Expression and characterization of the non-structural protein V of small ruminant morbillivirus

Abstract

Peste-des-petits ruminants is a transboundary viral disease of small ruminants caused by small ruminant morbillivirus (SRMV). In the present study, the full-length V gene of SRMV was constructed through site-directed mutagenesis from the P gene transcripts of the vaccine virus (Sungri/96 India) and expressed in a prokaryotic expression system. In animals, the seroconversion against this protein occurs from 14-days and is getting produced from 48 h in cell culture. An indirect ELISA developed using this protein has a relative sensitivity and relative specificity of 77.73% and 73.775%, respectively as compared to c-ELISA. In this ELISA, it was observed that most of the convalescent animals elicited higher level of antibodies than vaccinated animals.

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Peste-des-petits-ruminants (PPR) being a devastating viral disease of small ruminants causes havoc in the developing nations of Asia, Africa and Middle East. The disease is targeted for global eradication in 2030 by FAO and OIE [16]. The etiological agent, small ruminant morbillivirus (SRMV-formerly, peste-des-petits-ruminants virus) belongs to the genus Morbillivirus of the family Paramyxoviridae. Although, the SRMV is classified into four distinct lineages (I–IV) they are serologically indistinguishable. Many African countries report the presence of more than one lineage, whereas only lineage IV has been circulating across Asia [2, 13, 16]. The SRMV codes for six structural—N, P, M, F, H and L, and two non-structural proteins—C and V [1, 11]. The V protein of SRMV is produced by addition of one non-template “G” residue at a specific editing site of some fraction of P mRNA, which results in production of V protein with an N-terminus identical to the P protein with a different cysteine-rich C-terminus [8, 11]. The V protein is reported to block the IFN production [3, 5].

In India, the PPR is enzootic and occurs throughout the country in all seasons. However, the intensive use of live attenuated PPR vaccine and MAb based ELISAs developed in our laboratory in the ongoing PPR control program resulted in significant reduction of the disease incidence [12, 13]. As the PPR vaccine is live, the animal mounts strong antibody response which mimics the natural infection. This creates difficulties for serological differentiation of the infected animals from the vaccinated (DIVA) one. Developing DIVA enabled i.e. positively or negatively marked vaccine and a companion diagnostic assay will help the control program to a greater extent. Researchers have attempted to develop DIVA enabled vaccine and diagnostics using various techniques like reverse genetics, vectored vaccines or mAb resistant mutant with varying degree of success [6, 7, 9, 10, 14, 15]. Here we report the development of indirect ELISA using recombinant V protein and its reactivity with convalescent and vaccinated sera.

The PPR vaccine virus (Sungri/96) was propagated in Vero cells; viral RNA and cDNA was prepared [12]. For optimization of the indirect ELISA, sera samples with known SNT titers were used (negative < 1:2; positive > 1:8).

The V gene was constructed by site-directed mutagenesis using gene-specific primers containing the extra “G” residue at the editing site. The PCR amplicons were gel purified, cloned into pET32a vector and sequenced commercially. After verification, the cassette was transformed into E. coli BL21 cells and expressed as described earlier [4, 17]. The protein was purified under denaturing condition using nickel affinity columns (Qiagen) and the concentration was estimated calorimetrically. The recombinant protein was verified in SDS-PAGE and Western blotting. The purified protein was used for optimization of an indirect ELISA and for raising of hyperimmune sera in rabbits.

A total of 427 serum samples were used for optimization of indirect ELISA. These serum samples were already screened in competitive ELISA and classified into positive or negative. The rV protein based indirect ELISA was optimized using chequer board method and the highest dilution of positive serum over negative serum (P/N) differential was chosen as 1:5. To calculate the percent positivity strong positive (cELISA) sera was used. The ROC and cut-off values were determined using the MedCalc 12.5.0.

After the optimization of rV indirect ELISA, the performance of the assay was evaluated using 1000 serum samples which were collected from the mass vaccinated area. The samples also screened in cELISA for the validation of the assay. For evaluation of DIVA, serum samples from experimentally vaccinated (n = 26) and experimentally infected (n = 11) animals were used. From the above experimental animals samples were collected on 0, 7th, 14th, 28th and 45th days post inoculation. For DIVA, a second cut-off was determined from the mean OD of 45th day post-vaccinated sera plus 3SD (mean + 3SD).

