The vaccine potency testing experiment was carried out at ICAR-Indian Veterinary Research Institute Mukteshwar Campus as per the guidelines of Indian Pharmacopoeia-2014. The study was done after obtaining permission from Indian Veterinary Research Institute Animal Ethics Committee (IVRI-IAEC) under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The protocols were approved vide letter no 387/CPCSEA. Animals (ca. 1 year of age) for the experiment were initially tested to be negative for the presence of PPRV antibody by competitive ELISA (Singh et al., 2004b) and serum neutralization test (SNT; Dhinakar Raj et al., 2000). The animals were also found negative for PPRV antigen in nasal, ocular, buccal, and rectal swabs by sandwich ELISA (Singh et al., 2004c). A highly virulent PPRV (Izatnagar/94 - lineage IV) isolate maintained at PPR Laboratory, Division of Virology, Indian Veterinary Research Institute, Mukteshwar was used as a challenge virus (Sreenivasa et al., 2002). The accession number of this isolate is (KR140086.1; Sahu et al., 2017). Splenic suspension (10%) of virulent virus was inoculated subcutaneously (4 ml suspension, 2 ml each at two different sites). The unvaccinated infected group animals were monitored diurnally for, rectal temperature, any secretion from natural orifices, and feeding habit throughout the experimental period. The unvaccinated animals infected with the PPRV, developed symptoms characteristics of PPRV. The infected animals in which the temperature dropped subnormal were euthanized at 10 days post-infection. As PPRV is epitheliotropic and lymphotropic virus, the tissue samples—lung (epithelial) and spleen (lymphoid) were collected from PPRV infected sheep and goats (n = 2 for each of the species). The counterpart healthy tissues (control) were collected from nearby slaughter house from apparently healthy animals that were screened for the absence of PPRV antigen by sandwich ELISA and antibodies by competitive ELISA and SNT.

PPRV infection was confirmed in lung and spleen tissues by, RT-PCR, qRT-PCR, and sandwich ELISA.

Total RNA from each of the collected samples (lung and spleen) was isolated using the RNeasy Mini kit (Qiagen GmbH, Germany) according to the manufacturer’s protocol. The integrity and quantity of isolated RNA were assessed on a Bioanalyzer (Agilent Technologies, Inc). The RNA integrity number (RIN) value of all the samples was found greater than 8, which is considered suitable for further processing (Kukurba and Montgomery, 2015). The library was prepared using NEBNext Multiplex Small RNA Library Prep Kit (New England Biolabs Inc.) following the manufacturer’s protocol. Hundred nanograms of total RNA from each sample was used for small RNA library preparation. The quality of the libraries was assessed on Bioanalyzer. Libraries were quantified using a Qubit 2.0 Fluorometer (Life Technologies) and by quantitative real-time PCR (qPCR; Robin et al., 2016). The high-throughput sequencing was performed on Illumina – NextSeq500 (75 bp single-end) (manufacturer’s protocol).

The cattle genome sequence was obtained from ftp://ftp.ensembl.org/pub/release-89/fasta/bos_taurus/dna/ The genome was indexed using Bowtie short read aligner program (Langmead et al., 2009). miRNAs are conserved across species (Altuvia et al., 2005). Since there is no complete sheep and goats miRNAs dataset available in miRBase, known mature miRNAs and precursor sequences for cattle were obtained using a Perl script from miRBase database. Data were further processed using miRDeep2 software (Friedlander et al., 2008, 2012). After filtering (read length ≥18 nt) the set of collapsed, non-redundant, clean reads were mapped to the indexed cattle genome using a mapper module. To identify known miRNAs, clean reads were aligned against miRNA precursor sequences reported in the miRBase database using quantifier.pl module. Read counts for each miRNA identified using miRDeep2 were further used for the downstream analysis. The relative expression levels of miRNAs were normalized as TMM (trimmed mean of M-values) using edgeR R/Bioconductor package (Robinson et al., 2010) to identify differentially expressed miRNAs (DEmiRNAs) in lung and spleen.

To better understand the biological function of DEmiRNA, TargetScan tool (Aggarwal et al., 2015) with default parameters was used to predict target genes of the selected DEmiRNAs (six miRNAs selected based on their role). From these predicted genes, the dysregulated genes from the proteomics data were identified for the miRNA selected (downregulated proteins for upregulated miRNA and upregulated proteins for downregulated miRNA). These common target genes from TargetScan and proteomics data were considered for further analysis. The miRNA–protein network was created based on the expression profile of target genes and miRNAs using Cytoscape (ver. 3.1.1; Shannon et al., 2003).

Functional annotation of the selected DEmiRNAs in each tissue was performed using target genes governed by them in ClueGO (ver. 2.1.4; Bindea et al., 2009) in Cytoscape (ver. 3.1.1; Shannon et al., 2003). Immune system processes and KEGG pathways were selected to generate a functionally organized GO/pathway term networks.

