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Influence of mutation in nucleoprotein of Peste-des-petits-ruminants virus (PPRV) isolated from 2016 Indian outbreak
Journal Pre-proof Influence of mutation in nucleoprotein of Peste-des-petits-ruminants virus (PPRV) isolated from 2016 Indian outbreak Anurag R. Mishra, Prasana Kumar Rath, Susen Kumar Panda, Debasis Nayak
PII:
S0921-4488(20)30003-1
DOI:
https://doi.org/10.1016/j.smallrumres.2020.106048
Reference:
RUMIN 106048
To appear in:
Small Ruminant Research
Received Date:
6 November 2019
Revised Date:
7 January 2020
Accepted Date:
8 January 2020
Please cite this article as: Mishra AR, Kumar Rath P, Kumar Panda S, Nayak D, Influence of mutation in nucleoprotein of Peste-des-petits-ruminants virus (PPRV) isolated from 2016 Indian outbreak, Small Ruminant Research (2020), doi: https://doi.org/10.1016/j.smallrumres.2020.106048
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Influence of mutation in nucleoprotein of Peste-des-petits-ruminants virus (PPRV) isolated from 2016 Indian outbreak Anurag R. Mishraa✚, Prasana Kumar Rathb✚, Susen Kumar Pandab, and Debasis Nayaka* Affiliation Indian Institute of Technology Indore, MP 453 552, India
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College of Veterinary Science and Animal Husbandry, Bhubaneshwar, Odisha, India
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Equally contributing first authors of this work.
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Contact address of Corresponding Authors: The discipline of Bioscience and Biomedical Engineering
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Indian Institute of Technology Indore Khandwa Road, Indore
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MP 453 552, India Ph: +91-971 305 1845
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Corresponding Author: Dr. Debasis Nayak, Email: [email protected]
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Highlights
PPRV outbreak occurred in the Eastern Indian state of Odisha in the year 2016 was investigated
We observed 90% morbidity and 42% mortality rate with the current isolates.
Phylogenetic analysis based on the complete viral nucleocapsid (N) genes revealed that
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the circulating strain belonged to lineage IV along with the other Asian Isolates of
Interestingly we observed four unique mutations in N gene including one mutation in the RNA binding region of the protein.
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PPRVs.
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We postulate that change in N-RNA binding pattern could influence genome
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Abstract
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transcription, replication and overall viral pathogenesis in its natural host.
Peste des petits ruminants virus (PPRV) is an economically significant viral disease of small ruminants across the globe. Despite having efficacious vaccines and global eradication efforts, PRRV outbreaks still exist, particularly in developing countries. To better understand the viral isolates that circulate in India, we investigated clinical cases of PPRV outbreak that occurred in the Eastern Indian state of Odisha in the year 2016. We observed 90% morbidity and 42% mortality rate with the current isolates. Phylogenetic analysis based on the complete viral nucleocapsid (N) genes revealed that the circulating strain belonged to lineage IV along with the other Asian Isolates of PPRVs.
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Interestingly we observed four unique mutations in the N gene including one mutation in the RNA binding region of the protein. As N protein plays an essential role in the protection of viral genome and subsequently coordinates viral gene transcription and genome replication, we postulate that these mutations would influence overall viral pathogenesis in its natural host.
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Keywords: PPRV, Nucleocapsid protein, epidemiology, N-RNA interaction
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1. Introduction Peste des petits ruminants (PPR) is an acute, devastating viral disease of small ruminants with associated morbidity and mortality rates reaching up to 100% (Jones et al., 2016; LEFEVRE and DIALLO, 1990; Sen et al., 2010). The disease is characterized by pyrexia, conjunctivitis, mucopurulent ocular and nasal discharge, erosiveulcerative stomatitis, pneumonia, and severe diarrhea and often makes the host (sheep and goats) susceptible to secondary microbial infection (Balamurugan et al., 2014; Hammouchi et al., 2012). The causative agent, PPR virus (PPRV) is a non-segmented, negative-strand (ns) RNA virus that belongs to the genus Morbillivirus and placed
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under Paramyxoviridae family. This enveloped virion has a genome of approximately 16 kb length, which encodes six viral proteins; Nucleocapsid (N), Phosphoprotein (P), Matrix (M), Fusion (F), Haemagglutinin (H), and large
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Polymerase (L) protein. Among these, the N protein (also mRNAs) being at the N-terminus of the genome is the most abundant in the virally infected cells, which is valid for most of the nsRNA viruses (Bailey et al., 2007; Baron,
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2015; Diallo, 1990). The nucleocapsid protein is a structural component of negative-stranded RNA viruses that protects viral genomic and anti-genomic RNA (not mRNA) by forming a ribonucleocapsid complex that involves
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the N-N and N–RNA interaction. In the case of –ve strand RNA viruses, the genomic (and anti-genomic) RNA in
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the form of nucleocapsids is functionally active while the naked viral genome is biologically inactive. Though N protein enwraps the nascent genomic RNA primarily to protect it from cellular enzymatic degradation, while, it should be flexible enough to allow access of polymerase complexes to initiate transcription and replication
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activities(Ortín and Martín-Benito, 2015). Thus, the structure of nucleocapsids is dynamically controlled to facilitate viral replication in the host cells. While encapsidating these viral nucleic acids, the N protein units facilitate viral genome transcription and replication by recruiting viral polymerase (L) and accessory phosphoprotein (P) to the nucleocapsids(Kingston et al., 2005; Richard L. Kingston et al., 2004). Hence, any mutations or alternation to N
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protein has significant consequences with virus biology and pathogenesis. Although N function is central to virus biology, detailed knowledge on its structural information, molecular characterization, etc. is lacking which hampers the comprehension of the protein functions. The 525 aa long PRRV N protein is highly immunogenic. For this reason, the PPRV diagnosis is often
made by ELISA (Choi et al., 2003; Zhang et al., 2011) by using the respective monoclonal antibody (Bodjo et al., 2007). Based on sequence similarity with the other morbilliviruses, the PPRV N gene can be divided into four domains; region I (aa 1-120), II (aa 122-145), III (aa 146-398) and IV (aa 421-525) (Bailey et al., 2005; Diallo et al.,
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1994). Regions II and I are more immunogenic compared to that of regions III and IV. Region I is known to generate a much faster humoral immune response compared to region II (Choi et al., 2005). Region III and IV are the least conserved regions. This variability of region IV sequence is used for phylogenetic analysis and geographical origin identification. In Morbilliviruses, the N protein forms two distinct structures; NCORE and NTAIL. The NCORE binds to any non-specific RNA and is sufficient to form the nucleocapsid-like structure even in the absence of the NTAIL, but in the presence of P protein, it is highly specific to viral RNA (Bhella et al., 2002; Curran et al., 1995; Errington and
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Emmerson, 1997; Huang et al., 2002; Karlin et al., 2002; Spehner et al., 1997). The PRRV NTAIL(488-499 aa) known to interact with the P and P-L complexes (Karlin et al., 2002; Richard L Kingston et al., 2004). Besides this,
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the N also interacts with the other host proteins like Hsp72, IRF-3 and cell receptor those helps in viral replication, and it is tropism (Laine et al., 2003; Zhang et al., 2002). Study conducted by Bodjo et. al., to understand the
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structural basis of N-N interaction has demonstrated that two regions; an N-terminal (1-120 aa) and a central (146241 aa) regions are essential for the N- N interaction; whereas a short fragment spanning 121-145 aa is essential for
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the stability of the protein-protein interaction (Bodjo et al., 2008). However, information on the molecular
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mechanism of N-RNA interaction is lacking. The only known RNA binding motif which is conserved in the Morbilliviruses is F-X4-Y-X4-SYAMG (including PPRV). Understanding the structural and functional importance of the N concerning N-RNA interaction is of utmost essential to gain insight into the basis of viral replication and
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overall virus biology.
