2.1 | Animals Twelve healthy Black Bengal goats aged between 4 and 8 months with a history of no vaccination against PPR were purchased from local markets. Animals were kept in relative isolation for 2 weeks with adequate feed and water. The goats were dewormed using subcutaneous ‘Ivermectin’ injection as per manufacturer's recommendation. All goats were tested for anti-PPRV antibodies with a commercial competitive ELISA kit (ID Screen PPR Competition, IDVet, Montpellier, France) and found seronegative for PPRV.
2.2 | Ethics statement All applicable national and institutional guidelines for the care and use of animals were followed. The study was carried out in accordance with the recommendation of the Ethical Standard of Research Committee of Bangladesh Agricultural University, Mymensingh. The protocol and procedures employed were reviewed and approved by the Ethical Standard of the Research Committee (Ref. No. BAURES/ ESRC/699/2020; Dated: 28.06.2020). The authors also confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The US National Research Council's guidelines for the Care and Use of Laboratory Animals were followed.
2.3 | Preparation of PPR virus inoculum For experimental infection, PPR virus inoculum was prepared from a local isolate (BD/PPRV/2015/1; lineage IV) collected during a field outbreak in 2015. The virus was isolated in primary goat kidney cell culture. Briefly, a 20% tissue homogenate of lymph node was prepared from a PPR-infected goat using sterile PBS. A monolayer of primary goat kidney cells was then infected with 100 µl of the tissue homogenate and monitored daily for the development of any cytopathic effect (CPE). The CPE was first observed at 48 hr postinfection. The cell culture supernatant was harvested after three cycles of freeze-thawing and cleared by centrifugation at 3,000 rpm for 15 min. The presence of PPR virus in the cell culture supernatant was confirmed by real-time reverse transcription polymerase chain reaction (rtRT-PCR) as described below. The virus was passaged one more time in primary goat kidney cell culture and stored in aliquots at −80°C. Before use for experimental infection of goats, the concentration of virus in the inoculum was measured by end-point titration in primary goat kidney cell culture.
2.3 | Preparation of PPR virus inoculum For experimental infection, PPR virus inoculum was prepared from a local isolate (BD/PPRV/2015/1; lineage IV) collected during a field outbreak in 2015. The virus was isolated in primary goat kidney cell culture. Briefly, a 20% tissue homogenate of lymph node was prepared from a PPR-infected goat using sterile PBS. A monolayer of primary goat kidney cells was then infected with 100 µl of the tissue homogenate and monitored daily for the development of any cytopathic effect (CPE). The CPE was first observed at 48 hr postinfection. The cell culture supernatant was harvested after three cycles of freeze-thawing and cleared by centrifugation at 3,000 rpm for 15 min. The presence of PPR virus in the cell culture supernatant was confirmed by real-time reverse transcription-polymerase chain reaction (rtRT-PCR) as described below. The virus was passaged one more time in primary goat kidney cell culture and stored in aliquots at −80°C. Before use for experimental infection of goats, the concentration of virus in the inoculum was measured by end-point titration in primary goat kidney cell culture.
2.5 | Competitive enzyme-linked immunosorbent assay (ELISA) A competitive ELISA kit (ID Screen PPR Competition, ID-Vet, Montpellier, France) was used to measure the amount of PPRVspecific antibodies in sera of PPRV-infected and healthy goats. As mentioned in the kit, a sample having a competition percentage (CP) value of ≤35 was considered as seropositive.
2.6 | Quantification of PPR virus load in different tissues One PPRV-infected goat was euthanized at each time point of 5, 7, 14 and 18 dpi. Routine necropsy was performed and gross lesions were recorded. Tissues from the pre-scapular lymph node, liver, kidney and spleen were collected for quantification of PPRV RNA in tissues. To this end, a 20% tissue homogenate was prepared in sterile PBS. Total RNA was extracted with PureLink RNA Mini Kit (ThermoFisher Scientific, USA) and quantified by NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA). For normalization and internal control, 20 ng of total RNA was used per reaction in all rtRT-PCR. The rtRT-PCR was performed in Applied Biosystems 7500 Fast Real-Time PCR system using RevTrans QPCR One-Step EvaGreen (ROX) Kit (Bio-sell, Germany). The following primer pairs were used: NrF1 5′-TGA CCA GGG AAG AAG TCA CA-3′ and NrR1 5′-TCG TCT TCA GGC ATG ATC TC-3′ to amplify 120 bp product of nucleoprotein (N) gene of PPRV (Saravanan et al., 2004).
2.7 | Haematobiochemical analysis Routine haematological examination of whole blood samples collected at different time points post-infection was performed by the standard method (Lamberg & Rothstein, 1978). Routine haematological parameters such as haemoglobin, erythrocyte sedimentation rate, packed cell volume, total erythrocyte count, total leucocyte count and differential leukocyte count were tested. In addition, different serum biochemical constituents such as total protein, albumin, glucose, bilirubin, blood urea nitrogen, creatine kinase, alkaline phosphatase, alanine transaminase and aspartate transaminase, inorganic phosphorus and calcium were analysed using an automated T80 Ultraviolet-visible spectroscopy (UV/VIS) spectrophotometer (PG Instruments, UK). Furthermore, an automated electrolyte analyser GENLYTE 3000A (IVD) was used to analyse serum electrolytes (sodium, potassium and chloride ions) using a commercial kit (Electrolyte solution, Biogen, GmbH, Germany).
2.8 | Statistical analysis: Statistical analysis was performed using the software package GraphPad Prism Version 5.0. A one-tailed non-parametric Mann– Whitney U test was used to calculate the statistical differences in haematological and biochemical profiles between PPRV-infected and healthy goats.