Total RNA, including small RNA from the lung and spleen of control and infected sheep and goats were isolated using mirVana^TM miRNA isolation kit (Invitrogen). Reverse transcriptase reactions were performed using RT specific primers of miR-363, miR-760-3p, miR-21-3p, and U6snRNA by TaqMan^® MicroRNA Reverse Transcription Kit. Real-time PCR was performed using a standard TaqMan PCR kit protocol on an Applied Biosystems 7500 fast Sequence Detection System. The 10 μl PCR included 5 μl of 2× TaqMan Gene Expression Master Mix (Thermo Fisher Scientific Inc., Wilmington, DE, United States, Cat. No. 4369016), 0.5 μl of 20× TaqMan probe, 2 μl (0.134 ng) of RT product and 2.5 μl of NFW. The reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were run in triplicate. The threshold cycle (Aad et al., 2015) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The expression of the selected miRNAs described above was assayed taking the expression of U6snRNA as an internal control. The relative expression of each miRNA was calculated using the 2^-ΔΔCT method with a control group as calibrator (Schmittgen and Livak, 2008).

Viral infection in the lung and spleen of sheep and goats infected with PPRV was confirmed by RT-PCR of 351 bp N gene amplicon in lung and spleen (Supplementary Figure S1). The viral infection was further confirmed by sandwich ELISA and qRT-PCR in both lung and spleen of goats and sheep (data not shown).

In goats, miRDeep2 identified 298 and 283 miRNAs in control and PPRV infected lung, and 277 and 274 miRNAs in control and PPRV infected spleen. In sheep, 290 and 298 miRNAs in control and PPRV infected lung, and 274 and 256 miRNAs in control and PPRV infected spleen, respectively, were predicted. DEmiRNAs [FDR ≤ 0.05 and fold change (log₂FC) ≥ 1] in lung and spleen of PPRV infected sheep and goats are presented in Table 1. A total of 67 miRNAs (34 downregulated and 33 upregulated) were dysregulated in the lungs of PPRV infected goats. However, a relatively small number of DEmiRNAs—37 miRNAs (16 miRNAs downregulated and 21 miRNAs upregulated) were identified in the lungs of sheep (Table 1). In infected goat’s spleen, 50 miRNAs were dysregulated with 26 of them downregulated and 24 upregulated. In spleen of sheep, 56 miRNAs were differentially expressed after PPRV infection and of these, 26 miRNAs were downregulated and 30 miRNAs were upregulated (Table 1).

On comparing tissues across species, 20 and 11 miRNAs were found to be commonly differentially expressed in PPRV infected spleen and lung, respectively, in both sheep and goats. Among these 20 common DEmiRNAs in spleen, 11 DEmiRNAs (miR-199b, miR-1271, miR-217, miR-2887-1, miR-2887-2, miR-6119-3p, miR-221, miR-744, miR-30c, let-7a-5p-2, and miR-211) were downregulated and nine DEmiRNAs (miR-17-3p, miR-486, miR-146b, miR-363, miR-451, miR-193a-3p, miR-760-3p, miR-144, and miR-21-5p) were upregulated in goats, and in sheep, nine DEmiRNAs (miR-199b, miR-1271, miR-217, miR-6119-3p, miR-221, miR-744, miR-30c, let-7a-5p-2, and miR-211) were downregulated and 11 DEmiRNAs (miR-2887-1, miR-2887-2, miR-17-3p, miR-486, miR-146b, miR-363, miR-451, miR-193a-3p, miR-760-3p, miR-144, and miR-21-5p) were upregulated (Figure 1A and Table 2). Of these 20 common DEmiRNAs, miR-21-5p was the most upregulated (log₂FC = 2.35) and miR-199b was the most downregulated DEmiRNA (log₂FC = -3.03) in the spleen of goats. While in infected spleen of sheep the most upregulated DEmiRNAs were miR-451 (log₂FC = 2.75) and miR-144 (log₂FC = 2.63) and the most downregulated DEmiRNAs were miR-217 (log₂FC = -4.08) and miR-221 (log₂FC = -2.60).

The expression profile of the 11 common DEmiRNAs in the lung tissue varied in sheep and goats. miR-328 was found downregulated in goats but upregulated in sheep; two miRNAs—miR-2285f-2 and miR-27a-5p were found upregulated in goats but downregulated in sheep; six miRNAs—miR-320a-1, miR-320a-2, miR-1246, miR-363, miR-760-3p, and miR-21-3p were upregulated and two miRNAs—miR-34b and miR-150 were downregulated in both species (Figure 1B and Table 3). Among these 11 common DEmiRNAs, the expression of miR-21-3p, miR-760-3p, and miR-27a-5p was more abundant in lungs of PPRV infected goats with log₂ fold change of 5.82, 3.79, and 3.07, respectively, while miR-34b (log₂FC = -2.53) and miR-2285f-2 (log₂FC = -2.53) were found least abundant in lungs of goats and sheep, respectively.

