1. Introduction
Peste des petits ruminant’s virus (PPRV), a morbillivirus in the family Paramyxoviridae, causes an acute, extremely contagious disease. Ovine rinderpest or goat plaque is characterized by high fever, ocular and nasal discharges, pneumonia, necrotic and inflammation and ulcerative lesion of the mucosa in the gastrointestinal tract [
1]. PPRV was first reported in India in 1989 and subsequently spread all over the country [
2,
3,
4,
5]. In India, the disease is mainly controlled through the use of a Vero cell-attenuated Sungri 96 vaccine which elicits a protective antibody response forup to 78 months [
6]. PPRV infection is usually confined to populations of small ruminants with particular breeds of goats being reported as more susceptible than others [
7] and with more severe pathology compared to sheep [
8,
9]. Differential disease resistance to PPRV has been reported both at the species and breed levels; the Guinean breeds (West African dwarf (WAD), Iogoon, Kindi and Djallonke) are known to be highly susceptible [
7].
Although Cattle can become infected with PPRV, unlike the closely related rinderpest virus (RPV), they do not show clinical signs and are not susceptible to disease [
7,
10]. However, virus replication and sero-conversion does occur in large ruminants [
11]. Interestingly, a clinical case of PPRV infection was reported following experimental inoculation of calves [
12] and another report describes that PPRV was isolated from an RPV-like outbreak in Indian buffaloes [
13]. PPRV was also suspected to be involved in the epizootic disease that affected one-humped camels in Ethiopia in 1995–1996 [
14] with detection of PPRV antigen and nucleic acid in some of the pathological samples, but no live virus was isolated.
The genetics underlying this host-specific disease resistance to PPR is unknown. The two likely mechanisms are the differential presence or expression of viral specific receptors or the nature and type of the immune response. The signaling lymphocyte activation molecule (SLAM) a cellular receptor for PPRV, its expression level and PPRV replication rates have been shown to be highly correlated [
15]. Furthermore, different levels of SLAM mRNA correlated with virus replication in different species such as cattle, buffalo, goat and sheep. In addition to SLAM, ovine nectin-4 was identified as a novel epithelial receptor for PPRV, which determines tissue distribution and pathogenicity [
16]. The replication of PPRV in the PBMC of Indian domestic goats and water buffalo is influenced by the expression levels of TLR3, TLR7 and downstream signaling molecules. Upon stimulation of PBMC with synthetic TLR3 and TLR7 agonists or PPRV, the levels of pro-inflammatory cytokines were found to be significantly different across goats and water buffalo, a likely mechanism influencing differential susceptibility to disease [
17]. In contrast, immunosuppressive interleukin (IL) 10 levels were lower in PPR-resistant Kanni and Salem Black breeds of goat and water buffalo at the transcriptional level, correlating with reduced viral loads in infected PBMC. In addition, water buffalo also produced higher levels of interferon alpha (IFNα) in comparison with goats both at transcriptional and translational levels which were confirmed to be TLR7 mediated through inhibitor and pre-treatment studies [
17]. Thus, differential gene expression analysis can be a very powerful first attempt to correlate immune responses with gene regulation. Such approaches can also identify potential target genes for disease control.
Earlier studies used candidate gene-based approaches (individual genes or proteins one at a time) to understand the host and pathogen interactions. To gain a more global understanding of gene expression underlying differential responses to PPRV infection, we used an RNAseq approach to study the transcriptome of goat and cattle PBMC exposed to PPRV in vitro. This systems biology approach may be useful in understanding differences in susceptibility toPPR in different animal species, identifying early markers of infection, potential antiviral targets and for understanding the basic molecular mechanisms of host-virus interactions.
4. Discussion
PPRV is capable of replicating in a wide variety of ruminant species but disease susceptibility is confined to small ruminants. Understanding the host factors that lead to viral pathogenesis and disease susceptibility or resistance will help to identify novel targets and inform better control strategies. In this study, we used RNAseq to study the increased susceptibility of goats to PPRV-induced disease compared to cattle by confirming increased viral replication and the identification and predicted function of differentially expressed genes induced during infection. Here we report the interaction of functionally annotated immune-related pathway genes that are differentially regulated between goat and cattle after exposure to PPRV.
