Assessment of reference genes for qRT-PCR normalization to elucidate host response to African swine fever infection

Abstract

Viral infection disrupts the normal regulation of the host gene’s expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

Introduction

The infectious haemorrhagic fever (IHF) in pigs is caused by the African swine fever (ASF) virus, which is a member of the Asfivirus genus and Asfarviridae family. ASFV is a large dsDNA virus having approximately 170–194 kbp of the genome and it encodes a variable number of viral proteins, encompassing between 150 and 200, among which 68 are structural proteins and more than 100 non-structural proteins [1–4]. ASF is a transboundary disease and causes huge economic losses (up to 100% mortality) to the pig farmers globally. There is no effective vaccine against ASF which might be due to the complex structure of the virus or the pathways involved by the virus in the host. To know the infection mechanism of ASFV in pigs, it is important to have a good understanding of the host’s immune system, biological processes and molecular functions involved during infection (pig-ASFV interaction mechanism). This could be evaluated based on the differentially expressed host genes during infection by high throughput sequencing and quantitative real-time PCR. A stable and suitable housekeeping reference gene is very much essential in respect of appropriate qPCR analysis of gene expression for the study of ASFV infection in porcine tissues.

Gene expression analyses have become extremely important for uncovering gene function and the molecular mechanisms that regulate the different responses observed during the infection by various pathogens. The dysregulation (up- or down-regulation) of the reference genes should be consistent across tissues under a variety of experimental circumstances. If these requirements are not fulfilled, then normalization to varying internal references can lead to increased “noise” or erroneous results [5]. Generally, housekeeping genes are considered to be relatively stable and expressed in all cell types and physiological states [6]. However, some studies have indicated that the expression quantity of housekeeping genes in different cell types, tissues, experimental conditions and under different physiological states is inconsistent [7, 8]. Housekeeping genes, such as: Hypoxanthine-guanine phosphoribosyl transferase (HPRT1), Beta-2 microglobulin (B2M), 18 S ribosomal Ribonucleic acid (18 S rRNA), Phosphoglycerate kinase 1 (PGK1) and H3 histone family member 3 A (H3F3A) have been used as reference genes in different cells and experiments [9, 10]. The present study aimed to evaluate the stability of these five housekeeping genes in healthy and ASFV-infected porcine tissues for relative expression analyses and to select the most stable reference gene for further research on host protein-ASFV interactions during infection cycle.

Materials and methods

The whole experimental design of this study is shown in Fig. 1.

Fig. 1.

Outline of this study represents the samples sources, methodology and analysis for selection of suitable and stable housekeeping reference genes of different porcine tissues in African swine fever virus infected animals and healthy animal, as a control

Sample collection and molecular confirmation

The animals (Yorkshire breed) aged above 5 months were affected, and post-mortem tissue samples (spleen, liver, lymph node, kidney, and heart) were collected from five animals that died during the outbreak. Samples were collected aseptically immediately after death in RNAlater™ Stabilization Solution (Thermo Fisher Scientific) and brought to the laboratory (Animal Health Laboratory, ICAR-National Research Centre on Pig, Rani, Guwahati, Assam, India) and stored at -80 °C until RNA extraction for further investigation. The same type of tissue samples collected from five healthy slaughtered pigs were used as controls in the study (Fig. 1). The fresh 50 mg of each type of tissue sample was taken for isolation of DNA by DNeasy Blood & Tissue Kit (QIAGEN, Germany) as per the manufacturers protocol. The presence of ASFV in tissue samples was confirmed by using PCR as described in previous study [11].

