Detection of Newcastle disease virus minor genetic variants by modified single-stranded conformational polymorphism analysis

Abstract

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.

Figure 1

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.

Figure 2

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 2. Lanes: 1. Double-stranded pUC19 DNA/MspI Marker, 23 (Thermo Scientific, USA), 2. Oral swab nR La Sota, 3. Oral swab nR Roakin, 4. Cloacal swab nR La Sota, 5. Cloacal swab nR Roakin, 6. Plasmid nD La Sota, 7. Plasmid nD Roakin, 8. Allantoic fluid nR La Sota, 9. Allantoic fluid nR Roakin, 10. Transcribed RNA aR La Sota, 11. Transcribed RNA aR Roakin, 12. RT-PCR negative control, 13. PCR negative control. MSSCP was performed as described in Table 2 without the first two temperatures steps.

Figure 3

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 2.

Figure 4

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 2.