Sample Collection

During 2020–2021, the suspected outbreaks of PPR were reported by field veterinarians, within three different regions including Islamabad territory (33.6844° N, 73.0479°), Fateh Jang (33.8611° N, 72.4140° E), and Gilgit (35.9208° N, 74.3082°E). These animals were not vaccinated ever (Fig. 1). A total of 325 blood samples, including 120 sheep and 205 goats were randomly collected from apparently healthy as well as having typical PPRV signs (Table 1) following standard procedure [19].

Table 1 Details of PPR serum samples collected from selected locations

The clinical signs were consistent across all affected animals that have shown mouth erosion, nasal discharge, diarrhoea, or all the above symptoms. Blood samples from these animals were randomly collected in vacutainers (BD bioscience) without any anticoagulant for antibody detection.

Fig. 1

Locations of serum sample collections for sheep and goats during active outbreaks of PPRV reported from Pakistan, in 2020–2021 (colours indicate different divisions under provinces)

The serum was separated by centrifuging from the blood samples (n = 325) and stored at 4 °C for further research process. Postmortem examinations were also performed for dead animals and six tissue samples (n = 06) including spleen & lungs were collected. (Fig. 2). Other clinical samples and swabs (n = 19) were collected from live animals in a viral transportation medium (DMEM with 2 µg/ml pen-strep antibiotics) for antigen detection purposes. Aseptic precautions were taken during the sample collection and transportation. Every sample was labelled with a unique identification number and date. These were transported to the laboratory in cool boxes and stored at -80 °C till processed further (Table 2).

Table 2 Sample collection from different sources for the isolation of PPRV virus

Out of 25 clinical samples (swabs and tissues), only four samples were positive for PPRV when N-gene primer amplification was used with a positive rate of 16%, while the remaining 21 samples remained negative, as shown in Table 2.

Fig. 2

Animals presenting clinical signs of PPRV disease: a inflamed eye membranes; b evidence of “zebra striping” in the large intestine

RNA Extraction and cDNA Synthesis

All swabs and tissue samples were subjected to RNA extraction according to the manufacturer’s instructions using a commercial RNA extraction (Ambien Pure Link RNA) Mini Kit. Reagent contamination was detected using a negative control. Extracted RNA was eluted in 50 µL nuclease-free water, which was then stored at -70 °C until used., A NanoDrop(Thermo Scientific, Wilmington, DE, USA) measured the eluted RNA concentration and a conc. of ~ 2.0 or (100ng/ul)viral RNA was used for RT-PCR. A swab was obtained from clinically healthy sheep negative for cELISA and was considered a negative control. The synthesis of the cDNA of PPRV was performed according to the manufacturer’s instructions of Revert Ad First Strand cDNA(Thermo-Scientific) [20].

Optimization of Primers and PCR Amplification

A well-known diagnostic primer set NP3/NP4 by Couacy-Hymann was used to diagnose PPRV [21]. It was found useful in identifying, high-quality PPRV samples, like cell cultures grown viruses or any tissue samples from animals. The primer set NP3/NP4 has an N-coding gene between the positions 1232–1560 [21]. The primer set NP3/NP4, on the other hand, could be used to find the PPRV vaccine strain, but it was hard to find results when checking samples from an outbreak or with a low viral load [21]. Our investigation includes a primer set designed targeting N gene-based NP-F and NP-R from published PPRV sequences (GenBank Ac.no. KY967609 and KY967608) full genome sequences from Lahore and Faisalabad, Pakistan. These sequences were obtained from GenBank and accessed on 15th March 2018. (https://www.ncbi.nlm.nih.gov/tools/primer-blast). The BLAST tool from the NCBI database was used to search for sequenced data for gene fragments of PPRV. The CLUSTALX multiple-sequence alignment was used to target highly conserved N-coding gene’s region at nucleotide position 240–657 for further testing [22,23,24]. The primer sequences for these genes were NP-F (forward: 5’-AGTCACCCGGACAACTGATA-3’) and NP-R (reverse: 5’-CTTCTGCAATTCTGTTGCGG-3’) (Table 3). A few similar reports have been published [21, 25].