Among these 31 commonly DEmiRNAs, six DEmiRNAs were selected based on their role in viral infections, apoptosis, and fold change (Table 4). These miRNAs include miR-21-3p, miR-320a, miR-27a-5p, and miR-1246—expressed in lung of both species; miR-760-3p and miR-363—expressed in lung and spleen of both species (Figure 2). The miRNAs—miR-363 and miR-760-3p commonly present in PPRV infected lung and spleen of both species were identified to be upregulated. In lung, the expression of miR-363 and miR-760-3p was higher in goats with log₂ fold change of 2.42 and 3.79, respectively, than in sheep (miR-363, log₂FC = 1.04 and miR-760-3p, log₂FC = 1.24) after PPRV infection. In spleen, the expression profile of miR-363 was higher in goats (log₂FC = 1.55) than in sheep (log₂FC = 1.28), however, no difference in expression of miR-760-3p was observed in goats (log₂FC = 1.63) and sheep (log₂FC = 1.67) infected with PPRV.

A total of 1149 (714 downregulated, 435 upregulated) and 1565 (1041 downregulated, 524 upregulated) differentially expressed proteins were identified in lung of sheep and goats, respectively, and 944 (281 downregulated, 663 upregulated) and 909 (590 downregulated, 319 upregulated) differentially expressed proteins were identified in spleen of sheep and goats, respectively. The number of dysregulated proteins identified through mass spectrophotometry by each of these six miRNAs is shown in Figure 2. The miRNA–protein interactions for each species and tissue are represented in a network (Figure 3). In the miRNA–protein network of lung tissue of goats, three miRNAs—miR-21-3p, miR-363, and miR-320a mutually regulate EGFR (epidermal growth factor receptor), which is involved in immune response. Similarly, IGF1R (insulin like growth factor 1) protein, involved in regulation of immune response was the target of miR-27a-5p, miR-363, miR-320a, and miR-760-3p. The TRIM (tripartite motif family) family members TRIM24, TRIM36, and TRIM45 were identified to be modulated by miR-1246, miR-320a, and miR-21-3p, respectively. The expression level of NF-κB signaling-related molecules IRAK2 and TRAF4 was regulated by miR-1246 and miR-320; and miR-1246 and miR-760-3p, respectively (Figure 3A). In PPRV infected sheep lung, the upregulated miRNAs—miR-21-3p and miR-320a govern immune genes—TRAF6, EGFR, and ERBB4 and the downregulated miR-27a-5p potentially modulates the expression of genes—MAP3K7 and MAPK8IP3, involved in JNK signaling pathways (Figure 3B).

In PPRV infected spleen of goats, miR-363 and miR-760-3p regulate apoptotic molecules (NFATC2, NOX5, and MYO6) and target genes involved in immunological processes (NFATC2, IGF1R, KLHL21, and NOX5) (Figure 3C). The immune effector molecules IFIT5 and TRIM33 were targeted by miR-363, and CD244 and NFKBIE were regulated by miR-760-3p in infected spleen of sheep (Figure 3D).

Functional annotation of a total of 770 and 1226 target proteins governed by selected six miRNAs in infected sheep and goats infected lung, respectively, resulted in higher number of significantly enriched pathways and GO terms in goats than in sheep (Figure 4). The highly enriched common GO terms and pathways targeted by the miRNAs in the lung tissue of sheep and goats includes T cell receptor signaling pathway, Rap1 signaling pathway, Toll-like receptor TLR6:TLR2 signaling pathway, etc. (Figures 4A,B).

Similarly, functional annotation of 84 and 173 target proteins governed by two out of selected six miRNAs in infected sheep and goats spleen, respectively, identified enrichment of B cell receptor signaling and FC epsilon RI signaling pathways in both the species (Figures 4C,D). Furthermore, targets of DEmiRNAs in sheep spleen were also found enriched in FC-gamma R mediated phagocytosis and myeloid leukocyte mediated immunity. The targets of DEmiRNAs of goat’s spleen were found involved in NF-κB signaling pathway, alpha–beta T cell differentiation, ErbB signaling pathway and regulation of B cell activation. In addition, the number of significantly enriched pathways and GO terms were higher in lung and spleen tissue of goats than in sheep.

To further validate the expression of DEmiRNAs from high-throughput sequencing, qPCR was performed on three DEmiRNAs—miR-21-3p, miR-363, and miR-760-3p. The expression of miR-363 and miR-760-3p in sheep and goats in both the tissues was in concordance with small RNA sequencing results. The expression of miR-21-3p was found to be in concordance with the sequencing results in PPRV infected spleen of both the species. However, in infected lung miR-21-3p was found upregulated on qPCR though not found in small RNA sequencing data in both the species (Figure 5 and Table 5).