Research Article, J Virol Antivir Res Vol: 8 Issue: 1
Comparative Sensitivity of LFBK and LFBK αVβ6 Cell Lines for Isolation of Foot and Mouth Disease Virus from Riverine Buffaloes by Using Oro-Pharyngeal Fluids
*Corresponding Author : Ali A
Animal Health Research Laboratories, Animal Sciences Institute, National Agricultural Research Centre (NARC), Park Road Islamabad, Islamabad, Pakistan
Tel: + 972-883-6360
Received: 04 July 2018 Accepted: 17 December 2018 Published: 04 January 2019
Citation: Ali A, Zahur AB, Farooq U, Latif A, Naeem K, et al. (2019) Comparative Sensitivity of LFBK and LFBK αV β6 Cell Lines for Isolation of Foot and Mouth Disease Virus from Riverine Buffaloes by Using Oro-Pharyngeal Fluids. J Virol Antivir Res 8:1. doi: 10.4172/2324-8955.1000188
Foot and mouth disease (FMD) is a major Transboundary Animal Disease (TAD) which causes major economics losses to the developing countries. In Pakistan the disease is considered endemic and outbreaks are still being reported. Rapid diagnosis of the disease and Isolation of FMD virus is important to confirm viral subtyping and allow for the development of effective vaccines against the specific subtypes. Carrier animals are the major source of current outbreaks of FMD in Pakistan. Current study was planned and conducted for isolation of FMD virus from persistently infected animals by using LFBK cell line and comparison of LFBK and LFBK αVβ6 for isolation of FMDV isolates from recent FMD outbreaks. A total of 120 serum samples were collected from persistently infected riverine buffaloes and examined for the presence of FMD virus Non-Structural Proteins by using NSP-ELISA. Of 120 sera samples 23 animals were found positive for NSP’s. The Oro-pharyngeal fluids (OP) were collected from NSP-ELISA positive animals. The OP fluids samples were treated with Tri-cholo-Tri-flouro-Ethane (TTE) and inoculated onto LFBK cell line. Out of 23 OP fluid samples 11 exhibited CPE’s. A total of six (06) FMD viruses were confirmed by rRT-PCR and characterized by Indirect Sandwich ELISA as type O, Asia-1and A. The FMD virus isolates were acquired from FAO-UN project on FMD in Pakistan. All isolates were inoculated on both of the cell lines and observed for the development of CPEs. We found that the newly modified LFBK αVβ6 cell line exhibited CPEs more rapidly after 18-20 hours, while LFBK cell line CPEs developed after 24 to 48 hours. TCID50 calculated on LFBK αVβ6 was higher for the all the serotypes tested than LFBK cell line. Percentage of CPEs in LFBK αVβ6 per plate resulted higher than over LFBK cell line.
Keywords: FMDV; LFBK αVβ6; Persistent infection; Buffaloes
Foot and Mouth Disease (FMD) is highly infectious and contagious disease of large ruminants and wild animals with high morbidity and low motility rate. Foot and Mouth Disease (FMD is characterized by lameness, fever, vesicular lesions on feet, tongue, snout and teats . Sever morbidity in young susceptible animals may lead to death . Thus, FMD pose serious threats to livestock and limits their productivity [3,4]. In Pakistan, livestock contribute 12% of total Gross Domestic Product from Pakistan Economic Survey  and loss due to FMD may reach around 6.00 billion Rs. in Pakistan . In South Asian region, FMD remains endemic and has been common in subcontinent (indo-pak) . In Pakistan the disease is considered endemic and outbreaks are still being reported . Documented that number of outbreaks of the FMD is higher during January-March due to Eid-ul- Azha (Muslims’ religious festival) .
The causative agent of FMD is foot-and-mouth disease virus (FMDV), which is found in seven distinct serotypes such as O, A, C, Asia-1, SAT1, SAT2, SAT3 but within the serotypes, subtypes have been evolved . In Pakistan, four serotypes of FMDV A, O, Asia-I and C were detected [6,10-13]. Type C is no longer present in Asia, because the most recent strain was detected in 1995 in Pakistan . Individual contribution of serotypes A, Asia-1 and O for outbreak was 4.7%, 25% and 70% respectively . Despite of several drawbacks isolation of virus remains promising technique, due to its high sensitivity and it provides viral material for further studies . Moreover, isolation of virus is important to identify the viral subtyping and developing effective vaccines against the specific subtype and utility in epizootic- logical studies.
