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AUGMENTATION OF BIOLOGICAL TITER OF FOOT AND MOUTH DISEASE VIRUS IN IN VITRO CULTURE.

Byline: Q. Akram, K. Muhammad, M. Rabbani, J. Nazir, M. Nawaz, K. Hanif and Z. Shakoor

ABSTRACT

Foot and Mouth Disease (FMD) virus grew well on BHK-21 cell line. The virus showed poor tissue culture infective dose 50 (TCID50) that was log 103.2+-0.2 in BHK-21 cell line having Glasgow Minimal Essential Medium (GMEM) without Fetal Calf Serum (FCS). Addition of one per cent FCS in the medium @ one percent increased log 107.1+-0.2 units of virus TCID50. Incubation temperature of 350 C and 370 C supported the multiplication and maintenance of BHK-21 cells and yielded log106.6+-0.1 and log107.0+-0.2units of virus TCID50, respectively. Each serotype of FMD virus showed log106.29+-0.07 units of virus TCID50 in the stationary monolayer of BHK-21 cells in roux flask (75cm2), log107.66+- 0.02 units of virus TCID50 in roller bottles (490cm2) and log108.34 +- 0.07 units of virus TCID50 on 0.2 g of micro-carriers suspending in 200 ml of the growth medium in stirring bottle.

The infectivity titer/TCID50 of each of the virus serotypes was significantly higher in roller bottles than that achieved in roux flasks (pless than 0.05) and was significantly higher in stirring bottle containing micro-carriers suspending in the growth medium than that of harvested in roller bottle (pless than 0.05). It is concluded that the infectivity titer of the virus is directly proportional to number of BHK-21 cells in the culture system.

Key words: Anchorage dependant BHK-21 cells, roller bottles, infectivity titer, FMD virus

INTRODUCTION

FMD is a highly contagious viral disease of cattle, buffaloes, sheep, goats, etc. The disease is the main hurdle in the export of animal and their products. The disease spreads in a large population of susceptible animals (Awan et al., 2009) and is caused by an Aphthovirus of Picornaviridae (Grubman and Baxt, 2004). The virus has seven serotypes such as "A", "O", "C", "Asia-1", "SAT-1", "SAT-2" and "SAT-3" (Muhammad et al., 2012:). Out of these, only "A", "O" and "Asia-1" types of FMD virus are prevailing in Pakistan (Khuwaja et al., 2009: Awan et al., 2009). The virus grows in unweaned mice, foot pad of guinea pigs, bovine thyroid cells (BTY) and Baby Hamster Kidney cells (BHK-21) (Ferris et al., 2002: Khuwaja et al., 2009). The field FMD virus when grown first time on BTY cells show poor infectivity titer or biological titer. However, its infectivity titer is enhanced by repeated passage on BHK-21 cell line. BHK-21 cell lines are either anchorage dependent (adherent) or anchorage independent. Adherent

BHK-21 cells grow on roux flask, roller bottle or surface of micro-carrier (Giard et al., 1978). Each roux flask and roller bottle yields 1.7x107 and 4.4x107cells, respectively. However, cell yield in micro-carrier culture system depends upon number of micro-carrier / ml and total amount of growth medium in culture vessel yield (Akram et al., 2012). For vaccine production high biological titer of the virus is the basic requirement. In most of the biologics production units, FMD virus serotypes are grown in roux flasks (still culture system). This is a major impediment in production of vaccine to meet the requirements of the dairy industry. Present study is therefore designed to evaluate the factors (fetal calf serum, Temperature and culture system of adherent BHK-21 cells) augmenting biological titer of FMD virus serotypes in in vitro culture.

MATERIALS AND METHODS

Source of Baby Hamster Kidney (BHK-21) cells and its propagation: Baby Hamster Kidney (BHK-21) cells were procured from Department of Microbiology, University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan. The cryopreserved cells were revived using standard procedure (Altaf et al., 2012). BHK-21 cells were grown in filter sterilized Glasgow minimum essential medium (GMEM) with Earl's salts (Biomedical; USA) supplemented with 5 % fetal calf serum (FCS). The liquefied cells (107 cells) were transferred to roller bottle (480 cm2) containing 100 ml of the growth medium. The bottle was incubated at 37 degC with 5 % CO2 for 60 hours and the cells were harvested and transferred to roux flask (175 cm2) with 35 ml of the medium (Khuwaja et al., 2009), the roller bottle (Duran, GmbH, Germany) containing 100 ml of the medium and spinner flask containing 0.2g microcarriers/ 200 ml of the growth medium (Giard et al., 1978) in order to observe the effect of culture system on the biomass production of FMD virus.

Source of Foot and Mouth Disease (FMD) virus and its cultivation: Each of the serotype of FMD virus ("O", "A" and Asia-1") was obtained from the Department of Microbiology and cultivated on monolayer of BHK-21 cells in the above mentioned different culture systems.

medium were divided into six groups (A, B, C, D, E and F), each containing three bottles. Sterilized FCS was added @ 0, 1, 2, 3, 4, and 5 percent aseptically in each bottle of group A, B, C, D, E and F, respectively. Monolayer in each of the bottle was infected with 3ml of freshly grown the FMD "O" virus and was incubated at 370C for 48 hours.

medium were divided into six groups (A, B, C, D, E and F), each containing three bottles. Sterilized FCS was added @ 0, 1, 2, 3, 4, and 5 percent aseptically in each bottle of group A, B, C, D, E and F, respectively. Monolayer in each of the bottle was infected with 3ml of freshly grown the FMD "O" virus and was incubated at 370C for 48 hours.

