Isolation of peste des petits ruminants virus using primary goat kidney cell culture from kidneys obtained at slaughter

Abstract Background Traditionally isolation of peste des petits ruminant virus (PPRV) is performed in Vero cells that takes several blind passages before observing typical cytopathic effects (CPEs). As an alternate, researchers have been using lamb kidney (LK) cells but day‐old lambs are difficult to obtain and requires animal sacrifice. Objective We established a primary goat kidney (GK) cell culture from the kidneys obtained at slaughter. Methods The kidney of Black Bengal goats were collected from slaughter house and processed to make single cell suspension. The cells were resuspended in appropriate culture medium and maintained under optimum culture condition. Results The 80% confluent monolayer of GK cells was obtained after 15–20 days post seeding. Upon infection with a field isolate of PPRV, the well‐developed CPEs characterized by cell rounding, vacuolation in the cytoplasm and fusion of cells were observed after 48 hr post infection. Virus quantification in the culture supernatant revealed more viral RNA in GK cells than LK cells. The multicycle growth analysis of PPRV showed a steady increase in the virus loads in the culture supernatant of infected GK cells, suggesting an adaptation of the PPRV in GK cells. Conclusions The findings suggest that primary GK cells can be successfully prepared from the mature kidney cortical tissues and can be used for the isolation of PPRV. This system could reduce the unnecessary sacrifice of lambs or kids. Since kidneys of slaughtered goats are available throughout the year, using this protocol primary cell culture from mature goat kidney can provide primary cells to the laboratory throughout the year.

. The Vero cell line has been commonly used for the isolation and cultivation of morbilliviruses including PPRV and for vaccine production (Nanda et al., 1996). However, the isolation of PPRV using Vero cells usually requires several blind passages before observing the PPRV-induced cytopathic effects (CPE), and the CPE is sometime difficult to detect (Kumar et al., 2016;Sreenivasa et al., 2006). Moreover passaging of PPRV in Vero cells leads to the attenuation of the virus in susceptible hosts upon experimental infection (Pope et al., 2013;Truong et al., 2014).
A remarkable development in cell culture system of PPRV happened with the discovery of two cell surface receptors of morbilliviruses. The signalling lymphocyte activation molecule (SLAM) also known as CD150, a protein receptor expressed primarily on the lymphoid cell surface, is used preferentially by wild-type morbilliviruses to bind to the host (Tatsuo et al., 2001). Another receptor, the Nectin-4 has also been showed as putative receptor of morbilliviruses in epithelial cell surface (Noyce et al., 2011). The overexpression of recombinant SLAM or Nectin-4 in cell lines showed efficient replication of morbilliviruses in cell lines that showed limited replication previously. For example, the Vero/SLAM showed efficient replication of wild strains of CDV (Seki et al., 2003). Similarly, the monkey kidney cells CV1 overexpressing goat SLAM showed efficient replication of PPRV from pathological specimens (Adombi et al., 2011).
Beside kidney cells, lymphoid cell line (B95a) from marmoset, which carry the SLAM receptor on cell surface, were also shown to be very efficient in propagating PPRV (Sreenivasa et al., 2006). To our experiences, the limitation of using these recombinant cell lines in developing countries is transportation and maintenance of the cells using cool chain.
PPRV isolate losses its pathogenicity due to propagation in the unusual host cells (Adu et al., 1990). But pathogenesis study through experimental infection requires viruses with unaltered pathogenicity. Isolation of PPRV using primary lamb and goat kidney cell cultures could eliminate some of the limitations of cell line. The characteristics CPE in primary goat and lamb kidney cell cultures can be found during the one to two passages of virus infection and the pathogenicity of the propagated virus was found unaltered (Govindarajan et al., 2008;Taylor & Abegunde, 1979). However, kidneys of 1 to 2 weeks old lambs for the preparation of primary cell culture are often hard to obtain and require animal sacrifice. Therefore, we established primary goat kidney cell culture from kidneys of adult goats obtained at abattoir and successfully isolated PPRV using primary goat kidney cell culture.

| Preparation of primary goat kidney cell culture
Primary goat kidney cell culture was prepared using mature goat kidneys collected from slaughtered goats according to the procedure described by FAO (Rweyemamu, 1994) with minor modifications.
Briefly, fresh kidneys from adult goats were collected at slaughter, After 7 days, the growth medium was changed with similar growth medium with required additives. Simultaneously, the lamb kidney cells were also prepared following the above mentioned protocol.