Phylogenetic analysis is a bioinformatics tool used to get insight into the evolutionary relationship among the studied subjects and in particular molecular phylogenetic analysis approach based on gene and/or protein sequences is being extensively used to identify viral pathogens, viral strains and its relationship with global strains.
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In the case of PPRV, sequence analysis of a fragment of the N or F genes groups prevailing global strains to four lineages coupled to different geographic locations. Classification based on N reflects much better geographical origin than the classification based on F and HN (Diallo et al., 2007). Lineages I and II are found in West Africa, lineage III in Arabia, southern India, and East Africa, whereas lineage IV is predominantly present in the Middle East, Northern Africa, and Aisa (Parida et al., 2015). The Asian lineage IV is the most prevalent and is well reported in endemic areas of Africa and Asia. However, the reason for the increased pathogenicity and prevalence of lineage IV is still unknown. PPR outbreaks are continually being reported in India. Investigating the diversity in the
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circulating strains of PPRV is vital to understand and access the impact of the vaccine proficiency and disease mitigation controls. In this study, we have investigated a major PPRV outbreak (2016) occurred in the Eastern Indian state of Odisha and performed molecular characterization and phylogenetic analysis of the viral isolates based on viral N gene sequence information. The morbidity and mortality rates of this outbreak were observed to be ~90% and ~42%, respectively. Phylogenetic analysis grouped the current PPRV isolates into lineage IV. Interestingly, unique mutations (e.g., W333G) were observed in the RNA-binding domain of the N protein those might influence the stability and biological activities of N-RNA ribonucleoprotein complexes thus affecting the mutant virus biology
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and pathogenies in the host. 2. Materials and Methods
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Sample collection and RNA extraction. The local veterinary doctors reported a total of 45 suspected cases of PPRV in the respective regions during 2016. All suspected cases were clinically examined, samples were collected,
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and the data were recorded. Samples were then transported to the laboratory in cold condition and later saved in 80°C. Total cellular RNA was isolated from the cells using the Trizol method as per the manufacturer's protocol
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(Invitrogen, USA). Briefly, nasal or oral swab samples were dissolved in 1 ml of plain DMEM for 1 hour and centrifuged at 12,000 rpm for 10 min at 4°C. The cells were homogenized with 1 ml of Trizol, and the total RNA
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was extracted as per the manufacturer's protocol (Invitrogen, USA). The resultant RNAs were resuspended in 50 μl of nuclease-free water and stored at -80°C until further analysis.
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Vertebrate animal use statement
Authors declare that no live vertebrate animal experimentation was conducted for the work presented in this manuscript. Instead, clinical samples collected for routine laboratory examination were investigated. All the experiments were carried out as per the guidelines and regulations issued by the College of Veterinary Science and
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Animal Husbandry, Bhubaneswar, India.
Real-Time PCR. Purified RNA was quantified using Thermo scientific Nanodrop spectrophotometer at A260 and A280. Subsequently, cDNA was synthesized using the iScript cDNA synthesis kit (BioRad) as per manufacturer’s instructions. Following PCR cycle was used for cDNA synthesis: 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min and hold it at 4°C for 5-10 minute. Following cDNA synthesis, the real-time PCR assay was carried out in 25 µl reaction mixture containing 1 µl of cDNA from the sample, 150nM from each primer, and 12.5 µl of 2X POWER
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UP SYBR Green master mix (BioRad). The conditions for the real-time PCR was as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min followed by melting curve analysis. Construction of plasmids. The full-length PPR N gene was cloned into the pMD-20T vector using the TA cloning kit (TaKaRa). The cloning was confirmed by sequencing and double digestion with restriction enzymes. The concentration of the plasmid was estimated using Nanodrop and gel electrophoresis using densitometry method. The copy number was calculated as following: Copy number = [amount(ng) *6.022 * 1023] / length(Plasmid + Insert) * 1 * 109 * 650 (Lamien et al., 2011).
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Virus identification. The PCR reaction mixture of 50µl was prepared using the 5µl of Taq standard buffer, two µl of cDNA, 1.5µl of each forward and reverse primer (10µM), 1.5µl of dNTPs and nuclease-free water to make up the
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volume. The following PCR cycle was used: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing 54°C for 40 sec and extension at 68°C for 45 sec and final extension at
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68°C for 5 min. The PCR products were analyzed on a 1% agarose gel (for up to 500 base pairs and 0.8 % for more than 500 base pairs). Detail primer information is provided in Table S2.
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Sequencing and phylogenetic analysis. The PCR amplicons were sequenced with a set of three primers (Table 1).
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The consensus sequence was generated using the Bioedit 7.2.6.1 version software. The resultant sequence was submitted to the NCBI database. For phylogenetic analysis, the complete N gene sequence was retrieved from the GenBank database (Table S1). The retrieved sequences were aligned using the Cluster-W algorithm in MEGA 7.0.