Table 3 Oligonucleotide primers set used for the PPRV detection

A total of 20 µL reaction containing 4 µL of 5X PCR buffer, 0.8 µL of (10 mM) dNTPs, NP3-F (10 pmol/µl) 1.0 µL, and NP3-R (10 pmol/µl) 1.0 µL, 2 µLMgCl2 (100mM), 1.5 µL template cDNA, Taq polymerase (0.5 µl) and 9.2ul nuclease-free water was used. The RT-PCR cycle conditions have an initial denaturation phase at 94 °C for 10 min, 35 cycles of denaturation at 95 °C for 15 s, an annealing step at 59 °C for 1 min, extension at 72 °C for 1 min, lastly a final extension at 72 °C for 10 min and termination at 4 °C. All the tests were run at least twice.

Virus Isolation and Propagation

The swabs and tissue samples were processed following the standard procedures [26] and inoculum was prepared for PPR virus recovery. Briefly, sub-cultures were performed using pre-cultured Vero cells (ATCC-USA). Before inoculating the Vero cell line, the medium was decanted and inoculum was dispensed (1 ml) after filtering through 0.2 μm syringe filters (Millipore). Vero cells (ATCC, USA) were grown in Dulbecco Minimum Essential Medium (100 ml of DMEM) containing10% fetal bovine serum (FBS) and 2% of pen-strep antibiotics (Corning, USA) [26]. Confluent monolayers of Vero cells were infected with previously processed and filtered tissue and swab samples. These were then incubated at 37 °C for two hours before being washed with PBS and supplemented with DMEM (maintenance media). The infected Vero cells were maintained at 37 °C for seven days and evaluated daily for any cytopathic effects (CPE). The specimen was regarded as negative when the CPE was not observed after consecutive three blind passages. The PPRV isolates were confirmed based on specific CPE and by using ‘N’ gene-based RT-PCR. The CPE, induced by Morbillivirus, triggered cell rounding and syncytia formation. The cells undergo three cycles of freezing and thawing, followed by centrifugation. The resultant supernatants were used for RNA extraction.

Sequence Analysis of Selected Isolates

Sequencing was conducted and sequence data analysis was conducted following standard procedures [22, 26]. Sequence alignment was carried out using CLUSTALX software. In addition, sequenced data of Pakistani isolates were compared with other related sequences retrieved from the Gene Bank. The variations among N-gene sequences and the phylogenetic tree (analysis) of PPRV isolates used in the current study were constructed using the neighbour-joining technique in Mega-X (10.2.2). Bootstrap verification of the resulting phylogenetic tree was performed by analysis of 100 bootstraps. Given the expected similar genetic patterns, only three representative samples were randomly selected for sequencing (one from each sample collection area). Furthermore, the PCR amplicons were purified using the QIAEXII PCR purification kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Purified PCR products were sent for sequencing by Macrogen (Korea). Genius software was used to submit the sequencing to NCBI to obtain new accession numbers for our isolates from the current study.

After obtaining the N gene’s partial sequence through sequencing, different software (finch-TV software, BLASTN analysis and CLC Sequence Viewer 8) were used to analyze multiple sequence alignments to find single nucleotide polymorphisms (SNPs) (data not shown). A total of 8 sequences were selected and analyzed from various geographical locations, (including isolates from current studies), under Lineage IV of Small Ruminants Morbilliviruses (supplementary file1). Analysis of molecular variance (AMOVA) and a Nei’s distance-based cluster was also performed to examine genetic variation among populations [27], to illustrate the relationships between viral sequences from Islamabad and other regions, including previous isolates from Pakistan and other regions of world to understand its genetic evolution [28].

Antigen Preparation

One of our sequenced PPRV local strains (ABP3-Pak), from this study, was attenuated (45th passage) in Vero cells and used to prepare IELISA antigens, as previously described [29, 30]. The PEG 6000 at 8% (w/v) in sodium chloride at 2.3% (w/v) was used to precipitate the supernatant. Following overnight incubation at 4 °C, the mixture was centrifuged at 8500 g for 30 min. The pellet was dissolved, one-tenth of the original volume of supernatant, in a buffer containing Tris, NaCl, and EDTA at pH 7.4 (TNE buffer) [29]. This purified antigen was stored at -80 °C until used [29]. It was important to optimize the concentration of antigen that will be used to capture antibodies but avoid nonspecific binding. The concentration of the antigen was determined using Bio Spec-Nano spectrophotometers (Shimadzu) and TCID50 quantification [30].