RNA extraction and cDNA synthesis

Tissue samples from five infected subjects and five control subjects were individually collected and pooled by tissue type (spleen, liver, lymph node, kidney, and heart), forming composite samples for each group. Total RNA from each pulled tissue samples were extracted using RNeasy Mini kit (QIAGEN, Germany) as per manufacture’s protocol. The RNA pellet was suspended in 20 µl nuclease free water (NFW) followed by 5 µg of total RNA was treated with DNase I, RNase-free (ThermoScientific) as per the manufacturer’s protocol for complete removal of genomic DNA. To remove the residual DNase I reagents, the RNA sample was further precipitated by phenol: chloroform: Isoamyl alcohol at a concentration of 25: 24:1. Further the RNA pellet was suspended in 10 µl nuclease free water (NFW) and quantity and quality of the purified RNA was analysed in Nanodrop spectrophotometer. The reverse transcription reaction was performed using purified total RNA (1 µg) with a iScript cDNA synthesis kit, (BIO-RAD, USA) according to the manufacturer’s protocol. cDNA samples were stored at -20^oC till further used. In this study, we used oligo(dT) primers for reverse transcription, which specifically bind to the poly-A tail of mRNA, to ensure cDNA synthesis from mature mRNA transcripts. This approach was chosen to selectively amplify mRNA and to avoid the synthesis of cDNA from other types of RNA, providing more accurate and relevant gene expression data.

Candidate reference genes

Total five widely used reference genes such as HPRT1, B2M, 18 S rRNA, PGK1, and H3F3A were selected for this study [9, 12, 13]. Details of selected genes, primers and respective amplicon sizes are shown in Table 1. The nucleotide sequences of reference primers were validated through NCBI BAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and primers were synthesised from Integrated DNA Technologies, Inc. (https://www.idtdna.com/). Thereafter, each primer set for the candidate genes was assessed to identify the reference gene exhibiting the highest level of stability in both healthy and ASFV-infected porcine tissues.

Table 1.

The list of primers used in this study

Gene	Primer Name	Sequences (5’◊3’)	Bases	Product size (bp)	References
HPRT1	HPRT1-F	GGA CTT GAA TCA TGT TTG TG	20	91	[12, 13]
	HPRT1-R	CAG ATG TTT CCA AAC TCA AC	20
B2M	B2M-F	CAA GAT AGT TAA GTG GGA TCG	21	161	[12, 13]
	B2M-R	TGG TAA CAT CAA TAC GAT TTC	21
18 S rRNA	18 S-F	CCC ACG GAA TCG AGA AAG AG	20	125	[12, 13]
	18 S-R	TTG ACG GAA GGG CAC CA	17
PGK1	PGK1-F	GGG CTA AGC AGA TTG TAT G	19	180	[12, 13]
	PGK1-R	TGA CTT TAT CCT CCG TGT T	19
H3F3A	H3F3A-F	CTT TGC AGG AGG CAA GTG AG	20	72	[9]
	H3F3A-R	TGG CAT GGA TAG CAC ACA GG	20

Here, HPRT1 Hypoxanthine-guanine phosphoribosyl transferase, B2M Beta-2 microglobulin, 18S rRNA 18 S ribosomal Ribonucleic acid, PGK1 Phosphoglycerate kinase 1, H3F3A H3 histone family member 3 A

qRT-PCR study

The qRT-PCR study was performed to evaluate the expression pattern of selected candidate reference genes in healthy and ASFV-infected porcine samples. In qRT-PCR reactions, we utilized gene-specific primers as indicated in Table 1. Each 10 µl reaction, consisted of 5 µl of 2X Maxima SYBR/ROX Green qPCR MasterMix (Thermo Scientific, Lithuania), 0.5 µl each of forward and reverse primers at a concentration of 2.0 pm/µl, 3 µl of nuclease-free water, and 10ng (1 µl) of template cDNA. The experimental setup involved using a 96-well reaction plate, with each well receiving 0.01 ml of the respective treatment PCR reaction mixture. Real-time PCR was conducted for all experimental groups using the AriaMX Real-Time PCR System by Agilent Technologies, Inc. The qRT-PCR program was executed under the following conditions: an initial pre-denaturation step at 95 °C for 5 min, succeeded by 40 cycles of denaturation at 95 °C for 10 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s. Additionally, a melt analysis was conducted with temperatures at 95 °C for 30 s, 65 °C for 30 s, and 95 °C for 30 s. Each sample was analyzed in triplicate.