In the past few years there has been a steady increase in the spread of PPRV across different countries and around 63% of the small ruminants globally may be under threat [
18]. PPRV is considered small ruminant specific, with domesticated cattle and buffalo described as dead-end hosts. However, several surveillance programs have reported the presence of PPRV-specific antibodies in atypical hosts such as domesticated cattle (67%), buffalo (41%) and camel [
11,
19,
20]. The potential of the virus to overcome innate resistance in these atypical hosts to result in evident clinical signs or disease transmission has been little studied [
12,
21]. The toll-like receptors (TLR) 7 and TLR3 play a major role in innate recognition of ssRNA viruses. Upon sensing the viral pathogen associated molecular patterns (PAMPS), a complex network of intra-cellular signaling pathways are activated by these TLRs which results in the production of several antiviral cytokines. Previous data from our laboratory clearly provides evidence that the viral recognition by these innate immune receptors and the subsequent cytokine profiles results in a 2log
10 lower PPR virus titre in the PBMC of buffaloes when compared with goats [
17]. Experiments on the replication kinetics of PPRV in cattle and goat PBMC revealed an increased virus titre and fold change in PPRV M gene RNA expression (0.2 log
10 titre and 0.13 fold increase respectively) as early as 24 h post infection (PI) in goats (A 1.2 log
10 higher virus titre and 3.81 fold increase in expression at 120 h PI in goat PBMC; for details refer to
Supplementary Information—PPRV replication kinetics).
The genetic basis underlying differential host susceptibility or resistance is highly complex. Methods that enable the identification and analysis of gene interaction networksin a single experiment offer a powerful approach to understanding these complex processes. Systems biology approaches not only investigate the role of multiple genes contributing to a phenomenon but also generate information on their behavior and relationships in a quantitative, integrative and comprehensive manner [
22]. High-throughput sequencing generated RNAseq data can provide gene expression profiles to identify genes likely to contribute to host-virus interactions and potentially to differential resistance/susceptibility across different species [
23].
Among the cytokines, the interferons (IFNs) provide an anti-viral state to the host cells as well as help in modulating adaptive immune responses. Type I IFNs (IFNα/β) generate ananti-viral state by binding to the IFNα/β receptor which activates the transcription of interferon-stimulated genes (ISGs) via the JAK/STAT pathway while. Type II IFN (IFNγ) functions through a different receptor (IFNGR) to induce gamma activated factor (GAF) that activates the transcription of a distinct subset of cellular genes result in an antiviral state [
24,
25]. In addition to the interferon-induced genes, we also selected other significantly differentially regulated immune related genes with roles in the innate immune response, inflammatory response and defense response (
Figure 3A,B) [
17]. The classical inflammatory cytokines such as IFNβ, IFNγ, IL-4, IL-1β, IL-8, IL-10, IL-6 and IL-12 are induced in goats upon PPRV infection [
26,
27] even though the PPRV V protein possess the ability to inhibit the IFN signaling [
28,
29,
30]. In experiments with the Sungri/96 PPRV vaccine virus, IFN-α/β mRNA were not detected after the infection of PBMC [
31] and evasion of IFN-induced antiviral effects by PPRV have been shown to occur through miRNA mediated regulation of IRF3 and IRF7 [
32]. In contrast, studies with a virulent PPRV strain demonstrated that genes within the interferon and RIG-1 like receptor signaling pathway, IRF-7 and STAT-1 that regulate the expression of ISGs, were active in lymphocytes [
33]. Our previous report also indicates the contribution of TLR7 mediated higher IFN alpha induction as a factor for the lower replication of PPRV in cattle PBMC [
17].