To produce hyperimmune sera, 350 µg purified rV protein of was mixed with equal volume of Freund’s complete adjuvant (FCA) and immunized in two rabbits (0 day and 14th day). After the booster dose, blood samples were collected on 14, 21 and 28 days post-inoculation (dpi). Serum from healthy rabbit was used as negative control.

The expression kinetics of V protein in cell culture was studied using the dot blot assay. Briefly, Vero cells were grown in Leighton tubes and infected with 0.1 moi of PPRV. The cells were harvested at 0, 6, 12, 18, 24, 48, 72, 96, 120 and 144 h post infection (hpi). To determine the expression kinetics in dot blot assay, the cell lysates and hyperimmune sera raised in rabbits were used.

For the control of PPR, live attenuated vaccine (Sungri/96 or Nigeria/75/1) is being used. Various methods were evaluated to differentiate sera of the vaccinated and infected animals. In this study, the full-length V gene was constructed from the Sungri/96 through site-directed mutagenesis (GenBank Accession No. KJ956930). Optimum expression of the rV protein was obtained at 4 h. In SDS-PAGE and Western blot analysis with the hyperimmune sera a ~ 63 kDa protein was obtained (Fig. 1). The rV protein was purified under denaturing condition with a yield of 0.5–3.4 mg/mL.

Fig. 1.

a SDS-PAGE analysis of PPRV V protein expressed E. coli. Lane 1: protein marker and Lane 2: rV protein and b Western blot analysis of PPRV V protein reactivity with rabbit HIS. Lane 1: rV protein and Lane 2: protein marker

The optimal working dilution of rV antigen was found to be 1:1000. The optimum single dilution for convalescent, vaccinated and healthy serum from goats was chosen using maximum positive–negative (P/N) differential plot (Fig. 2a). The serum dilution of 1:5 was found to be sufficient for all type of sera in large scale screening. After optimization, the assay performance was tested using random sera samples (n = 1000) from a mass vaccinated area. The results indicate that the assay has a relative sensitivity (rSn) and relative specificity (rSp) of 77.72% and 73.77%, respectively as compared to c-ELISA (Table S1).

Fig. 2.

a Reactivity of the Log₂ serially diluted negative, vaccinated and convalescent goat sera against the rV protein. The highest dilution with maximum difference between positive and negative samples (P/N differential; 1:5) was chosen for further testing. NG1—negative goat 1; NG2—negative goat 2; Vaccinated—vaccinated goat sera and Convalescent—convalescent goat sera. b The reactivity of the negative (n = 2), vaccinated (n = 26) and convalescent goat sera (n = 11) in the rV protein based indirect ELISA. The dotted, dashed and solid graph lines denote the negative, vaccinated and convalescent sera. The horizontal solid line indicates the cut-off between negative and vaccinated sera and the horizontal dashed line indicates the cut-off between vaccinated and convalescent sera

The reactivity of the rV protein with convalescent sera (n = 11), vaccinated sera (n = 26) and sera from two healthy goats indicated that the sero-conversion for the V protein occurs from 14th day post infection/vaccination and was maintained up to the study period of 45th day (Fig. 2b) which is very similar to our earlier observation in C protein [17]. The reactivity of one serum from vaccinated (GN14) goat was found to be falling in the negative range and another convalescent serum was found to fall in the vaccinated sera range (GN 93). The levels of antibody production in convalescent animals are found to be two to three folds higher than that of vaccinated animals. The reactivity pattern of V protein with convalescence and vaccinated sera was stronger than the C protein. This may be due to the cross reactivity of the P protein as N terminal half of the V is identical to P protein. As such there is no data available on expression kinetics of V protein in vivo or ex vivo, in the present study the expression time course analyses has been studied in Vero cells and the V protein is found to express 48 h onwards (Fig. 3). The data generated in this study will be useful in evaluating V protein defective mutants as DIVA candidates.

Fig. 3.

The dot-blot analysis of the expression kinetics of V protein in PPRV infected Vero cells. The results indicate that the earliest reactivity of V protein appeared at 48 h

In conclusion, we have developed an indirect ELISA targeting the non-structural protein V of SRMV and shown the reactive trend of sera from vaccinated and convalescent animals. To get a clear picture on the usage of this assay for DIVA purpose, more number of samples from vaccinated and convalescent animals needs to be tested. Further, the V protein has identical N terminus with P protein and the cross-reactivity of the P protein in the indirect ELISA needs further investigation.

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Conflict of interest

The authors declare that they have no conflict of interest.