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Phylogenetic analysis was carried out using the Neighbour-Joining method using the Kimura 2-parameter nucleotide substitution model in MEGA version 7.0 with 1000 bootstrap replicates. 3. Results
3.1 Virus identification and clinical pattern in PRRV affected animals
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In December 2016, a PPR outbreak was reported in the Ganjam district of Odisha (India). The affected animals (31 goats and 14 sheep) exhibited predominantly nasal discharge, diarrhea, and mouth lesion along with the symptoms of fever, bronchopneumonia, and other clinical symptoms suggestive of PPR (Fig. 1). All goats belong to the native “Ganjam” breed, which is a mid-sized animal primarily reared for meat purposes. Whereas the sheep breeds are nondescriptive in nature. Further, these animals lack any record of prior vaccination against PPRV. All animals are pasture-fed. The outbreak revealed morbidity and mortality of ~90% and ~42%, respectively, in the affected herd. In the affected herd, overall 60% of the infected animals showed all five types of clinical signs of PPR
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whereas the rest of them did not show at least one clinical sign. Out of the 45 infected animals, only 26 animals survived (18 goats and eight sheep). Clinical data showed that the mortality in goats is higher (60%) with severe clinical signs compared to sheep mortality (43%) (Fig. 2.A), which is similar to previously reported cases of PRRV lineage IV outbreak (Lefèvre and Diallo, 1990). However, the overall mortality rate is lower than the recently reported outbreaks (45% mortality) (Senthil Kumar et al., 2014). This difference might be due to the host characteristics and environmental factors or attributed to a mutation in the viral genome. 3.2 Virus detection and quantification
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Out of 45 affected animals, samples from 16 cases were analyzed for PCR detection of viral nucleic acids. Primers targeting partial (352bp) and full length (1764bp) N gene were used for PCR amplification. Of these tested
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samples, all (n=16) were found PCR positive (Fig. 2B) and subsequently confirmed by sequencing. The gene sequence information was submitted to the NCBI database (GenBank accession no. MG748708, MG748709,
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MG748710, and MG748711). To estimate the viral load in the clinical samples, we performed the SYBR Greenbased real-time PCR assay targeting the 502-604 region of the N gene. Interestingly, significantly (<0.01) higher
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viral load (1.5x109 genome/ml) was found in the nasal samples of deceased animals compared to those who survived
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(2.67x108 genome/ml) (Fig. 2.C). Similarly higher viral load was observed in the animals with severe symptoms compared to animals having mild symptoms (Fig. 2C).
3.3 Phylogenetic relationship and comparative sequence analysis of PPRV isolates
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To obtain the epidemiological information and genetic relatedness of the circulating strains, a phylogenetic tree was constructed by taking consideration of all available (n=61) full-length N gene sequences downloaded from the Genebank database (Table S1) including four representative sequences of the current isolates. First, we analyzed full-length N gene sequences (Fig. 2) along the side with partial N (result not shown) gene sequences. Interestingly
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both the methods (partial and full length) revealed similar clustering patterns. The current isolates were clustered to Asian lineage IV with a close relation to earlier reported Indian strains such as Delhi (KX033350.1) and Tamil Nadu (KT860065.1 and KT860064.1) (Fig. 2). A full-length N gene sequence analysis was done to understand the pattern of an amino acid (aa) variation.
The result revealed that the current isolate is 98.8 %-100% identical with previously reported Indian isolates. It has > 95% identity with lineage IV and 91% -94 % identity with lineage I, II and III (Table 1). The overall divergence calculated for the current study was less than 10%. Lineage IV differs primarily at 13 aa positions compared to other
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lineages. Out of these 13, two are specific to previously reported Indian origin-specific (I471F and N514D) (Fig. 3). Here, we observed four unique mutations (E297D, W333G, G461R, and E512G). Out of which, the first two mutations (E297D and W333G) in region III of N and are restricted to the goat isolates, whereas other mutations (G461R and E512G) in region IV of N are present in both sheep and goat isolates. These results show the emergence of a new mutation in the N protein compared to previously reported cases, which might have altered biological functions. Since N protein plays a critical role in virus biology, which is closely associated with template function
modeling analysis and in vitro analysis is required to conclude this phenomena.
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4. Discussion
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of viral nucleocapsids, to better understand the effect of mutations on its biological functions, computational
Small ruminant (sheep and goats) rearing is a critical livelihood asset, particularly for the lower-income groups
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across the globe. Since sheep and goats are vulnerable to PPRV infection, global efforts are on to control and mitigate PPR outbreak. Further jointly various global agencies aim for the complete elimination of PPR by 2030.
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This necessitates due to scrutiny about the nature of viral isolates and its ecological impact. Globally, PPRV isolates
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are grouped into four distinct lineages based on the N and F gene sequences. The Asian region often witnesses the outbreak of PPRV of lineage IV referred to as Asian lineage, which was first reported in India in 1989, and since then it is persistently reported (Muthuchelvan, 2015; Shaila et al., 1996). The current study describes the first PPR
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outbreak in the Eastern Indian state of Odisha, occurred during 2016. To better understand the biological effect of N mutations in the circulating PPRV isolates, the molecular characterization was performed. For the first time, we performed a full-length N gene-based phylogenetic analysis to get a better picture of its relationships with global strain. Although perfect cross-protection against PPRV from different lineages is possible, a lineage classification is
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must for monitoring PPRV and tracing the source of the outbreak. Phylogenetic analysis of the circulating strains using the N gene fell into four separate lineages as reported earlier (Kock et al., 2015; Kumar et al., 2014). The current circulating strains (Asian lineage IV) were clustered with Tamil Nadu and Delhi strains. This cluster forms a single robust clade with no outlying sequence or regional intermingling. Lineage IV is considered to be a newly emerging virus with much more virulence and overwhelming the other lineages in an African country (Kwiatek, 2011; Libeau et al., 2014; Muniraju et al., 2016) and the persistent of lineage IV in India is a matter of concern (Muthuchelvan, 2015). We have now learned that Indian strains are divided into two sub-clusters. Isolates of Tamil
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Nadu, Delhi, and current strain belong to one subcluster, whereas the rest of the isolates were grouped to a separate subcluster. This indicates either two sources of origin for PPRV in the country or the strain is evolving into two subclusters independently. However, the reason for this is yet to be determined. The amino acid sequence analysis revealed two unique mutations in the RNA binding domain of the N protein. The PPRV is a non–segmented, negative-stranded (ns) RNA virus. Typically, the nucleocapsid (NP) of the negative-stranded viruses consists of a single viral genomic RNA wrapped with multiple N protein subunits. Thus, for a functional NP, in addition to N-RNA interaction, proper N-N interaction would be required to form a stable,
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biologically active structure for template functions. Further, NP structure should be flexible enough to allow access to viral polymerase during genome transcription and replication process.
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For this reason, any mutation in the N protein poses significance to virus biology. Prior studies with vesicular stomatitis virus (VSV), a prototypic nsRNA virus shows that the N terminal arms or C terminal loops that
(Leyrat et al., 2011; Zhang et al., 2008).