Various cell cultures viz ., Calf Thyroid (CTh) and Porcine Kidney, Secondary Porcine Kidney Cells, Primary Lamb Kidney (LK), Bovine kidney and Lamb Testicle LT have been used to isolate FMDV [16-19]. Established cell lines, such as BHK-21, continuous Pig Kidney Cells used for FMDV propagation . But there is urgent need to find susceptible cell line to all FMDV serotypes and subtype and which can be maintained easily .
Some primary cells lines, such as bovine thyroid (BTY) are highly susceptible to a wide range of FMDV serotypes Snowdon et al. , but some drawbacks are associated with these cell lines such as low sensitivity, costly preparation and losing susceptibility after multiple passages . The most sensitive cells for the isolation of FMDV are BTY which have highly levels of αV β6 integrin expression . LFBK is well documented cell line of fetal porcine kidney which has susceptibility to all FMDV serotypes and remains unchanged over several passages. Bovine thyroid (BTY) cells are more susceptible to FMDV serotypes as compare to other cells like, IBRS-2 and Fetal Bovine Kidney Cells . Furthermore, two established Cell lines ZZR 127 and LFBK αV β6 reported by different research groups up till now to have highly sensitivity to all FMDV serotypes.
FMDV can survive at 4°C (39.2°F) on wool and in bovine feces for approximately two to three months which is the potential source of FMD infection to the susceptible animals. Hence, FMD may be spread to other FMD free countries through trade of contaminated meat, milk, semen and animal byproducts. FMDV can severely affect the economy of a country by limiting the animal and their products trade in international market. The virus must be inactivated in these products by exposure to temperature acidic pH or by using different chemical treatments without compromising nutritious value. Several environmental factors inactivate the FMD virus in animal products.
Keeping in view the above scenario the objectives of planned study were isolation and identification of FMDV from persistently infected animals by using cell culture techniques, Comparative Sensitivity of LFBK and LFBKαV β6 cell line for FMDV isolates and to check the physiochemical properties of FMDV.
Materials and Methods
The current study was planned to isolate FMDV from persistently infected buffaloes and comparison of LFBK and LFBK-αVß6 cell lines for isolation of FMD viruses. The study was conducted at Animal Health Research Laboratories, Animal Sciences Institute (ASI), National Agriculture Research Centre (NARC), Islamabad, Pakistan.
Experimental animals and study plan
The animals included in the study were riverine buffaloes kept at peri-urban dairy farms of Islamabad Capital Territory (ICT). A total of one hundred and twenty (n=120) buffaloes having a history of FMD during last six months were bled for sera collection. The sera were examined for the presence of FMD virus non-structural proteins by using NSP-ELISA. The NSP positive animals were sampled for Oro- Pharyngeal (OP) fluid using probang cups. The OP fluid samples were treated with Tri-cholo-Tri-flouro-Ethane (TTE) for isolation of FMDV. The isolated FMD viruses were confirmed by reverse transcriptase Real time PCR (rRT-PCR) and characterized using indirect sandwich ELISA (Is-ELISA). For comparison of LFBK and LFBK-αVß6 cell lines FMD field viruses were provided by Progressive Control of Foot and Mouth disease in Pakistan (GCP/PAK/123/USA), Food and Agriculture Organization (FAO) .
Non-structural protein ELISA (NSP-ELISA)
The non-structural protein antibodies of FMD virus were detected using NSP-ELISA. The NSP-ELISA kit (CHEKIT FMD-3ABC boovIDEXX, USA) according to the manufacturer’s protocol , the standardized reagents, assay protocol and manual were supplied with the kit.
Oro-Pharyngeal (OP) samples were collected from the NSP positive animals by using probang cups form the anterior part of oral cavity and specialized treatment called Tri-cholo-Tri-flouro-Ethane (TTE) treatment was done of OP samples to emulsify and neutralize antibodies for virus liberation. With TTE treatment of suspected samples for virus, the titer of virus increased 10-100 folds. The end product of the TTE treatment was stored at -80°C and used as inoculum for virus isolation.
Isolation of FMD viruses
LFBK cells were cultured in Dulbecco Modified Essential Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (Hyclone) and antibiotics Amphotericin B (100 IU), penicillin (100 IU) and streptomycin (100 μg/ml). The LFBK cells were maintained using DMEM supplemented with 2% fetal bovine serum at 37°C in the presence of CO2 and pH ranging from 6.5 to 7.5.