Culture system: Nine culture vessels in each of the culture systems (roux flask, roller bottle and spinner flask with micro-carrier) were divided into three groups (A, B and C), each containing three flasks of each of the culture system. Each of the culture vessels of each group was infected with 3, 5 and 7 ml of each serotype of FMD virus, respectively. Each of the vessels was incubated at 37o C for 48 hours Biological Titration of the Virus (TCID50): After incubation periods, cytopathic effects (CPE) of the virus was observed under inverted microscope and the harvested culture fluid was processed for biological titration (TCID50) (Alhaji et al., 2011).

Statistical Analysis: Log value of each TCID50 value of the virus was calculated and was processed for calculation of mean and SD values. The data thus recorded as a result of each of the above mentioned parameters was compared using one way analysis of variance (ANOVA) at 5% level of probability (SPSS 13.0).

RESULTS AND DISCUSSION

Cell culture adapted FMD virus grew well on BHK-21 cell line (Figure 1-3). There are many cell lines including BHK-21 cells, lamb kidney cells and bovine thyroid (BTY) cells used for growth of the virus. The virus has ligand molecules and host cells have specific receptors. The virus serotypes from field do not grow directly on monolayer of BHK-21 cells. This could be possibly due to coating of secretary antibodies on receptors of the cells (Jackson et al., 1996). However, the virus can directly grow on primary monolayer of BTY cell then after 2nd, 3rd passages can adapt on BHK-21 cells (Ferris et al., 2002: Khuwaja et al., 2009). First time growth of the virus on BHK-21 cells after passages on BTY gives 103titer. Repeated passages on BTY cells give increase TCID50 of FMD up to 10 (Khuwaja et al., 2009). The virus showed poor growth in BHK-21 cell line having cell culture medium without FCS (Figure 1).

This could be due to rapid death of cells in the maintenance medium that don't support replication of the virus. Addition of FCS in GMEM supported the maintenance of BHK-21 cells and also supported the replication of FMD virus.

Addition of FCS in GMEM @ 1 and 2 % concentration proportionally increased the infectivity titer of FMDV, i.e., 103, 107, 107, respectively (Figure 4). However, further increase in FCS in GMEM didn't improve the replication of FMDV and hence didn't show significant increase in the infectivity titer of the virus. FCS neutralizes the residual amount of trypsin in inoculums and supports the attachment of cells with substrate and also support the maintenance of BHK-21 cells. This could be possible reason of having higher infectivity titer of virus at 1% FCS in the cell culture medium. Further increase of the serum in the GMEM improved the multiplication of BHK-21 cells but may not support the replication of the virus. Incubation temperature of 330C and 390C did not support the multiplication of BHK-21 cell line in the presence of GMEM supplemented with FCS and hence didn't support the replication of FMD virus.

This could be possible reason of having poor infectivity titer of the virus at above mentioned temperature. However, incubation temp of 350C and 370C supported the multiplication and maintenance of BHK-21 cells that might had supported the replication of FMD virus (Figure 5). This might be possible reason of high infectivity titer of 10 6.6 and 10 7.2 at 350C and 370C respectively. The replication/infectivity titer of virus is directly proportional to number of cells (Razdan et al., 1996).

Anchorage dependant BHK-21 cells are grown in three ways. The cells are grown in roux flasks, dishes, and multiwell plates in stationary cultures, in inner surface of roller bottles which are rotated (3 rpm) on motorized racks and on micro-carriers suspended in growth medium in mechanically stirred vessels. These rolling culture vessels (Roller bottles) are widely used for producing large quantities of cells (Kretzmer et al., 2002). Although BHK-21 cells are adapted to grow on micro-carriers to achieve high cell density but the roller bottles are better choice for growing monolayer of adherent cells (Kunitake et al., 1997: Kretzmer et al., 2002).

In the present study, FMD virus type "O" serotypes showed 106.4 +- 0.15units of infectivity titer (Tissue culture infective dose 50-TCID50) in the stationary monolayer of BHK-21 cells in roux flask (75cm2), 107.2 +- 0.2units of TCID50 in roller bottles (490cm2) and 108.13+-0.1units of TCID50 on 0.2 g of micro-carriers suspending in 200 ml of GMEM growth medium in stirring bottle (Figure 3).

FMD virus type "A" serotype showed 106.0 +- 0.2units of infectivity titer (Tissue culture infective dose 50-TCID50) in the stationary monolayer of BHK-21 cells in roux flask (75cm2),107.2+- 0.1units of TCID50 in roller bottles (490cm2) and 108.2 +- 0.05units of TCID50 on 0.2 g of micro-carriers suspending in 200 ml of GMEM growth medium in stirring bottle (Figure 4). FMD virus type "Asia-1" serotypes showed 106.1 +- 0.2units of infectivity titer (Tissue culture infective dose 50-TCID50) in the stationary monolayer of BHK-21 cells in roux flask (75cm2), 107.0 +- 0.2units of TCID50 in roller bottles (490cm2) and 108.0+-0.3units of TCID50 on 0.2 g of micro-carriers suspending in 200 ml of GMEM growth medium in stirring bottle (Figure 5).

The infectivity titer of the virus serotype was significantly higher in roller bottles than that achieved in roux flasks (pless than 0.05) and was significantly higher in stirring bottle containing micro- carriers suspending in growth medium than that of harvested in roller bottle (pless than 0.05). The infectivity titer of the virus is directly proportional to number of BHK-21 cells in the culture system (Altaf et al., 2012: Khuwaja et al., 2009: Salivac et al., 2006).

It is concluded that 1 - 2% of FCS, 35-370 C incubation temperature and surface area of roller bottles for attachment of BHK-21 cells are critical factors for supporting maximum infectivity titer of FMD virus serotypes. However, infectivity titer of the FMD virus depends on number of micro-carriers suspended in growth medium in stirring vessels.

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Publication:Journal of Animal and Plant Sciences
Date:Jun 30, 2013
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