Development and Evaluation of a Nested PCR for Improved Diagnosis and Genetic Analysis of Peste des Petits Ruminants Virus (PPRV) for Future Use in Nascent PPR Eradication Programme

Abstract

Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants caused by PPR virus (PPRV). PPR is endemic in Asia, the Middle East and across large areas of Africa and is currently targeted for global eradication by 2030. The virus exists as four different lineages that are usually limited to specific geographical areas. However, recent reports of spread of PPRV, in particular of lineage IV viruses to infection-free countries and previously PPR endemic areas are noteworthy. A rapid and accurate laboratory diagnosis and reports on its epidemiological linkage for virus spread play a major role in the effective control and eradication of the disease. Currently, molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) are usually used for diagnosis of PPR while the sequencing of part of the nucleocapsid gene is usually carried out for the viral lineage identification. However, it is difficult to diagnose and sequence the genetic material if the animal excreted a low level of virus at the initial stage of infection or if the PPRV is degraded during the long-distance transportation of samples to the reference laboratories. This study describes the development of a novel nested RT-PCR assay for the detection of the PPRV nucleic acid by targeting the N-protein gene, compares the performance of the assay with the existing conventional RT-PCR and also provides good-quality DNA suitable for sequencing in order to identify circulating lineages. The assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. This assay provides a solution with an easy, accurate, rapid and cost-effective PPR diagnostic and partial genome sequencing for use in resource-limited settings.

Keywords: PPR; RT-PCR; diagnostic; eradication program; genetic analysis; peste des petits ruminants virus; rapid detection.

Figure 1

Gel images of PCR products using RNA of Nigeria/75/1 vaccine strain. (A,B): PCR products obtained from first round of PCR using primer pair NP3/NP4 (even number lane) and N1197F/N1658R (odd number lane), (C): nested PCR products using primer pair NP3/NP4. M: 1 kb plus marker DNA; lanes 1–8: PCR products corresponding to dilution 10⁻³ to 10⁻¹⁰, respectively. All the odd and even lanes in (A,B) shows products of primer pair N1197F/N1658R and NP3/NP4, respectively using neat, 10⁻¹–10⁻¹⁰ RNA. Lanes 1–8 in C represents 10⁻³–10⁻¹⁰ RNA, respectively.

Figure 2

Gel images of PCR products using RNA of lineage I virus Ivory Coast (A–C) and lineage II virus Nigeria/76/1 (D–F). (A,D): PCR products obtained from first round of PCR using primer pair NP3/NP4, (B,E): PCR products obtained from first round of PCR using primer pair N1197F/N1658R, (C,F): PCR products obtained from nested PCR using primer pair NP3/NP4. M: 1 kb plus marker DNA; lanes 1–11 in (A,B,D,E) correspond to neat, 10⁻¹ to 10⁻¹⁰ RNA, respectively. Lanes 1–10 in C correspond to 10⁻¹ to 10⁻¹⁰ RNA, and lanes 1–8 in (F) correspond to 10⁻¹ to 10⁻⁸ RNA, respectively.

Figure 3

Gel images of PCR products using RNA of Linage III viruses Sudan/Sinar 1972 (A–C) and IBRI-Oman 1982 (D–F). (A,D): PCR products obtained from first round of PCR using primer pair NP3/NP4, (B,E): PCR products obtained from first round of PCR using primer pair N1197F/N1658R, (C,F): PCR products obtained from nested PCR using primer pair NP3/NP4. M: 1 kb plus marker DNA; lanes 1–12 in (A,B,D,E) correspond to neat, 10⁻¹ to 10⁻¹¹ RNA, respectively. Lanes 1–7 in (C) correspond to 10⁻³ to 10⁻⁹ RNA, and lanes 1–11 in (F) correspond to neat, 10⁻¹ to 10⁻¹⁰ RNA, respectively.

Figure 4

Gel images of PCR products using RNA of lineage IV viruses Sungri/96 (A–C) and Morocco/2008 (D–F). (A,D): PCR products obtained from first round of PCR using primer pair NP3/NP4, (B,E): PCR products obtained from first round of PCR using primer pair N1197F/N1658R, (C,F): PCR products obtained from nested PCR using primer pair NP3/NP4. M: 1 kb plus marker DNA; lanes 1–7 in (A,B) correspond to neat, 10⁻¹–10⁻⁷ RNA, respectively. Lanes 1–4 in (C) correspond to 10⁻³–10⁻⁶ RNA, respectively. Similarly, lanes 1–12 in (D,E) correspond to neat, 10⁻¹–10⁻¹¹ RNA, respectively. Lanes 1–9 in (F) correspond to 10⁻³–10⁻¹¹ RNA, respectively.

Figure 5

Gel images of PCR products using in-vitro transcribed RNA standards (A–C) and field/experimental samples (D–F). (A): PCR products obtained from first round of PCR using primer pair NP3/NP4, (B): PCR products obtained from first round of PCR using primer pair N1197F/N1658R, (C): PCR products obtained from nested PCR using primer pair NP3/NP4, (D): PCR products obtained from first round of PCR using primer pair NP3/NP4 (lanes 1–4) and N1197F/N1658R (lanes 5–8) of samples from Democratic Republic of Congo (DRC) (correspond to samples 1–4 in Table 1), (E): nested PCR products of DRC samples using primer pair NP3/NP4, (F): nested PCR products of field or experimental samples (correspond to samples 5–16 in Table 1) using primer pair NP3/NP4. M: 1 kb plus marker DNA; lanes 1–10 in (A,B) correspond to 10⁹–10⁰ genome copies, respectively. Lanes 1–7 in (C) correspond to 10⁶–10⁰ genome copies, respectively.

Figure 6

Gel images of PCR products using field/experimental (correspond to samples 17–21 in Table 1) samples (A–D) and environmental (correspond to samples 22–26 in Table 1) samples (E,F). (A): PCR products obtained from first round of PCR using primer pair NP3/NP4, (B): PCR products obtained from first round of PCR using primer pair N1197F/N1658R, (C): nested PCR products using primer pair NP3/NP4, (D): nested PCR products using primer pair NP3/NP4 when template DNA was purified, (E): PCR products obtained from first round of PCR using primer pair NP3/NP4 (lanes 1–5) and N1197F/N1658R (lanes 6–10) of environmental samples from Nepal (correspond to samples 22–26 in Table 1), (F): nested PCR products of environmental samples using primer pair NP3/NP4. M: 1 kb plus marker DNA.