LFBK cells were sub-cultured using split ratio method following Burleson in class II biosafety cabinet. Flasks with complete monolayer of LFBK cells were washed with Phosphate Buffer Saline (PBS) by gentle shaking to remove all cell debris, after washing PBS was discarded. About 1 ml of trypsin (0.05%) solution was added into the cell culture flask and incubated for 10-15 min. Flask was observed for complete detachment of cells from flask. About 21 ml growth media (DMEM with 10% fetal bovine serum) was added and shacked gently to break up clumps and made a homogenized suspension. This suspension medium was equally distributed into 3 tissue culture flask (25 cm2) each of the flask contained 7 ml of suspension culture. The flask was incubated at 37°C containing 5% CO2.
Infection of LFBK cells for isolation of FMDV
Tissue culture flasks (25 cm2) which are 80% confluent was used for infection. The growth media was discarded and cells were washed with PBS. The cell culture flask was inoculated with 500 μl of inoculums (TTE treated OP fluid samples) filtered through 0.22 μm filter (Sartorious). The inoculated flask was incubated for 40 min till the adsorption of virus. About 7-10 ml DMEM medium was added for maintenance of cells. Negative control flask was inoculated with Sterile PBS. The flask was incubated at 37°C containing 5% CO2.
Flask was examined regularly for appearance of Cythopathic Effects (CPE) after every 24 hours for 72 hours. The sample was considered positive when 80% of CPE cells were observed in the flask, Samples showed no CPE after third passage considered as negative. The same procedure was followed for all the TTE treated OP fluid samples.
Detection of FMD virus isolates
Each FMD virus isolate was confirmed by Real-time Polymerase Chain Reaction (rRT-PCR) as described by (Challens et al, 1997). RNA was extracted using QIAamp Viral RNA Mini Kit following the manufacturer instructions (Qiagen, GmBh, Germany). Reagents and washing buffers were prepared according to the kit manual. To elute the extracted RNA 40 μl of RNA’s free water was used. The extracted RNA was places at -200°C until further used. The quantity and purity of extracted RNA was determined using Nano drop (Nano Drop 1000, Thermo scientific, Wilmington, DE, USA).
Real time RT-PCR
For the detection of FMD virus, real time PCR was performed as described by Challahan et al. (2002). Taq Man core reagents kit was used (Taq Man, EZ-RT-PCR CORE REAGENTS). The reaction mixture having 5X TaqMan EZ Buffer, 25 mM Mn (Oac), 10 mM dNTPs Mix, 50 μM of each primers, 10 μM Probe, 2.5 U/μl rTth DNA Polymerase, 10.5 μl of RNAse-free water. Template RNA was used in real time polymerase chain reaction. Reaction was carried out in a machine ABI Prism 7500 real time PCR machine (Applied Biosystem). All conditions were maintained as described by Challahan et al. .
For the detection of Serotype of FMDV Indirect Sandwich ELISA was performed following the kit manual (BDSL-UK), the standardized reagents, assay protocol and manual were supplied with the kit.
Preparation of 96 well cell culture plate of LFBK and LFBK- αVß6 cell line
LFBK and LFBK-αVß6 flask were prepared as described earlier. A flask with confluent monolayer was taken. Flasks were sub-cultured (trypsinized) as mentioned earlier. Took a 96-well, flat bottom micro titer plate. About 100 μl of cells from sub-cultured flask contained cells count of 4 × 105 cells/ml were added to each well of the plate.
For virus titration 180 μl infectious DMEM media (without serum) was added to each well from row A–H in U bottom 96 well micro titer plate thus made virus serial dilution 4-8 replicate of each virus mostly been used. Six (06) replicates were made for each of the virus, the side wells of the plate were not used to avoid contamination, 20 μl original virus was added to first well of all rows A1-H1 properly mixed it with multichannel pipeter, done serial dilution to column 11th and lift 12th column as control.
According to plate layout each well of the plate was inoculated with 50 μl of virus dilution. Precaution was taken not to touch tip to the walls or the bottom of each well to avoid contamination. Plate was covered with the lid, marked both lid and plate with a marker for plate identification. Incubated at 37°C for 48-72 hours in CO2 (5%) incubator and examined the CPE after every 24 h for 72 h under an inverted microscope .
