Comparative analysis of peste des petits ruminants virus tropism in Vero and Vero/SLAM cells

Abstract

A comparative study was conducted to investigate tropism of peste des petits ruminants (PPR) virus in Vero and Vero/SLAM (signaling lymphocyte activation molecule) cells. PPR virus was isolated from tissues and blood samples of sheep and goats following second passage in both the cell line. Intensive cytopathic effects and higher antigen load (by sandwich enzyme linked immuno sorbant assay [ELISA]) were observed in Vero/SLAM cells as compared to Vero cells. A significantly higher virus titer (i.e. ranging from 4.5 to 6.5 log₁₀TCID₅₀/ml) was detected in Vero/SLAM than Vero cell isolates (i.e. ranging from 3.5 to 4.5 log₁₀TCID₅₀/ml). The present study has demonstrated that Vero cells expressing the SLAM receptors are highly efficient for isolating PPR virus (PPRV) from pathological samples, as it offers a substantial improvement including faster viral replication resulting in primary viral isolation in a shorter period of time, and higher yield of virus than traditional cell culture methodologies.

Keywords:

• PPR virus
• Vero
• SLAM
• sandwich ELISA
• TCID₅₀

Introduction

The etiological agent, peste des petits ruminants (PPR) virus, has been classified under family Paramyxoviridae, order Mononegavirales, and genus Morbillivirus (van Regenmortel et al. 2000). Diagnosis of PPR virus (PPRV) infection relies on laboratory techniques such as virus isolation, demonstration of PPRV antigen, and nucleic acid. Definitive identification of PPR virus isolates can be made by immunological techniques like enzyme immunoassay (Nanda et al. 1996) and molecular techniques like reverse transcription polymerase chain reaction (RT-PCR) (Ozkul et al. 2002). Virulence and biological properties of the virus can be detected by titration method (Mariner et al. 1990). Vero cells have been the most commonly used host for isolation and cultivation of morbilliviruses including PPRV (Diallo et al. 1989). Like in case of certain marine morbilliviruses (Nielsen et al. 2008), canine distemper virus (Seki et al. 2003), and measles virus (Tatsuo et al. 2000), Vero cells expressing SLAM (signaling lymphocyte activation molecule also known as CD150) receptor could be the suitable host system for cultivation of PPRV. PPR virus as any other morbilliviruses has the propensity to utilize two receptors, namely, CD46 and CD150 (Sarkar et al. 2009). CD46 is a complement regulatory protein expressed on all cells except red blood cells (Liszewski et al. 1991) and SLAM is a glycoprotein belonging to the CD2 subset of the immunoglobulin super family and is expressed on the surface of a proportion of primary B cells, Epstein-Barr virus (EBV) transformed B cells (B95a cells), activated T cells, memory T cells, T cell clones, immature thymocytes, mature dendritic cells, and activated monocytes (Tangye et al. 2000). Wild-type PPRV strains might use an alternative receptor(s) in Vero/SLAM cells, probably more efficiently than the CD46 (in plain Vero cells), for binding the virus (Adombi et al. 2011). Keeping the above facts in mind, present study was undertaken to compare tropism of PPRV in Vero and Vero/SLAM cells by observing cytopathic effect (CPE), PPRV antigen load, and virus titer.

Materials and methods

Pooled field samples comprising of six-tissue sample and one whole blood sample from field outbreak (north Gujarat, India). Cell line (Vero and Vero/SLAM) and PPR sandwich enzyme linked immuno sorbant assay (ELISA) kit available at National Morbillivirus Referral Laboratory, Division of Virology, IVRI, Mukteswar, were used in the present work. The cells were propagated in a growth medium containing Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FCS; Sigma-Aldrich, St. Louis, USA). The isolation of PPR virus from tissue and blood samples was carried out using protocol given by Nanda et al. (1996) with little modification. After two blind passages, virus was isolated and confirmed by PPR sandwich ELISA. Sandwich ELISA was performed strictly as per the protocol outlined in the user's manual supplied with the kit.

Titration of PPR virus was performed to study the infectivity titer of PPR virulent virus present in Vero and Vero/SLAM cells following second passage. Virus titration was carried out in 96 well microtiter plates using Vero cells as per the technique described by Mariner et al. (1990). Briefly, Vero cells were subcultured and 100 ¼l of the cell suspension was added to each well (0.5 × 10⁶ cells/ml) in 96 well tissue culture plates. Plates were incubated at 37^°C in the presence of 5% CO₂. Following day, virus was diluted 10-fold in EMEM containing 2% FCS in deep well plate. An aliquot of 100 µl of virus suspension was added to each well keeping four replicates per dilution. One well was kept as control (without virus) per replicate. The plates were incubated at 37^°C in the presence of 5% CO₂. Media were replaced each alternate day with EMEM containing 2% FCS. Reading was taken on the sixth day of titration. End points were calculated using Reed and Muench (1938) method.

Results and discussion

Characteristic CPE of PPR virus was observed following second passage. In Vero cells, CPE was characterized initially by rounding and ballooning of cells and later on aggregation of cells followed by formation of fusion mass and syncytia. In Vero/SLAM, CPE was characterized initially by rounding of cells and clumping of cells followed by formation of granulation foci and cell lysis. In few cases syncititiation was also observed. Development of intense CPE in Vero/SLAM compared with Vero cell is in agreement with the finding of Sarkar et al. (2009). Nielsen et al. (2008) isolated and characterized marine mammal morbilliviruses in DogSLAM tag cells and observed that it offered a substantial improvement including faster viral replication, resulting in primary viral isolation in a shorter period of time and higher yield of virus than traditional cell culture methodologies.