| Subculture of primary kidney cells and infection with local a PPRV isolate
The monolayer of first passage of primary goat kidney cell culture at about 80% confluency was trypsinized using 0.25% Trypsin (Gibco, Cat: 15090046)-Ethylenediaminetetraacetic acid (EDTA) solution. Cells were suspended at 1:3 ratios with the growth medium containing 12% FCS and other additives and dispersed into new culture flasks. At 80% confluency, cells were trypsinized as above, resuspended and used for next passages. Five of such subcultures were performed consecutively. For the isolation of PPRV, the 80% confluent monolayer of goat kidney cells was used. Initially, a 20% lymph node homogenate (RT-PCR confirmed for PPRV) obtained from natural PPR infected goats was used as inoculum. The culture supernatant was removed and the cells were washed with warmed PBS twice. Then 100 µl inoculum was added to the culture flask and incubated for 1 hr at 37°C. PBS was used as mock infection in the control flask. Then culture medium containing 5% FCS was added to the flask and incubated again. The cells were monitored twice daily under microscope for the development of CPE. The effects of carryover toxins, if any, present in inoculum was monitored up to 48 hr by observing cell morphology. The infected cells were frozen and thawed 5 times when maximum CPE were observed at 96 hpi.
Then the culture fluid was centrifuged at 1,200 g for 15 min. The supernatant was collected, divided into small aliquots and stored frozen (−80°C) until used. For consecutive passages, the cell culture monolayers was prepared and inoculated with the virus suspension using above mentioned frozen inoculum. When maximum CPE manifested, the virus suspension was harvested and stored as above. The serial passaging of the virus in primary goat kidney cell culture was continued until the 3rd passage and the quantification of virus titre in the culture supernatant was performed using 96-well flat bottom tissue culture plates as per description of Reed and Muench (Reed & Muench, 1938). Similar infection protocol was also performed for primary lamb kidney cell culture. For multicycle growth analysis, confluent monolayers of primary goat kidney cells were infected with PPRV as above and the culture supernatants were harvested at 24, 36, 48, 60, 72 and 96 hpi and the amount of PPRV RNA in the culture supernatants was quantified by real time quantitative RT-PCR (RT-qPCR).

| Culture of goat kidney cells on coverslip
A sterile plastic petri dish containing glass coverslip was used to propagate the primary goat kidney cells. The confluent monolayer of goat kidney cells was harvested using 0.25% trypsin and resuspended in M-199 medium containing 12% FCS. About 4 ml (one million cells/ml) of cell suspension was dispensed into petri dishes and incubated at 37°C in a CO 2 incubator. Upon confluency, 100 µl of the PPRV was inoculated into the goat kidney cells and observed for the development of the PPRV-induced CPE. When approximately 80% of the cells developed CPE, the cells on coverslip were fixed with ice-cold methanol and stained with haematoxylin and eosin (H&E) stain as per standard method.

| Detection and quantification of PPRV
Viral RNA was extracted from both cell lysates and culture supernatants using PureLink™ Viral RNA/DNA Mini Kit (Invitrogen). The presence of PPRV RNA in the cell lysates was detected by an established conventional RT-PCR method (Forsyth & Barrett, 1995). A 448 bp fragment of F gene of PPRV was amplified using Superscript III OneStep RT-PCR kit (Invitrogen) as per the manufacturer's instructions. The quantity of the PPRV RNA in the cell lysates and supernatants was determined by RT-qPCR method using Rev Trans QPCR One-Step EvaGreen® (ROX) (Bio&SELL, Germany). For RT-qPCR, the forward primer NP3 (5ʹ-TCTCGGAAATCGCCTCAC AGACTG-3ʹ) and reverse primer NP4 (5ʹ-CCTCCTCCTGGTCCTCC AGAATCT-3ʹ) was used as described by Couacy-Hymann and colleagues (Couacy-Hymann et al., 2002). For internal control of RNA input, equal amount (20 ng/reaction) of RNA was used for all samples. The result was analysed by ΔCt (delta cycle thresold) method using the following formula: ΔCt = (Ct target-Ct uninfected control).
The change in the viral gene expression was calculated as 2 -ΔCt and the value indicated in an n-fold difference relative to the uninfected control.