Data analysis

One tissue sample each was collected and the experiments were run in triplicates and Mean ± SE of CT values were calculated using standard method [14]. Stability analysis of reference genes expression was performed using the Comparative delta CT [15], geNorm [16], BestKeeper [17] and NormFinder [15] algorithm available at RefFinder (www.heartcure.com.au/reffinder/) for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder [18].

Results

Screening of porcine tissue samples for ASFV by PCR

All porcine tissues were screened against ASFV by PCR and all the infected tissue samples revealed the presence of ASFV (amplicon size of 478 bp of B407L gene) in 1% agarose gel electrophoresis whereas tissue samples from healthy pigs were negative for ASFV (Fig. 2).

Fig. 2.

Confirmation of ASFV infection in porcine tissues. Agarose gel electrophoresis shows a specific PCR amplified product (478 bp size) in each ASFV infected porcine tissue and positive control sample. In healthy tissue samples, no ASFV amplicon was found. Here, L: 100 bp Plus DNA marker, PC: Positive control, Sample 1: infected liver tissue, Sample 2: healthy liver tissue, Sample 3: infected kidney tissue, Sample 4: healthy kidney tissue, Sample 5: infected heart tissue, Sample 6: healthy heart tissue, Sample 7: infected spleen tissue, Sample 8: healthy spleen tissue, Sample 9: infected lymph node tissue, Sample 10: healthy lymph node tissue and NC: negative control

Assessment of CT values for housekeeping genes

The boxplot presented in Fig. 3 displays the distribution of studied CT values for each endogenous gene and the mean CT values of each genes calculated in all groups (ASF–infected and healthy group) are given in Table 2. The mean CT value of the reference genes ranged from 10.32 ± 0.05 (18 S rRNA in Kidney) to 37.88 ± 0.33 (HPRT1 in Heart) in healthy group and 11.39 ± 0.25 (18 S rRNA in Spleen) to 37.78 ± 1.12 (HPRT1 in Lymph node) in ASFV-infected group.

Fig. 3.

Distribution of CT values of five housekeeping genes in healthy and ASFV-infected pig tissue samples. The boxes represent the upper and the lower quartiles of cycle thresholds range with medians (www.boxplot.tyerslab.com)

Table 2.

Mean CT values of candidate reference genes measured by qRT-PCR

Group	Mean CT value ± SEM of candidate reference genes
	HPRT1		B2M		18 S rRNA		PGK1		H3F3A
	Healthy	Infected	Healthy	Infected	Healthy	Infected	Healthy	Infected	Healthy	Infected
Liver	33.82 ± 0.23	33.49 ± 0.35	27.66 ± 0.33	27.67 ± 0.33	12.30 ± 0.10	12.20 ± 0.27	28.53 ± 0.85	27.95 ± 1.20	18.02 ± 0.14	20.43 ± 0.13
Kidney	37.23 ± 0.40	34.44 ± 0.35	36.00 ± 1.35	29.00 ± 0.22	10.32 ± 0.05	12.19 ± 0.22	35.55 ± 0.59	29.29 ± 0.28	18.94 ± 0.04	20.69 ± 0.10
Heart	37.88 ± 0.33	32.60 ± 0.15	37.27 ± 0.94	22.57 ± 0.45	10.54 ± 0.14	12.97 ± 0.57	27.99 ± 0.23	28.14 ± 0.40	18.03 ± 0.13	21.21 ± 0.11
Spleen	36.33 ± 0.18	36.36 ± 0.19	26.15 ± 0.46	25.76 ± 0.46	13.62 ± 0.09	11.39 ± 0.25	25.71 ± 0.22	26.51 ± 0.54	21.61 ± 0.79	18.20 ± 0.31
Lymph Node	29.72 ± 0.75	37.78 ± 1.12	22.60 ± 0.16	28.27 ± 0.25	10.97 ± 0.12	11.90 ± 0.13	21.79 ± 0.35	31.02 ± 0.36	17.20 ± 0.10	23.30 ± 0.09

Evaluation of stability of candidate reference genes expression

This study was conducted to identify the most stable housekeeping gene in porcine tissues under natural infection of ASFV. The CT values of each group were sorted in the RefFinder format and ranks of genes were generated. Based on the rankings of genes from each programme (geNorm, NormFinder, BestKeeper, and the comparative Delta-CT technique), the RefFinder algorithm generated the geometric mean of their weights for the overall final ranking (Figs. 4 and 5). The present study revealed the presence of stable reference genes in different tissue samples under investigation.