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interact with the neighboring subunits can disrupt the nucleocapsid structure and impact N-RNA complex formation Even a single mutation in (Y289A) can completely abrogate the
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encapsidation, transcription, and replication of the virus in the host cells (Nayak et al., 2009). The mutation could
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result in a change in the side chain of the residue making the virion of a temperature-sensitive phenotype. As no crystal structure is available for PPRV N protein, we could evaluate the exact role of mutation on RNA binding and N-N interaction. As the N-RNA complex partly regulates the N-RNA template functions. The exact impact of
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mutation on stabilizing/destabilized N-RNA complex on genome replication and transcription yet to be determined and requires a detail in vivo study. However, we speculate that instability of the N-RNA complex could positively influence the degree of virulence and properties of current isolates. This theory is guarded and needs further experimental validation, which is beyond the scope of the study. The multiple sequence alignment has shown a
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unique tryptophan mutation in the RNA binding region of the nucleocapsid protein. Understanding the importance of the accumulation of these amino acid changes in the virus requires more experiments, which will help us under infectivity, pathogenicity, and transmission in sheep and goats. Availability of the reverse genetics system to manipulate viral genome is a powerful tool to study the mutational effect on various biological parameters of the virus replication and pathogenesis characteristics. Given the role of N-RNA complex formation central to PPRV biology, more studies are required to conduct in vivo assays such as alanine scanning mutagenesis with the help of the viral reverse genetics (RG) system to delineate the exact functions. Thus understanding the structural and
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functional importance of the N gene in the context of N-N and N-RNA interactions in different lineages would give us an insight into the essential viral replication and may provide a window of opportunities for development of candidate vaccine or therapeutic intervention in the coming future.
Declaration of interest statement
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The authors declare no competing financial and non-financial interests.
Acknowledgment
Declaration of interest statement
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The authors declare no competing financial and non-financial interests.
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ARM is thankful to the Department of Science and Technology (DST, Govt India) INSPIRE graduate fellowship.
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Kwiatek, O., 2011. Asian Lineage of Peste des Petits Ruminants Virus, Africa. Emerging Infectious Diseases 17, 1223–1231. https://doi.org/10.3201/eid1707.101216 Laine, D., Trescol-Biémont, M.-C., Longhi, S., Libeau, G., Marie, J.C., Vidalain, P.-O., Azocar, O., Diallo, A., Canard, B., Rabourdin-Combe, C., Valentin, H., 2003. Measles virus (MV) nucleoprotein binds to a novel cell surface receptor distinct from FcgammaRII via its C-terminal domain: role in MV-induced immunosuppression. Journal of virology. https://doi.org/10.1128/JVI.77.21.11332 Lamien, C.E., Le Goff, C., Silber, R., Wallace, D.B., Gulyaz, V., Tuppurainen, E., Madani, H., Caufour, P., Adam,
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Parida, S., 2016. Emergence of Lineage IV Peste des Petits Ruminants Virus in Ethiopia: Complete Genome Sequence of an Ethiopian Isolate 2010. Transboundary and emerging diseases 63, 435–442. https://doi.org/10.1111/tbed.12287
Muthuchelvan, D., 2015. Peste-Des-Petits-Ruminants: An Indian Perspective. Advances in Animal and Veterinary Sciences 3, 422–429. https://doi.org/10.14737/journal.aavs/2015/3.8.422.429 Nayak, D., Panda, D., Das, S.C., Luo, M., Pattnaik, A.K., 2009. Single-Amino-Acid Alterations in a Highly Conserved Central Region of Vesicular Stomatitis Virus N Protein Differentially Affect the Viral
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Nucleocapsid Template Functions. Journal of Virology 83, 5525–5534. https://doi.org/10.1128/JVI.02289-08 Ortín, J., Martín-Benito, J., 2015. The RNA synthesis machinery of negative-stranded RNA viruses. Virology 479– 480, 532–544. https://doi.org/10.1016/j.virol.2015.03.018 Parida, S., Muniraju, M., Mahapatra, M., Muthuchelvan, D., Buczkowski, H., Banyard, A.C.C., 2015. Peste des petits ruminants. Veterinary Microbiology 181, 90–106. https://doi.org/10.1016/j.vetmic.2015.08.009 Sen, A., Saravanan, P., Balamurugan, V., Rajak, K.K., Sudhakar, S.B., Bhanuprakash, V., Parida, S., Singh, R.K., 2010. Vaccines against peste des petits ruminants virus. Expert Review of Vaccines 9, 785–796.
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Molecular characterisation of lineage IV peste des petits ruminants virus using multi gene sequence data.
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Veterinary Microbiology 174, 39–49. https://doi.org/10.1016/j.vetmic.2014.08.031
Shaila, M.., Shamaki, D., Forsyth, M. a, Diallo, A., Goatley, L., Kitching, R.., Barrett, T., 1996. Geographic
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distribution and epidemiology of peste des petits ruminants viruses. Virus Research 43, 149–153.
Spehner, D., Drillien, R., Howley, P.M., 1997. The assembly of the Measles Virus Nucleoprotein into Nucleocapsidlike Particules is Modulated by the Phosphoprotein. Virology 232, 260–268.
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Zhang, G.R., Zeng, J.Y., Zhu, Y.M., Dong, S.J., Zhu, S., Yu, R.S., Duoji, C., Lei, Z.H., Li, Z., 2011. Development of an indirect ELISA with artificially synthesized n protein of PPR virus. Intervirology 55, 12–20. https://doi.org/10.1159/000322220
Zhang, X., Glendening, C., Linke, H., Parks, C.L., Brooks, C., Udem, S.A., Oglesbee, M., 2002. Identification and
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characterization of a regulatory domain on the carboxyl terminus of the measles virus nucleocapsid protein. Journal of virology. https://doi.org/10.1128/JVI.76.17.8737
Zhang, X., Green, T.J., Tsao, J., Qiu, S., Luo, M., 2008. Role of Intermolecular Interactions of Vesicular Stomatitis Virus Nucleoprotein in RNA Encapsidation. Journal of Virology 82, 674–682. https://doi.org/10.1128/JVI.00935-07
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Figure Legends Figure 1. Clinical Score of the PPR virus infection in the small ruminant. Clinical scores were assigned to
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the other signs (bronchopneumonia, mouth lesions, diarrhea and fever).
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infected animals based on the severity of the primary clinical sign (nasal and ocular discharge) and the presence of
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Figure 2. PPR virus mortality, detection, and viral load determination. A) The mortality rate in the goats and sheep in percentages. B) Agarose gel electrophoresis of the positive samples amplified using primers specific to partial N gene sequences (352bp) (lane 2-4) and for full-length N gene (1764) (lane 6-8). Negative control lanes (lane 5 & 9), lane 1 and 10 are 1 Kb DNA ladder. C) Real-time PCR targeting 123 bp of the N gene was done to assess the viral copy number in comparison to plasmid standard. Viral copy number was determined by comparing it
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with a standard curve.
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Figure 3. Phylogenetic analysis of PPRV N gene. Phylogenetic tree showing clustering of N gene of Peste des petits ruminant virus constructed using GenBank available strains by the bootstrap test of phylogeny using the neighbor-joining method. The bootstrap confidence tested on using the 1000 replicates and the major cluster values are indicated on the node/branch of a tree.