Infected cell lysate was tested after each passage level by PPR sandwich ELISA for PPRV antigen. All the samples after first passage both in Vero and Vero/SLAM were detected negative for PPRV antigen. During second passage, PPR viral antigen load as measured by optical density (OD) value was higher in infected Vero/SLAM lysate as compared to infected Vero cell lysate (Table 1). However, following third passage, sandwich ELISA OD value was more or less similar in both cells infected with PPR virulent virus. In a study, Adombi et al. (2011) also reported that monkey CV1 cells expressing goat SLAM are highly efficient for isolating PPRV from pathological samples; this further suggests that SLAM, as is in the case for measles, canine distemper and rinderpest virus, is also a receptor for PPRV.

Table 1. Comparative antigen load, measured by sandwich ELISA in Vero/SLAM and Vero cell lines infected with PPR virulent virus.

	Passage 1		Passage 2 (average OD; positive control = 1.988)		Passage 3 (average OD; positive control = 2.058)
PPRV isolates	Vero	Vero/SLAM	Vero	Vero/SLAM	Vero	Vero/SLAM
1	Negative	Negative	0.45	0.81	1.155	1.197
2	Negative	Negative	0.466	1.145	1.214	1.365
3	Negative	Negative	0.455	1.11	1.382	1.393
4	Negative	Negative	0.44	1.05	1.61	1.995
5	Negative	Negative	0.404	1.125	1.592	1.603
6	Negative	Negative	0.475	1.175	1.204	1.452
7	Negative	Negative	0.271	0.845	0.99	1.312

Infectivity titers in Vero cell PPRV isolates were lower (i.e. ranging from 3.5 to 4.5 log₁₀TCID₅₀/ml) as compared to Vero/SLAM PPRV isolates (i.e. ranging from 4.5 to 6.5 log₁₀TCID₅₀/ml). Observation clearly indicated a significant titer difference of 2.0 between Vero and Vero/SLAM cells (Table 2). Sarkar et al. (2009) also reported higher PPRV antigen load in Vero/SLAM cell PPRV isolates. Subsequently in the same experiment, they observed that Vero and Vero/SLAM cells infected with attenuated PPR vaccine virus at a 0.001 multiplicity of infection produced the highest quantitable titer difference, approximating to 1.2 log₁₀TCID₅₀, indicating the higher virus yield in Vero/SLAM as compared to Vero cell. Difference in antigen and viral load between Vero and Vero/SLAM isolates needs to be explained.

Table 2. Comparative viral load, measured by virus titration assay following second passage in Vero/SLAM and Vero cell lines infected with PPR virulent virus.

	Titer (log10TCID50/ml)
PPRV isolates (passage 2)	Vero/SLAM	Vero	Titer difference
PPRV 1	6.5	4.5	2
PPRV 2	5.5	3.5	2
PPRV 3	5.5	4.5	1
PPRV 4	6	4.5	1.5
PPRV 5	5.5	3.5	2
PPRV 6	6.5	3.5	3
PPRV 7	4.5	3.5	1

Tissue tropism in vivo can be explained. SLAM mRNA has been detected at high levels in spleen, mesenteric lymph node, hilar lymph node, mandibular lymph node, superficial cervical lymph node, nasal mucosa, duodenum, heart, gallbladder, thymus, and blood; this is similar to the tissue tropism of PPR virus. However, expression of SLAM is low in lungs, colon, and rectum. In addition, very low levels are being detected in larynx, tongue, and esophagus, which suggest the possible presence of an alternative receptor (CD46) for PPRV (Meng et al. 2011). Pawar et al. (2008) reported in a study that the SLAM mRNA expression levels could be one of the determinants of different susceptibility of ruminant species to PPRV. The possible reason may be related to the more efficient entry of the virus through homologous host species SLAM molecule when compared to its counterpart (Vero).Vero/SLAM cells have caprine SLAM (i.e. CD150) besides CD46 on their surface (Sarkar et al. 2009). Viruses utilizing SLAM were generally lymphotropic in nature and are better adapted to grow on cells expressing SLAM receptor like Vero/SLAM while epitheliotropic virus is adapted to grow at conditions that permit the exclusive use of CD46 in Vero cells (Yanagi et al. 2006). The virus that ordinarily uses the CD46 receptor for entry is drawn by the CD150 receptor. Entry by CD150 is not simultaneous and presumably entry by CD150 is not inhibitory for a subsequent entry by CD46 (Sarkar et al. 2009). High viral antigen and virus titer during second passage in Vero/SLAM suggests that the replication of the virus in Vero/SLAM cells was initially more efficient when compared to native Vero cells. After the initial difference there was more or less uniform quantity between the virus yields in Vero and Vero/SLAM during third passage.

In conclusion, it may be inferred that Vero cell line expressing SLAM receptors is a highly sensitive in vitro system for the cultivation of PPRV.

Acknowledgments

This study was supported by National Morbillivirus Referral Laboratory, Division of Virology, Indian Veterinary Research Institute, Campus Mukteswar – 263138, Nainital District, Uttarakhand, India.