Viral infection-induced interferons induce an exclusive and moderately overlapping set of interferon stimulated genes (ISG) and studies have identified 50 to 1000 ISGs (using microarrays) that are typical to some cells types with effector functions limited to the classical ISGs, namely protein kinase (PKR; also known as eukaryotic translational initiation factor 2-alphase kinase -EIF2AK2), Myxovirus Resistance Protein 1 (MX1), 2′-5′-oligoadenylate synthetase 1 (OAS1), Tripartite Motif Containing 5 (TRIM5), Interferon-stimulated gene 15 (ISG15), adenosine deaminase acting on RNA (ADAR), interferon-induced transmembrane protein 1/2/3 (IFITM1/2/3 also called CD225), Tetherin (also known as BST2 or CD17), Viperin (also known as Radical S-Adenosyl Methionine Domain Containing 2 -RSAD2) [
34,
35] and ATP-dependent dsRNA helicase DHX58 also known as RIG-I-like receptor LGP2 (RLR) [
36]. Up-regulation of the ISGs (ISG15, Mx1, Mx2, RSAD2, IFIT3 and IFIT5) that play a role in antiviral response and the viral sensors MDA5, LGP2 and RIG1 were found to up-regulated in lymphocytes upon exposure to a virulent strain of PPRV [
33]. Our RNAseq data also reveal that the IFN type I responses are highly up-regulated in cattle. This increased type I interferon results in up-regulation of the ISG effector molecules such as Mx, OAS, PKR and related genes in cattle which have a crucial role to play in viral defense. One of the major effects of the IFN is induction of Oligoadenylate synthetase (OAS) that is capable of polymerizing ATP in a template-dependent fashion. OAS results in 2′,5′-linked oligoadenylate polymers (2-5A) which is not found in mRNA and DNA, resulting in the activation of RNase-L that leads to cleavage of viral genomes. This pathway is also regulated by enzymes encoded by virus and the host that degrades 2-5A. One such enzyme is phosphodiesterase 12 (PDE12) which degrades 2-5A and thus inhibition/decreased levels of PDE12 might up-regulate viral-infection-induced OAS/RNase-L pathway resulting in increased resistance to viral pathogens. Our results indicated that PDE12, a negative regulator of the innate immune response, is up-regulated in goats which could be a major reason for the decreased resistance to viral replication.
This host antiviral response is also potentiated by the increased levels of the interferon-induced IFIT family genes (IFIT1, IFIT2, IFIT3, IFIT5) in cattle (
Table 6). The up-regulation of these genes was confirmed in the RT-qPCR experiments. In addition, it was observed that the DHX58/ RLR gene is also up-regulated in cattle. DHX58/ RLR induces RIG-I which helps in intra-cytoplasmic sensing of dsRNA and results in type I interferon release through the IRF-7 pathway. The stimulation of the IRF-7 gene has been shown toprovide host defense against viral infection by inducing Viperin/RSAD2 and our data shows that Viperin/RSAD2 are highly up-regulated in cattle. Gene profiling in IFN-α/βR-deficient mice indicates that Viperin is induced by several different viruses, dsRNA and poly-dAdT [
37,
38]. This pathway inhibits replication of Flaviviruses (HCV), West Nile virus (WNV) and Dengue virus, herpesviruses (HCMV), a paramyxovirus (Sendai virus), a rhabdovirus (VSV) an Orthomyxovirus (Influenza A virus) and a retrovirus (HIV-1) [
39,
40,
41,
42,
43].
The fundamental aim of our study was to better understand differential resistance/ susceptibility to PPRV between small and large ruminants. Earlier reports from our work indicated that PPRV induced increased interferon levels in cattle and buffalo and were likely to be a major factor causing decreased PPRV replication. This study reveals a complex interaction of ISGs and their effector molecules is likely a major factor leading to host defense and decreased PPRV replication in cattle, confirming our earlier report. In summary, these results indicate the potential role of ISGs and their interacting proteins in the differential replication of PPRV in PBMC between goats and cattle. If the host immune responses in small ruminants are sufficiently suppressed, especially the interferon-stimulated genes, this could in part explain clinical disease in particular breeds and species. Further in-vitro experiments are being planned on selected target genes identified using a siRNA/ CRISPR Cas9 system approach and over expression to understand their contribution to the differential replication of PPRV between these two species. In addition, efforts to generate small molecule inhibitors to some of the targets identified, e.g., PDE12, could help in improving the immune responses to PPRV in the susceptible species.