Calculation of Tissue Culture Infective Dose (TCID50)
Results were interpreted by the observation of CPEs under the microscope, marked and noted the number of wells showed CPEs. The Tissue Culture Infective Dose (TCID50) was calculated by Spearmankarber method
A total of n=120 sera were analyzed for the detection of FMD virus NSP antibodies. 23/120 sera were found positive by NSP-ELISA with a positive percentage of 19.1%. NSP positive animals (n=23) were sampled for OP fluid using Probang Cups. A total of 23/23 NSP positive sera were subjected to TTE treatment for isolation of FMD virus.
Isolation of FMD Viruses
A total of n=23 TTE treated OP fluid samples were subjected to isolation of FMDV using LFBK cell line. A total of 11/23 samples showed CPE’s on LFBK cell line. The CPE’s were characterized by detachment of cell, cell rounding and clumps formation of cells.
Real time RT-PCR
A total of 11/11 CPE showed samples were analyzed by Real time PCR and 6/11 found positive for FMD viral RNA.
Indirect Sandwich ELISA for Sero-typing
Real-time PCR positive sample were further tested by sandwich ELISA for serotyping of FMDV
Comparative sensitivity of LFBK and LFBK-αVß6 cell lines
A total of n=8 FMD virus isolates were provided by UN-FAO project on FMD Islamabad. These all isolates were subjected to TCID50 using LFBK and LFBK-αVß6 cell lines showing the FMD isolates received.
While TCID50 of different serotypes of FMD viruses on LFBK cell line in comparison with LFBK αV β6 cell line, results expressed in percentage of positive well of both cell lines, represents percentage of positive wells on LFBK αV β6.
Foot and Mouth Disease (FMD) is highly contagious disease of large ruminants including wild animals. It is characterized by high morbidity and low motility rate. The clinical signs associated are lameness, fever, vesicular lesions on feet, tongue, snout and teats . Isolation of FMD virus is important to confirm the viral subtyping and allow for the development of effective vaccines against the specific subtype and amplify live virus from original tissue samples suitable for subsequent studies. Although new advance techniques have been developed and designed but virus isolation is irreplaceable.
In this study efforts were directed to isolate FMD viruses on LFBK cell line using Oro-pharyngeal (OP) fluid samples from persistently infected buffaloes. During this study it was found that 19% sera were found positive for NSP antibodies against FMD virus collected from persistently 120 infected animals. Our results showed that out of 23 OP fluid samples 11 exhibited CPE’s, out of which only six (54.5%) were confirmed by Real-time RT-PCR and Is-ELISA. These results were complete concurrence with Verin et al. .
Interestingly, LFBK αV β6 was found more sensitive cell line for the isolation of FMD virus. BHK21 cell line was documented the most commonly used cell line for the propagation and isolation of FMDV worldwide but now a days the most sensitive cell line for the isolation of FMDV is LFBK cell line after the transduction of αV β6 receptors. Our results for LFBK αV β6 are complete agreed with LaRocco et al. .
Finally, comparative studies for sensitivity of LFBK and LFBK-αV β6 were investigated for the isolation of FMDV (Table 1). Results have depicted the LFBK-αV β6 cells were more susceptible to all FMDV serotypes derived from persistently infected animals as well as field isolates compare to commonly used cells for FMDV isolation (Table 2). Virus titer was higher with LFBK-αV β6 cell line when compared with LFBK cell line and these observations corroborated with (LFBK and LFBK-αV β6 ) as reported by Zabal et al.  for BHK21 (6.74 OVK (7.25) and BFK (6.6,) TCID50/ml but the cell line in our study exhibited CPEs more rapidly and we can get results within 48 hours while they reported results after 72 h.
|S.No||Sample I.D||Ct Values||Results|
|1||Cell Culture 1||16.23||Positive|
Table 1: Result of real time RT-PCR.
|S.No||Sample I.D||Ct Values||Results|
|1||Cell Culture 1||16.23||Positive|
Table 2: Result of Sandwich ELISA.
According to King et al.  the most sensitive and supreme primary cells for the isolation of FMDV are bovine thyroid cells (BTY) which have highly levels of αV β6 integrin expression. In current we didn’t examined bovine thyroid cells (BTY) as reported by LaRocco et al.  that BTY is less susceptible than LFBK αV β6 on the basis of their results. Brehm et al.  claimed the most sensitive cell line fetal goat tongue cell line for the isolation of FMDV but the main drawback in this cell line is that several days are needed to become confluent as comparison with LFBK αV β6 and while current study showed LFBK was ready to be used after 24-30 h. LoRacco et al. , reported that BHK-21 cell are not highly susceptible for FMDV isolation as compared to LFBK αV β6 . However in our study, LFBK-αV β6 cells showed higher sensitive than LFBK and BHK-21 cells. This clearly indicates that LFBK αV β6 cell line can be useful in rapid isolation and for the high yield isolation (Table 3).