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Figure 4. Schematic diagrams presenting mutation in PPRV N gene A) Reference Sequence (NC_006383.2) B) Current isolates with site-specific mutations Sequence 1 (MB748708, MB748710, and MB748711) C) Sequence 2
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(MB748709).
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Table 1. Percent identity and divergence of different PPRV published sequences from various lineages.
Percentage Identity 4
5
6
7
8
9
10
11
12
13
95.4
94.9
93.3
95.6
94.1
95.1
95.1
95.1
93.3
93.9
94.5
94.7
1
EU267273.1
97.9
93.2
95.8
98.5
99.6
99.6
99.6
97.9
98.5
99
99.2
2
EU360596.1
93.9
96.4
97.1
97.5
97.5
97.5
95.8
96.4
97
97.1
3
KC609745.1
94.5
92.4
92.8
92.8
92.8
91.1
91.8
92.2
92.4
4
KJ867543.1
94.7
95.4
95.4
95.4
93.7
94.5
94.9
95.1
5
KR781449.1
98.1
98.1
98.1
96.4
97
97.5
97.7
6
KT633939.1
100
100
98.3
98.9
99.4
99.6
7
KT860064.1
100
98.3
98.9
99.4
99.6
8
KT860065.1
98.3
98.9
99.4
99.6
9
KX033350.1
98.9
98.7
10
MG748710
99.4
99.2
11
MG748711
99.8
12
MG748708
13
MG748709
3
5.3
2.1
4
7
7.2
6.4
5
4.5
4.3
3.7
5.7
6
6.1
1.5
2.9
8
5.5
7
5.1
0.4
2.5
7.6
4.7
1.9
8
5.1
0.4
2.5
7.6
4.7
1.9
0
9
5.1
0.4
2.5
7.6
4.7
1.9
0
0
10
7
2.1
4.3
9.5
6.6
3.7
1.7
1.7
1.7
11
6.4
1.5
3.7
8.7
5.7
3.1
1.1
1.1
1.1
12
5.7
1
3.1
8.2
5.3
13
5.5
0.8
2.9
8
5.1
1
2
3
4
5
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4.7
98.7
2.5
0.6
0.6
0.6
1.1
0.6
2.3
0.4
0.4
0.4
1.3
0.8
0.2
6
7
8
9
10
11
12
1.3
13
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NCBI Acc. No.
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Divergence
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PII:
S0921-4488(20)30003-1
DOI:
https://doi.org/10.1016/j.smallrumres.2020.106048
Reference:
RUMIN 106048
To appear in:
Small Ruminant Research
Received Date:
6 November 2019
Revised Date:
7 January 2020
Accepted Date:
8 January 2020
Please cite this article as: Mishra AR, Kumar Rath P, Kumar Panda S, Nayak D, Influence of mutation in nucleoprotein of Peste-des-petits-ruminants virus (PPRV) isolated from 2016 Indian outbreak, Small Ruminant Research (2020), doi: https://doi.org/10.1016/j.smallrumres.2020.106048
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Influence of mutation in nucleoprotein of Peste-des-petits-ruminants virus (PPRV) isolated from 2016 Indian outbreak Anurag R. Mishraa✚, Prasana Kumar Rathb✚, Susen Kumar Pandab, and Debasis Nayaka* Affiliation Indian Institute of Technology Indore, MP 453 552, India
b
College of Veterinary Science and Animal Husbandry, Bhubaneshwar, Odisha, India
✚
Equally contributing first authors of this work.
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Contact address of Corresponding Authors: The discipline of Bioscience and Biomedical Engineering
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Indian Institute of Technology Indore Khandwa Road, Indore
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MP 453 552, India Ph: +91-971 305 1845
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Corresponding Author: Dr. Debasis Nayak, Email: [email protected]
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Highlights
PPRV outbreak occurred in the Eastern Indian state of Odisha in the year 2016 was investigated
We observed 90% morbidity and 42% mortality rate with the current isolates.
Phylogenetic analysis based on the complete viral nucleocapsid (N) genes revealed that
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the circulating strain belonged to lineage IV along with the other Asian Isolates of
Interestingly we observed four unique mutations in N gene including one mutation in the RNA binding region of the protein.
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PPRVs.
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We postulate that change in N-RNA binding pattern could influence genome
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Abstract
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transcription, replication and overall viral pathogenesis in its natural host.
Peste des petits ruminants virus (PPRV) is an economically significant viral disease of small ruminants across the globe. Despite having efficacious vaccines and global eradication efforts, PRRV outbreaks still exist, particularly in developing countries. To better understand the viral isolates that circulate in India, we investigated clinical cases of PPRV outbreak that occurred in the Eastern Indian state of Odisha in the year 2016. We observed 90% morbidity and 42% mortality rate with the current isolates. Phylogenetic analysis based on the complete viral nucleocapsid (N) genes revealed that the circulating strain belonged to lineage IV along with the other Asian Isolates of PPRVs.
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Interestingly we observed four unique mutations in the N gene including one mutation in the RNA binding region of the protein. As N protein plays an essential role in the protection of viral genome and subsequently coordinates viral gene transcription and genome replication, we postulate that these mutations would influence overall viral pathogenesis in its natural host.