| Establishment of primary goat kidney cell culture
The goat kidney cells settled down and started to grow after 4-5 days of seeding and became morphologically distinct after 7 days (Figure 1a). The 80% confluent monolayer of primary goat kidney cells was obtained after 15 days post seeding ( Figure 1b) and full confluency was found at day 20 post seeding ( Figure 1c).
The primary goat kidney cells were maintained up to 30 days post seeding although the cells were overgrown and appeared to die (Figure 1d). However, the lamb kidney cells took about 15 days for full confluency (data not shown). During the 2nd and 3rd subculture, goat kidney cells grew relatively faster than the 1st culture and became confluent by day 10 post seeding.
However, the growth rate of primary goat kidney cells decreased from the 4th subculture, although the cell morphology remained unchanged.

| Infection morphology of PPRV in primary goat kidney cells
During the first passage, the initial CPE characterized by cell round-    Such differences could be due to the species and age differences as the kidney cells from lamb were collected from kid at sacrifice (Govindarajan et al., 2008). However, the subsequent passages of goat kidney cells took shorter duration (10 days) to become confluent which could be explained by the juvenile nature of the cells once they divide. The early cell rounding in goat kidney cells was visible after 36 hpi whereas it took about 48 hpi for lamb kidney cells (data now shown). The kinetics of PPRV-induced CPEs depend on multiple factors such as host species, cells types, genetic differences of virus strains, etc. Several studies reported increase susceptibility of goats to PPRV infection than sheep (Fakri et al., 2017;Wernike et al., 2014). PPRV shows CPE as early as 2 days post infection in marmoset B lymphoblastoid cells (Sreenivasa et al., 2006). The influence of virus strains on cell culture permissiveness was also shown in another study where a locally circulating PPRV-AR/87 strain showed very rapid onset of CPEs (18 hpi) as compared to the vaccine strain PPRV-Sungri/96 (72 hpi) (Singh et al., 2010). The early CPEs found in PPRV-infected primary goat kidney cells in this study could be explained by the origin of cells and characteristics of virus strain used. Natural PPR affected Black Bengal goats showed severe pathological changes in kidneys and elevated serum creatine kinase (Begum et al., 2018), and large amount of PPRV was detected in kidneys of Black Bengal goats experimentally infected with a local PPRV strain (Begum et al., 2020), suggesting an increase permissiveness of goat kidney cells to PPRV infection. Moreover the PPRV strain used in this study was collected from a local field outbreak in Black Bengal goat with severe lesions in kidneys. It suggests a high adoption of PPRV in kidney cells of Black Bengal goats.

| D ISCUSS I ON
In line with this, we also found a relatively higher quantity of viral RNA in the supernatant of goat kidney cells. The multicycle growth analysis of PPRV in the primary goat kidney cells also showed a steady increase in the PPRV in the culture supernatant with the increase of time and passages. The characteristics CPEs induced by PPRV found in this study have also been described elsewhere (Adu et al., 1990;Saeed et al., 2004). The developed primary kidney cells are being routinely used for the isolation of field strains of PPRV in our laboratory and the pathogenicity of the propagated virus remained unaltered during subsequent experimental infection (Begum et al., 2020).
For the isolation of PPRV, kidneys cell lines such as MDBK, BHK-21, Vero and CV1 have been used with some limitations. However, the cell lines overexpressing the SLAM or Nectin-4 have many advantages over conventional cell lines and greatly enhanced the replication of PPRV in cell culture (Adombi et al., 2011;Birch et al., 2013;Fakri et al., 2016;Seki et al., 2003). The primary goat kidney cells developed in this study also showed efficient replication of field strains of PPRV. Therefore, the primary goat kidney cells can be readily used where the SLAM or Nectin-4 expressing cell lines are not available.

| CON CLUS IONS
In conclusion, we established a primary goat kidney cell culture using mature goat kidney cells which showed acceptable replication efficiency for PPRV. Such method can be applied for the isolation of PPRV from field outbreaks and fit well for the preparation of PPRV inoculum for experimental infection study. Furthermore, since kidneys of slaughtered goats are available throughout the year, it can be suggested that using this protocol primary cell culture from mature goat kidney can provide primary cells to the laboratory throughout the year. This could reduce the unnecessary sacrifice of lambs or kids.

E TH I C S S TATEM ENT
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 The funder had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

CO N FLI C T O F I NTE R E S T S
None declared.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.413.