3. Australian velogenic strain 99-0655, R. Vaccine mesogenic Roakin strain. MSSCP was performed in 13,5% polyacrylamide gel with addition of 3% glycerol as described in Table
Low pathogenic strains of NDV in feces of infected birds can remain active for more than two months, and the virus can persist for over two years in bird population [
The Australian case gave rise to the discussion whether it is possible to detect such events in advance. To cope with the problem we need to establish a method that will meet some basic requirements. This method must be able to detect numerous genetically altered variants in the pool of progenitor viruses as well as it must be able to distinguish between two sequences with only single nucleotide mismatch. Additional advantage of such method should be low cost and simplicity.
The combination of Reverse Transcription real-time, digital PCR with Sanger sequencing or applications of Next Generation Sequencing and oligonucleotide microarrays can give great results, but they are expensive and highly demanding for trained technician and elaborate equipment [
During our study, we developed a method, based on RT-PCR followed by MSSCP analyses, that meets the requirements described in the second paragraph. In our opinion, fast diagnostics of a new genetic variant that appears in previously stable population could alert veterinary services and lead, after confirmation, to preventive actions for disease eradication. MSSCP can differentiate between lentogenic vaccine, mesogenic vaccine, circulating velogenic, lentogenic natural occurring, and their highly pathogenic mutated NDV variants. This can be achieved by comparison of unknown pattern of silver stained bands to previously established database of known isolates. Even without direct information about pathotype of the unknown sample, it is easy to distinguish potentially unwanted strain of NDV. The technique described above does not require highly sophisticated and expensive tools and is easy to perform by laboratory technical personnel. Additionally it is relatively inexpensive and takes about 5 minutes per sample from the end of one-step RT-PCR until visualization. Main disadvantage (that has no significance for a technician performing the assay) of MSSCP is a lengthy and arduous optimization of gel run conditions like selection of optimum conditions (temperature, voltage, and time) and gel percentage of polyacrylamide and other additives.
Many methods have been developed that allow for detecting different pathotypes of Newcastle disease virus. One of the most popular and validated molecular techniques is real-time PCR. The disadvantage of this method is difficulty in detection of multistrain infections and minute changes in sequence that can alter virulence. In this study, we described a method based on a modification of single-stranded conformational polymorphism at specially programmed temperatures (MSSCP) that can be easily applied for NDV screening. We also showed that to prepare a library of MSSCP patterns it is not necessary to use isolated genomic material but only short synthetized nucleotides. In other words, it is possible to create a database of ssDNA band patterns for all-known NDV F gene sequences (e.g., available in NCBI) without need to isolate their genetic material but by synthetizing dsDNA analogs and reverse transcription to RNA. They can be used as a template for RT-PCR and produce exactly the same MSSCP ssDNA band patterns (Figure
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research was cofunded by the National Centre for Research and Development within the Applied Research Programme Grant no. PBS1/B8/2/2012.