Fig. 4.

Graphical representation of comparative gene stability in RefFinder of each experimental group (healthy tissue: lymph node, heart, kidney, spleen, liver and all healthy tissue; ASFV infected tissue: lymph node, heart, kidney, spleen, liver and all ASFV infected tissue). Comparative gene stability represents the most stable to least stable reference genes that analysed by normFinder, ΔCT method, Genorm, BestKeeper in each experimental group

Fig. 5.

Graphical representation of Comparative gene stability in RefFinder of experimental group combination of CT values of all healthy and ASFV infected tissues. Comparative gene stability represents the most stable to least stable reference genes that analysed by normFinder, ΔCT method, Genorm, BestKeeper in each experimental group

The stability of reference genes by comparative delta CT analysis revealed that 18 S rRNA was most stable for healthy liver and kidney; H3F3A was most stable for healthy heart and lymph node, ASFV-infected kidney, heart, and lymph node; and HPRT1 was found stable for healthy spleen, ASFV-infected liver and spleen and combination of all healthy and ASFV-infected tissues of pig (Table 3).

Table 3.

List of most stable endogenous control genes in different analysis groups as suggested by RefFinder algorithms (overview ranking results of reference genes)

Group/ Category		Ranking of best stable reference genes based on
		Comparative delta CT	BestKeeper	NormFinder	geNorm	Recommended comprehensive ranking by RefFinder
A	Healthy liver	18 S rRNA	18 S rRNA	18 S rRNA	18 S rRNA| H3F3A	18 S rRNA
	Healthy kidney	18 S rRNA	H3F3A	18 S rRNA	18 S rRNA| H3F3A	18 S rRNA
	Healthy heart	H3F3A	18 S rRNA	18 S rRNA	PGK1| H3F3A	H3F3A
	Healthy spleen	HPRT1	18 S rRNA	HPRT1	HPRT1| PGK1	HPRT1
	Healthy lymph node	H3F3A	H3F3A	18 S rRNA	18 S rRNA| H3F3A	H3F3A
B	Infected liver	HPRT1	H3F3A	HPRT1	B2M| H3F3A	H3F3A
	Infected kidney	H3F3A	H3F3A	H3F3A	18 S rRNA| PGK1	H3F3A
	Infected heart	H3F3A	H3F3A	HPRT1	B2M| PGK1	H3F3A
	Infected spleen	HPRT1	HPRT1	HPRT1	HPRT1| H3F3A	HPRT1
	Infected lymph node	H3F3A	H3F3A	H3F3A	18 S rRNA| H3F3A	H3F3A
C	All Healthy and Infected Liver + Kidney + Heart + Spleen + Lymph Node	HPRT1	18 S rRNA	HPRT1	18 S rRNA| H3F3A	HPRT1

Here, Group A: Analysed CT values of healthy individual samples; Group B: Analysed CT values of ASFV-infected individual samples; Group C: Analysed CT values of combined healthy and ASFV-infected samples

The BestKeeper algorithm identified 18 S rRNA as the most stable gene for healthy liver, heart, spleen, and combination of all CT values of healthy and ASFV-infected tissues of pigs. H3F3A was found to be most stable for healthy kidney and lymph node, ASFV-infected liver, kidney, heart and lymph node and HPRT1 was found to be most stable for only ASFV infected spleen (Table 3).