Development of Indirect ELISA (IELISA)

The ELISA plates were coated with a partially purified attenuated PPR virus as the coating antigen [31]. A 96-well flat-bottom ELISA plate (NUNC Maxisorp, Hamburg, Germany) was coated with 100 ul/well of PPRV virus antigen at an optimal 1:10 dilution (104.8 TCID50/ml) in carbonate–bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C in a humid environment under constant orbital shaking conditions. Unbound antigen was removed by washing plates thrice with wash buffer, PBS-T (PBS containing 0.05% Tween-20) at pH 8.0. Every well-received 100 µL of blocking buffer (PBS-T containing 5% Bovine serum albumin) to block the remaining sites in each well. After two hours at 37 °C, the plates were again washed thrice with PBS-T wash buffer (PBS-T). Each serum sample was diluted individually (1:50) in a blocking buffer. The diluted test sample was added in 100 µL volume to individual wells in duplicates, including goat-produced positive antiserum (VNT titer > 1:4) and a negative serum (VNT titer < 1:2) from one of our previous studies (unpublished data). These positive and negative controls were implemented as previously described by Balamurugan and colleagues [31].This was followed by 2 h of humid incubation conditions at 37 °C. Anti-goat IgG HRPO-conjugated horseradish peroxidase (Abcam, UK), dissolved in 1:1000 conc. of blocking buffer, 100ul volume was added to each well and the plate was incubated for 1 h at 37 °C [32, 33].

A 100ul of substrate solution, tetra-methyl benzidine substrate (TMB), Cambridge, UK was added to each well, followed by 15 min of dark, room-temperature incubation conditions. The reaction was terminated with 100 µl of 1 M H2SO4 added to each well. ELISA readers measured the absorbance values (OD) at a wavelength of 450 nm. The mean sample/positive ratio (S/P) was calculated using OD values obtained from each tested sample. The results are expressed as percent positivity (PP) value. The PP values greater than 50% were considered positive samples [7, 34].

$$Negative \,control\,\left( \right)\, = \,Mean \,of\,Negative\, Control$$

$$Positive\, control\left( \right)\, = \,Mean\, of\,Positive \,Control$$

$$\fracvalue=\frac X100$$

Initial Validation and Data Analysis of both Diagnostic Assays

In the indirect IELISA test, checkerboard titration was used to optimize the working dilution of antigens and antibodies. As previously described [30], the antigen and serum dilutions that presented the maximum difference between positive and negative absorbance values at 450 nm (P/N) were carefully selected. For this investigation; the reference negative serum (VNT titer < 1:2) from healthy non-vaccinated animals, and positive serum (VNT titer > 1:4) from PPRV-vaccinated animals, were tested in twofold dilutions starting from 1:2 dilutions. The antigen and serum dilutions that gave maximum difference in absorbance at 450 nm between positive and negative (P/N) were selected for testing the serum samples on larger scales. Test sera also included standard controls such as true and false positive and negative samples. According to the manufacturer’s instructions, for the cELISA kit, cutoff values of 50 and 60% were used. In contrast, a cutoff value of 50% positivity was used for the IELISA kit. The data were analyzed using a two-way contingency table using the statistical package Graph Pad Prism 5.01 [35]. Specificity and sensitivity values were determined for both I-ELISA and c-ELISA assays using the previously mentioned statistical formula [36] to compare the results. The notations presented above are explained as follows:

$$Sensitivity \left(\%\right)=a/a+c x 100$$

$$Specificity \left(\%\right)=\frac+b x 100$$

a = true positive (T.P.), b = false positive (F.P.), c = false negative (F.N.), d = true negative (T.N.)

Commercial ELISA (c-ELISA) Test

PPRV antibodies were detected using a commercial c-ELISA test (ID Vet® for PPR). The manipulation of the c-ELISA test was based on the manufacturer’s instructions. The ELISA microplate was read using an ELISA reader with a 450 nm filter (Bio-Rad, IMark™, Microplate reader) [37].

Virus Neutralization Test (VNT)

The ability of anti-PPV antibodies to neutralize the virus was tested in Vero cells in the manner described in a previous study [29]. Briefly, the serum samples were individually incubated with 100 TCID-50 of PPRV viruses in two-fold dilutions. They were incubated at 4 C for 24 h before being poured onto the Vero cells to determine the virus infectivity rate in duplicate. These cells were then monitored daily to assess their specific cytopathic effects. Final readings were calculated on the seventh post-infection day.

Ethical Considerations

Approval to conduct the study was received from the National Agriculture Research Center (NARC) from the Institutional Biosafety Committee (I.B.C. reference No.: NIGAB/NARC/02/05-01-2021).