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Keywords: PPRV, Nucleocapsid protein, epidemiology, N-RNA interaction
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1. Introduction Peste des petits ruminants (PPR) is an acute, devastating viral disease of small ruminants with associated morbidity and mortality rates reaching up to 100% (Jones et al., 2016; LEFEVRE and DIALLO, 1990; Sen et al., 2010). The disease is characterized by pyrexia, conjunctivitis, mucopurulent ocular and nasal discharge, erosiveulcerative stomatitis, pneumonia, and severe diarrhea and often makes the host (sheep and goats) susceptible to secondary microbial infection (Balamurugan et al., 2014; Hammouchi et al., 2012). The causative agent, PPR virus (PPRV) is a non-segmented, negative-strand (ns) RNA virus that belongs to the genus Morbillivirus and placed
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under Paramyxoviridae family. This enveloped virion has a genome of approximately 16 kb length, which encodes six viral proteins; Nucleocapsid (N), Phosphoprotein (P), Matrix (M), Fusion (F), Haemagglutinin (H), and large
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Polymerase (L) protein. Among these, the N protein (also mRNAs) being at the N-terminus of the genome is the most abundant in the virally infected cells, which is valid for most of the nsRNA viruses (Bailey et al., 2007; Baron,
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2015; Diallo, 1990). The nucleocapsid protein is a structural component of negative-stranded RNA viruses that protects viral genomic and anti-genomic RNA (not mRNA) by forming a ribonucleocapsid complex that involves
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the N-N and N–RNA interaction. In the case of –ve strand RNA viruses, the genomic (and anti-genomic) RNA in
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the form of nucleocapsids is functionally active while the naked viral genome is biologically inactive. Though N protein enwraps the nascent genomic RNA primarily to protect it from cellular enzymatic degradation, while, it should be flexible enough to allow access of polymerase complexes to initiate transcription and replication
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activities(Ortín and Martín-Benito, 2015). Thus, the structure of nucleocapsids is dynamically controlled to facilitate viral replication in the host cells. While encapsidating these viral nucleic acids, the N protein units facilitate viral genome transcription and replication by recruiting viral polymerase (L) and accessory phosphoprotein (P) to the nucleocapsids(Kingston et al., 2005; Richard L. Kingston et al., 2004). Hence, any mutations or alternation to N
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protein has significant consequences with virus biology and pathogenesis. Although N function is central to virus biology, detailed knowledge on its structural information, molecular characterization, etc. is lacking which hampers the comprehension of the protein functions. The 525 aa long PRRV N protein is highly immunogenic. For this reason, the PPRV diagnosis is often
made by ELISA (Choi et al., 2003; Zhang et al., 2011) by using the respective monoclonal antibody (Bodjo et al., 2007). Based on sequence similarity with the other morbilliviruses, the PPRV N gene can be divided into four domains; region I (aa 1-120), II (aa 122-145), III (aa 146-398) and IV (aa 421-525) (Bailey et al., 2005; Diallo et al.,
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1994). Regions II and I are more immunogenic compared to that of regions III and IV. Region I is known to generate a much faster humoral immune response compared to region II (Choi et al., 2005). Region III and IV are the least conserved regions. This variability of region IV sequence is used for phylogenetic analysis and geographical origin identification. In Morbilliviruses, the N protein forms two distinct structures; NCORE and NTAIL. The NCORE binds to any non-specific RNA and is sufficient to form the nucleocapsid-like structure even in the absence of the NTAIL, but in the presence of P protein, it is highly specific to viral RNA (Bhella et al., 2002; Curran et al., 1995; Errington and
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Emmerson, 1997; Huang et al., 2002; Karlin et al., 2002; Spehner et al., 1997). The PRRV NTAIL(488-499 aa) known to interact with the P and P-L complexes (Karlin et al., 2002; Richard L Kingston et al., 2004). Besides this,
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the N also interacts with the other host proteins like Hsp72, IRF-3 and cell receptor those helps in viral replication, and it is tropism (Laine et al., 2003; Zhang et al., 2002). Study conducted by Bodjo et. al., to understand the
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structural basis of N-N interaction has demonstrated that two regions; an N-terminal (1-120 aa) and a central (146241 aa) regions are essential for the N- N interaction; whereas a short fragment spanning 121-145 aa is essential for
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the stability of the protein-protein interaction (Bodjo et al., 2008). However, information on the molecular
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mechanism of N-RNA interaction is lacking. The only known RNA binding motif which is conserved in the Morbilliviruses is F-X4-Y-X4-SYAMG (including PPRV). Understanding the structural and functional importance of the N concerning N-RNA interaction is of utmost essential to gain insight into the basis of viral replication and
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overall virus biology.
Phylogenetic analysis is a bioinformatics tool used to get insight into the evolutionary relationship among the studied subjects and in particular molecular phylogenetic analysis approach based on gene and/or protein sequences is being extensively used to identify viral pathogens, viral strains and its relationship with global strains.
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In the case of PPRV, sequence analysis of a fragment of the N or F genes groups prevailing global strains to four lineages coupled to different geographic locations. Classification based on N reflects much better geographical origin than the classification based on F and HN (Diallo et al., 2007). Lineages I and II are found in West Africa, lineage III in Arabia, southern India, and East Africa, whereas lineage IV is predominantly present in the Middle East, Northern Africa, and Aisa (Parida et al., 2015). The Asian lineage IV is the most prevalent and is well reported in endemic areas of Africa and Asia. However, the reason for the increased pathogenicity and prevalence of lineage IV is still unknown. PPR outbreaks are continually being reported in India. Investigating the diversity in the
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circulating strains of PPRV is vital to understand and access the impact of the vaccine proficiency and disease mitigation controls. In this study, we have investigated a major PPRV outbreak (2016) occurred in the Eastern Indian state of Odisha and performed molecular characterization and phylogenetic analysis of the viral isolates based on viral N gene sequence information. The morbidity and mortality rates of this outbreak were observed to be ~90% and ~42%, respectively. Phylogenetic analysis grouped the current PPRV isolates into lineage IV. Interestingly, unique mutations (e.g., W333G) were observed in the RNA-binding domain of the N protein those might influence the stability and biological activities of N-RNA ribonucleoprotein complexes thus affecting the mutant virus biology
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and pathogenies in the host. 2. Materials and Methods
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Sample collection and RNA extraction. The local veterinary doctors reported a total of 45 suspected cases of PPRV in the respective regions during 2016. All suspected cases were clinically examined, samples were collected,
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and the data were recorded. Samples were then transported to the laboratory in cold condition and later saved in 80°C. Total cellular RNA was isolated from the cells using the Trizol method as per the manufacturer's protocol
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(Invitrogen, USA). Briefly, nasal or oral swab samples were dissolved in 1 ml of plain DMEM for 1 hour and centrifuged at 12,000 rpm for 10 min at 4°C. The cells were homogenized with 1 ml of Trizol, and the total RNA
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was extracted as per the manufacturer's protocol (Invitrogen, USA). The resultant RNAs were resuspended in 50 μl of nuclease-free water and stored at -80°C until further analysis.
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Vertebrate animal use statement
Authors declare that no live vertebrate animal experimentation was conducted for the work presented in this manuscript. Instead, clinical samples collected for routine laboratory examination were investigated. All the experiments were carried out as per the guidelines and regulations issued by the College of Veterinary Science and
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Animal Husbandry, Bhubaneswar, India.
Real-Time PCR. Purified RNA was quantified using Thermo scientific Nanodrop spectrophotometer at A260 and A280. Subsequently, cDNA was synthesized using the iScript cDNA synthesis kit (BioRad) as per manufacturer’s instructions. Following PCR cycle was used for cDNA synthesis: 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min and hold it at 4°C for 5-10 minute. Following cDNA synthesis, the real-time PCR assay was carried out in 25 µl reaction mixture containing 1 µl of cDNA from the sample, 150nM from each primer, and 12.5 µl of 2X POWER
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UP SYBR Green master mix (BioRad). The conditions for the real-time PCR was as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 sec, 60°C for 1 min followed by melting curve analysis. Construction of plasmids. The full-length PPR N gene was cloned into the pMD-20T vector using the TA cloning kit (TaKaRa). The cloning was confirmed by sequencing and double digestion with restriction enzymes. The concentration of the plasmid was estimated using Nanodrop and gel electrophoresis using densitometry method. The copy number was calculated as following: Copy number = [amount(ng) *6.022 * 1023] / length(Plasmid + Insert) * 1 * 109 * 650 (Lamien et al., 2011).