According to the NormFinder analysis, 18 S rRNA was identified as the most stable reference gene for each of the healthy tissues (healthy liver, kidney, heart and lymph node) except for healthy spleen; H3F3A was found to be most stable for ASFV-infected kidney and lymph node. HPRT1 was found stable for healthy spleen, ASFV-infected liver, spleen and combination of all healthy and ASFV-infected tissue of pig group (Table 3).

The geNorm program was used to calculate the stability values (M value) for the five candidate genes and it revealed that 18 S rRNA and H3F3A were most stable for healthy liver, healthy kidney, healthy lymph node, ASFV infected lymph node and combination of all healthy and ASFV infected tissues of pig; PGK1 and H3F3A were found to be stable for healthy heart; HPRT1 and PGK1 were found to be stable for healthy spleen; B2M and H3F3A were found to be stable for infected liver; 18 S rRNA and PGK1 were found to be stable for infected kidney; HPRT1 and H3F3A were found to be stable for infected spleen and B2M and PGK1 were found to be stable for ASFV-infected heart (Table 3).

Based on RefFinder’s recommended comprehensive ranking, 18 S rRNA gene was determined to be the most stable for both healthy liver and kidney tissues; H3F3A gene expression was found to be the most stable in healthy heart and lymph node and also for liver, kidney, heart and lymph node of ASFV-infected group; HPRT1 was found to be the most stable gene both for healthy and ASFV-infected spleen as well as for combination of CT values of all healthy and ASFV-infected tissues (Table 3).

Discussion

The housekeeping endogenous genes of a cell continue to be expressed in order to maintain the body’s physiological functions. During viral infection, host gene expression profiles, including those of housekeeping genes are modulated, which changes the cellular pathways [10, 19, 20]. However, the presence of a universal housekeeping gene with a stable expression pattern in various tissues and organs with different factors is not very common [21–23]. Therefore, it is very much essential to select the most stable housekeeping genes only after validation, particularly during infections. In the present work, housekeeping genes with distinct roles were chosen to prevent genes belonging to the same biological pathways. Different porcine tissues such as skeletal muscle [24, 25]; intramuscular stromal-vascular cells [26]; intestine of piglets [13]; dorsal root ganglia and spinal cord [27]; lung, liver, heart, stomach, intestine, spleen and kidney of minipig [28]; inguinal ring/canal [9]; ovary and uterus [29]; articular cartilage [30]; adult mesenchymal stem cells [31]; liver, lung, kidney, spleen, stomach, small intestine and large intestine of Berkshire, Landrace, Duroc and Yorkshire pigs [32] have been evaluated for reference stable genes. Post-mortem sample collection inherently poses a risk of RNA degradation, which can affect the accuracy of gene expression analysis. In this study, to minimize degradation, samples were collected promptly after death, immediately preserved in RNAlater, and stored at -80 °C. These precautions were essential for preserving RNA integrity and ensuring that the gene expression data accurately represented the biological state at the time of death.

ASFV infection significantly impacts the expression of various gene groups, particularly those involved in immune response, apoptosis, and cellular stress pathways [33, 34]. During acute ASFV infections, elevated levels of pro-inflammatory IL-1α, IL-1β, IL-6, TNF, CCL2, CCL5, and CXCL10 were detected in the palatine tonsil, lymph nodes, spleen, and kidney using qPCR assay [34]. These changes underscore the necessity for careful selection of housekeeping genes for normalization in gene expression studies.