|2||HFZ-480||O||Pan Asia 2 Ant10|
|3||OKR-520||A||Iran 05 sis-12|
|5||MRP-580||A||Iran 05 sis-12|
|6||RHK-618||O||Pan Asia 2 (unnamed)|
|7||AJK-628||A||Iran 05 FARS-11|
|8||SG-699||A||Iran 05 sis-12|
Table 3: Virus isolates received from UN-FAO project on FMD in Pakistan.
Current study showed that LFBK and LFBKαV β6 cell lines are susceptible to all FDMV serotypes. There are many cell culture system have been used for the propagation and isolation of FMDV. Some strains of FMDV do not grow properly in primary bovine thyroid cells (BTY). Its isolation requires swine cells like IB-RS-2 . But it is reported that IB-RS-2 cell less sensitive to some FMDV serotypes as compare to BTY cells , due to αV β6 low level on cell surface . Also, LFBK cell line is susceptible to all FDMV serotypes.
For the first time this comparative study of LFBK and LFBK αV β6 cell line for the isolation of FMDV was designed to identify the capabilities of LFBK αV β6 for rapid and high yield isolation of FMDV in comparison with LFBK cell line. Moreover, we have standardized the chemical requirement of LFBK and LFBK-αV β6 cell line by providing the different quantities of serum to the cells in growth maintenance media (DMEM). It was observed that the growth cell was optimal when growth medium is supplemented with 10% serum and for maintenance of cells 2% serum medium showed good results. Both of cell lines showed same kind of CPEs but CPEs were appeared on LFBK αV β6 more rapidly as compared with LFBK cell (Table 4).
|2||HFZ-480||O||Pan Asia 2 Ant10||5.80|
|3||OKR-520||A||Iran 05 sis-12||4.80|
|5||MRP-580||A||Iran 05 sis-12||4.13|
|6||RHK-618||O||Pan Asia 2 (unnamed)||4.3|
|7||AJK-628||A||Iran 05 FARS-11||ND*|
|8||SG-699||A||Iran 05 sis-12||2.8|
Table 4: TCID50 of FMD virus serotypes on LFBK cell line, Result observed after 48 hours of infection. *ND=Not Detected.
Although the reason is still unknown that how LFBK and LFBK αV β6 support the growth of FMD virus but as reported by La Rocco et al.  that it might be these cell lines lake factor which restricted by other bovine origin cell line (Table 5). Further studies are needed to explore the underlying mechanism .
|2||HFZ-480||O||Pan Asia 2 Ant10||6.47|
|3||OKR-520||A||Iran 05 sis-12||6.63|
|5||MRP-580||A||Iran 05 sis-12||6.30|
|7||AJK-628||A||Iran 05 FARS-11||ND|
|8||SG-699||A||Iran 05 sis-12||5.30|
Table 5: TCID50 of different serotypes on LFBKαVβ6 CELL LINES.
Our results are highly Expressive that persistent infection in riverine buffaloes can serve as the potential source of new FMD outbreaks (Table 6).
|2||HFZ-480||O||Pan Asia 2 Ant10||5.80||6.47|
|3||OKR-520||A||Iran 05 sis-12||4.80||6.63|
|5||MRP-580||A||Iran 05 sis-12||4.13||6.30|
|6||RHK-618||O||Pan Asia2 (unnamed)||4.3||6.47|
|7||SG-699||A||Iran 05 sis-12||2.8||5.30|
Table 6: TCID50 on LFBK cell line in comparison with LFBK αVβ6 cell lines.
The results of current study concluded that use of LFBK-αV β6 harvest high yield of FMDV. Our results strongly recommend LFBK- αV β6 is the sensitive most cell line for rapid FMDV diagnostic purposes due to high level of αV β6 (Table 7).
|2||HFZ-480||O||Pan Asia 2 Ant10||55%||62.97%|
|3||OKR-520||A||Iran 05 sis-12||44.45%||64.8%|
|5||MRP-580||A||Iran 05 sis-12||37%||61%|
Table 7: Percentage of positive well of both cell line.
Authors are very thankful to the UN-FAO Project on FMD “Progressive Control of FMD in Pakistan GCP/PAK/123/USA” for providing recent isolates of FMD viruses for this study.
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