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Virus identification. The PCR reaction mixture of 50µl was prepared using the 5µl of Taq standard buffer, two µl of cDNA, 1.5µl of each forward and reverse primer (10µM), 1.5µl of dNTPs and nuclease-free water to make up the
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volume. The following PCR cycle was used: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing 54°C for 40 sec and extension at 68°C for 45 sec and final extension at
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68°C for 5 min. The PCR products were analyzed on a 1% agarose gel (for up to 500 base pairs and 0.8 % for more than 500 base pairs). Detail primer information is provided in Table S2.
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Sequencing and phylogenetic analysis. The PCR amplicons were sequenced with a set of three primers (Table 1).
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The consensus sequence was generated using the Bioedit 7.2.6.1 version software. The resultant sequence was submitted to the NCBI database. For phylogenetic analysis, the complete N gene sequence was retrieved from the GenBank database (Table S1). The retrieved sequences were aligned using the Cluster-W algorithm in MEGA 7.0.
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Phylogenetic analysis was carried out using the Neighbour-Joining method using the Kimura 2-parameter nucleotide substitution model in MEGA version 7.0 with 1000 bootstrap replicates. 3. Results
3.1 Virus identification and clinical pattern in PRRV affected animals
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In December 2016, a PPR outbreak was reported in the Ganjam district of Odisha (India). The affected animals (31 goats and 14 sheep) exhibited predominantly nasal discharge, diarrhea, and mouth lesion along with the symptoms of fever, bronchopneumonia, and other clinical symptoms suggestive of PPR (Fig. 1). All goats belong to the native “Ganjam” breed, which is a mid-sized animal primarily reared for meat purposes. Whereas the sheep breeds are nondescriptive in nature. Further, these animals lack any record of prior vaccination against PPRV. All animals are pasture-fed. The outbreak revealed morbidity and mortality of ~90% and ~42%, respectively, in the affected herd. In the affected herd, overall 60% of the infected animals showed all five types of clinical signs of PPR
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whereas the rest of them did not show at least one clinical sign. Out of the 45 infected animals, only 26 animals survived (18 goats and eight sheep). Clinical data showed that the mortality in goats is higher (60%) with severe clinical signs compared to sheep mortality (43%) (Fig. 2.A), which is similar to previously reported cases of PRRV lineage IV outbreak (Lefèvre and Diallo, 1990). However, the overall mortality rate is lower than the recently reported outbreaks (45% mortality) (Senthil Kumar et al., 2014). This difference might be due to the host characteristics and environmental factors or attributed to a mutation in the viral genome. 3.2 Virus detection and quantification
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Out of 45 affected animals, samples from 16 cases were analyzed for PCR detection of viral nucleic acids. Primers targeting partial (352bp) and full length (1764bp) N gene were used for PCR amplification. Of these tested
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samples, all (n=16) were found PCR positive (Fig. 2B) and subsequently confirmed by sequencing. The gene sequence information was submitted to the NCBI database (GenBank accession no. MG748708, MG748709,
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MG748710, and MG748711). To estimate the viral load in the clinical samples, we performed the SYBR Greenbased real-time PCR assay targeting the 502-604 region of the N gene. Interestingly, significantly (<0.01) higher
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viral load (1.5x109 genome/ml) was found in the nasal samples of deceased animals compared to those who survived
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(2.67x108 genome/ml) (Fig. 2.C). Similarly higher viral load was observed in the animals with severe symptoms compared to animals having mild symptoms (Fig. 2C).
3.3 Phylogenetic relationship and comparative sequence analysis of PPRV isolates
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To obtain the epidemiological information and genetic relatedness of the circulating strains, a phylogenetic tree was constructed by taking consideration of all available (n=61) full-length N gene sequences downloaded from the Genebank database (Table S1) including four representative sequences of the current isolates. First, we analyzed full-length N gene sequences (Fig. 2) along the side with partial N (result not shown) gene sequences. Interestingly
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both the methods (partial and full length) revealed similar clustering patterns. The current isolates were clustered to Asian lineage IV with a close relation to earlier reported Indian strains such as Delhi (KX033350.1) and Tamil Nadu (KT860065.1 and KT860064.1) (Fig. 2). A full-length N gene sequence analysis was done to understand the pattern of an amino acid (aa) variation.
The result revealed that the current isolate is 98.8 %-100% identical with previously reported Indian isolates. It has > 95% identity with lineage IV and 91% -94 % identity with lineage I, II and III (Table 1). The overall divergence calculated for the current study was less than 10%. Lineage IV differs primarily at 13 aa positions compared to other
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lineages. Out of these 13, two are specific to previously reported Indian origin-specific (I471F and N514D) (Fig. 3). Here, we observed four unique mutations (E297D, W333G, G461R, and E512G). Out of which, the first two mutations (E297D and W333G) in region III of N and are restricted to the goat isolates, whereas other mutations (G461R and E512G) in region IV of N are present in both sheep and goat isolates. These results show the emergence of a new mutation in the N protein compared to previously reported cases, which might have altered biological functions. Since N protein plays a critical role in virus biology, which is closely associated with template function
modeling analysis and in vitro analysis is required to conclude this phenomena.
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4. Discussion
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of viral nucleocapsids, to better understand the effect of mutations on its biological functions, computational
Small ruminant (sheep and goats) rearing is a critical livelihood asset, particularly for the lower-income groups
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across the globe. Since sheep and goats are vulnerable to PPRV infection, global efforts are on to control and mitigate PPR outbreak. Further jointly various global agencies aim for the complete elimination of PPR by 2030.
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This necessitates due to scrutiny about the nature of viral isolates and its ecological impact. Globally, PPRV isolates
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are grouped into four distinct lineages based on the N and F gene sequences. The Asian region often witnesses the outbreak of PPRV of lineage IV referred to as Asian lineage, which was first reported in India in 1989, and since then it is persistently reported (Muthuchelvan, 2015; Shaila et al., 1996). The current study describes the first PPR
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outbreak in the Eastern Indian state of Odisha, occurred during 2016. To better understand the biological effect of N mutations in the circulating PPRV isolates, the molecular characterization was performed. For the first time, we performed a full-length N gene-based phylogenetic analysis to get a better picture of its relationships with global strain. Although perfect cross-protection against PPRV from different lineages is possible, a lineage classification is
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must for monitoring PPRV and tracing the source of the outbreak. Phylogenetic analysis of the circulating strains using the N gene fell into four separate lineages as reported earlier (Kock et al., 2015; Kumar et al., 2014). The current circulating strains (Asian lineage IV) were clustered with Tamil Nadu and Delhi strains. This cluster forms a single robust clade with no outlying sequence or regional intermingling. Lineage IV is considered to be a newly emerging virus with much more virulence and overwhelming the other lineages in an African country (Kwiatek, 2011; Libeau et al., 2014; Muniraju et al., 2016) and the persistent of lineage IV in India is a matter of concern (Muthuchelvan, 2015). We have now learned that Indian strains are divided into two sub-clusters. Isolates of Tamil
9
Nadu, Delhi, and current strain belong to one subcluster, whereas the rest of the isolates were grouped to a separate subcluster. This indicates either two sources of origin for PPRV in the country or the strain is evolving into two subclusters independently. However, the reason for this is yet to be determined. The amino acid sequence analysis revealed two unique mutations in the RNA binding domain of the N protein. The PPRV is a non–segmented, negative-stranded (ns) RNA virus. Typically, the nucleocapsid (NP) of the negative-stranded viruses consists of a single viral genomic RNA wrapped with multiple N protein subunits. Thus, for a functional NP, in addition to N-RNA interaction, proper N-N interaction would be required to form a stable,
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biologically active structure for template functions. Further, NP structure should be flexible enough to allow access to viral polymerase during genome transcription and replication process.