Our analysis revealed that ASFV infection can alter the expression stability of commonly used reference genes. In the current investigation, a series of housekeeping genes, including HPRT1, B2M, 18 S rRNA, PGK1, and H3F3A, were assessed in tissues from healthy and ASFV-infected pigs using four distinct computational tools, including Normfinder, BestKeeper, geNorm, and ΔCT in RefFinder. In the present study the ranking by RefFinder provided a robust and accurate analysis of the stability exhibited by the candidate housekeeping genes. These housekeeping genes were selected due to their convenient, most abundant and invariant expression across tissues, cells and experimental treatments. In this study, 18 S rRNA was identified as stable for healthy liver and kidney in comprehensive ranking by RefFinder. Ribosomal RNAs are essential for protein synthesis. rRNA functions as the catalytic core for protein synthesis within the 40 S ribosomal subunit. The assumption is that elevations in the quantity of ribosomes, resulting in higher RNA transcription and protein synthesis, are directly related to the abundance of 18 S rRNA [35]. However, the high abundance of 18 S rRNA can complicate the accurate quantification of low-expression genes and its stability may vary under different experimental conditions and stages of ASFV infection, potentially affecting its reliability as a normalization control. H3F3A was identified as stable in most of the experimental group such as healthy spleen and lymph node and ASFV-infected liver, kidney, heart and lymph node etc. H3F3A is crucial for preserving genome integrity throughout mammalian development as it contributes to the formation of the histone hetero-octamer complex [36]. H3F3A aids in the maintenance of chromosomal heterochromatic structures throughout the entire cell cycle [37]. In the present study, the reference gene HPRT1 was found as stable in spleen tissue of both healthy and ASFV-infected pig. HPRT has been widely employed as an internal control for evaluating alterations in gene expression in qRT-PCR studies [10, 38, 39]. HPRT, in the salvage pathway, efficiently recycles approximately 90% of free purines in humans, synthesizing guanine and inosine [40, 41]. This enzyme transfers phosphoribose from phosphoribosyl pyrophosphate (PRPP) to hypoxanthine or guanine bases to form inosine monophosphate (IMP) and guanosine monophosphate (GMP), respectively [41, 42]. Because GTP is consistently required for DNA synthesis and serves as an energy source in the cell, HPRT functions as a reliable housekeeping gene present in all somatic tissues but in minimal quantities [43, 44].

Overall, the current study revealed that each selected reference gene was present with varying degree of expression in different healthy and ASFV-infected tissues. It was also observed that these housekeeping genes (H3F3A, 18 S rRNA and HPRT1) are tissue-specific. The reference gene H3F3A was found as most stable gene equally in healthy and ASFV infected tissues of heart and lymph node. Whereas, HPRT1 was identified as most stable gene equally in Healthy and ASFV infected spleen. It was also observed that in tissues such as liver and kidney of healthy and infected pig, the reference genes were found to be different. However, when the CT values of all the tissues from ASF infected pigs as well as from healthy pigs were analysed by the RefFinder algorithm for overall ranking, the HPRT1 was found as the most stable gene. As HPRT1 was found as the most stable housekeeping gene in combination of all tissues from both ASFV-infected and healthy pigs, this gene can be used as a reference endogenous control gene in normalization of qRT-PCR during ASFV infection.

In conclusion, this study revealed that ASFV infection might have caused significant changes in expression pattern of frequently utilised HKGs in various organs of pig. Results of comparative delta CT, BestKeeper, NormFinder, geNorm and recommended comprehensive ranking by RefFinder analyses showed that 18 S rRNA, H3F3A and HPRT1 genes were optimal endogenous control reference genes in healthy and ASFV-infected different porcine tissue samples. Using the approach, recommended comprehensive ranking by RefFinder (overall ranking from Comparative delta CT, BestKeeper, NormFinder, geNorm), HPRT1 was identified as the most stable housekeeping gene for normalization of expression studies in all combinations of CT values of both healthy and ASFV-infected porcine tissues (liver, kidney, heart, lymph node). To the best of our knowledge, this study for the first time addresses the selection and validation of stable housekeeping genes through qPCR method in porcine tissues collected from naturally occurring ASFV outbreaks. Thus, the findings of this study can be applied to have better understanding on ASFV infection in pigs.

Author contributions

SR and JS contributed to the conception and design of the study. JS and GSS performed the experiments and analysis of the data. JS, SR, RD, and SRP contributed to the interpretation of results and manuscript writing. PJD, VKG, JD, and SP supervised the research work. All authors read and approved the final manuscript.

Funding

This work was supported by funding from the Department of Biotechnology, Govt. of India in the form of project entitled “Establishment of a consortium for One Health to address Zoonotic and Transboundary diseases in India, including NE region”.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.