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For this reason, any mutation in the N protein poses significance to virus biology. Prior studies with vesicular stomatitis virus (VSV), a prototypic nsRNA virus shows that the N terminal arms or C terminal loops that
(Leyrat et al., 2011; Zhang et al., 2008).
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interact with the neighboring subunits can disrupt the nucleocapsid structure and impact N-RNA complex formation Even a single mutation in (Y289A) can completely abrogate the
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encapsidation, transcription, and replication of the virus in the host cells (Nayak et al., 2009). The mutation could
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result in a change in the side chain of the residue making the virion of a temperature-sensitive phenotype. As no crystal structure is available for PPRV N protein, we could evaluate the exact role of mutation on RNA binding and N-N interaction. As the N-RNA complex partly regulates the N-RNA template functions. The exact impact of
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mutation on stabilizing/destabilized N-RNA complex on genome replication and transcription yet to be determined and requires a detail in vivo study. However, we speculate that instability of the N-RNA complex could positively influence the degree of virulence and properties of current isolates. This theory is guarded and needs further experimental validation, which is beyond the scope of the study. The multiple sequence alignment has shown a
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unique tryptophan mutation in the RNA binding region of the nucleocapsid protein. Understanding the importance of the accumulation of these amino acid changes in the virus requires more experiments, which will help us under infectivity, pathogenicity, and transmission in sheep and goats. Availability of the reverse genetics system to manipulate viral genome is a powerful tool to study the mutational effect on various biological parameters of the virus replication and pathogenesis characteristics. Given the role of N-RNA complex formation central to PPRV biology, more studies are required to conduct in vivo assays such as alanine scanning mutagenesis with the help of the viral reverse genetics (RG) system to delineate the exact functions. Thus understanding the structural and
10
functional importance of the N gene in the context of N-N and N-RNA interactions in different lineages would give us an insight into the essential viral replication and may provide a window of opportunities for development of candidate vaccine or therapeutic intervention in the coming future.
Declaration of interest statement
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The authors declare no competing financial and non-financial interests.
Acknowledgment
Declaration of interest statement
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The authors declare no competing financial and non-financial interests.
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ARM is thankful to the Department of Science and Technology (DST, Govt India) INSPIRE graduate fellowship.
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Figure Legends Figure 1. Clinical Score of the PPR virus infection in the small ruminant. Clinical scores were assigned to
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the other signs (bronchopneumonia, mouth lesions, diarrhea and fever).
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infected animals based on the severity of the primary clinical sign (nasal and ocular discharge) and the presence of
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Figure 2. PPR virus mortality, detection, and viral load determination. A) The mortality rate in the goats and sheep in percentages. B) Agarose gel electrophoresis of the positive samples amplified using primers specific to partial N gene sequences (352bp) (lane 2-4) and for full-length N gene (1764) (lane 6-8). Negative control lanes (lane 5 & 9), lane 1 and 10 are 1 Kb DNA ladder. C) Real-time PCR targeting 123 bp of the N gene was done to assess the viral copy number in comparison to plasmid standard. Viral copy number was determined by comparing it
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with a standard curve.
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Figure 3. Phylogenetic analysis of PPRV N gene. Phylogenetic tree showing clustering of N gene of Peste des petits ruminant virus constructed using GenBank available strains by the bootstrap test of phylogeny using the neighbor-joining method. The bootstrap confidence tested on using the 1000 replicates and the major cluster values are indicated on the node/branch of a tree.
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Figure 4. Schematic diagrams presenting mutation in PPRV N gene A) Reference Sequence (NC_006383.2) B) Current isolates with site-specific mutations Sequence 1 (MB748708, MB748710, and MB748711) C) Sequence 2
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(MB748709).
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Table 1. Percent identity and divergence of different PPRV published sequences from various lineages.
Percentage Identity 4
5
6
7
8
9
10
11
12
13
95.4
94.9
93.3
95.6
94.1
95.1
95.1
95.1
93.3
93.9
94.5
94.7
1
EU267273.1
97.9
93.2
95.8
98.5
99.6
99.6
99.6
97.9
98.5
99
99.2
2
EU360596.1
93.9
96.4
97.1
97.5
97.5
97.5
95.8
96.4
97
97.1
3
KC609745.1
94.5
92.4
92.8
92.8
92.8
91.1
91.8
92.2
92.4
4
KJ867543.1
94.7
95.4
95.4
95.4
93.7
94.5
94.9
95.1
5
KR781449.1
98.1
98.1
98.1
96.4
97
97.5
97.7
6
KT633939.1
100
100
98.3
98.9
99.4
99.6
7
KT860064.1
100
98.3
98.9
99.4
99.6
8
KT860065.1
98.3
98.9
99.4
99.6
9
KX033350.1
98.9
98.7
10
MG748710
99.4
99.2
11
MG748711
99.8
12
MG748708
13
MG748709
3
5.3
2.1
4
7
7.2
6.4
5
4.5
4.3
3.7
5.7
6
6.1
1.5
2.9
8
5.5
7
5.1
0.4
2.5
7.6
4.7
1.9
8
5.1
0.4
2.5
7.6
4.7
1.9
0
9
5.1
0.4
2.5
7.6
4.7
1.9
0
0
10
7
2.1
4.3
9.5
6.6
3.7
1.7
1.7
1.7
11
6.4
1.5
3.7
8.7
5.7
3.1
1.1
1.1
1.1
12
5.7
1
3.1
8.2
5.3
13
5.5
0.8
2.9
8
5.1
1
2
3
4
5
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4.7
98.7
2.5
0.6
0.6
0.6
1.1
0.6
2.3
0.4
0.4
0.4
1.3
0.8
0.2
6
7
8
9
10
11
12
1.3
13
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2
NCBI Acc. No.
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Divergence
1
2
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1
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