Phylogenetic And Sequence Variability Analyses Of Vp1 Protein Of Foot And Mouth Disease Viruses In Cattle In Amhara Region Of Ethiopia

Background: Foot-and-mouth disease (FMD) is one of the contagious and the most economically devastating viral diseases caused by the FMD virus. The present study was conducted with the aim of molecular characterization of the FMD virus isolated from outbreaks that occurred in cattle in two different districts of Amhara regional state of Ethiopia. Samples collected from outbreaks were isolated on BHK-21 cell and serotype were detected using antigen detection ELISA. Phylogenetic and amino acid variability analyses of isolated viruses were undertaken after sequencing the VP1 gene in World Reference Laboratory, Pirbright, UK. Result: In the present study only serotypes O was found in the samples analysed using antigen detection ELISA and in 10 out of 13 (76.9%) cultured samples, virus were isolated on BHK-21 cells. Phylogenetic analysis of these isolates revealed that, they belonged to East Africa topotype-3 (EA-3) and shared 96.6% nucleotide similarity with Sudan’s isolates. A total of 10.3% amino acid variations were recorded between VP1 gene sequences of the field isolates of serotype O FMDV and vaccine strain (O/ETH/38/2005) used in Ethiopia for vacccine production. Conclusion: The phylogenetic analysis serotype O detected in this study revealed that the virus was clustered with East African topotype-3 (EA-3) and exhibited high genetic similarity with isolates from Sudan. A number of amino acid variations were also noted at different sites of VP1 gene when comparing field isolates with the vaccine strain. Thus, to enhance control of FMD in Ethiopia, detailed molecular analysis of the field isolates along with in-vitro vaccine matching tests need to be undertaken at frequent intervals to assess the protective potential of the vaccine strain in use.

Foot and mouth disease is one of the most important livestock diseases in the world in terms of its economic impact [16]. The annual economic impact of FMD in terms of visible production losses and vaccination costs in endemic regions of the world is estimated between US$ 6.5 and 21 billion, while outbreaks in FMD free countries and zones cause losses of more than US$1.5 billion a year [17]. The presence of FMD in Ethiopia leads to considerable economic losses through production loss, morbidity and mortality of affected animals [16,18,19]. The economic losses of FMD outbreaks in this country due to milk loss, draft power loss and mortality were on average USD 76 per affected herd and USD 9.8 per head of cattle in the affected herds in crop-livestock mixed system; and USD 174 per affected herd and USD 5.3 per head of cattle in the affected herds in the pastoral system [20] . The diseases also the major constraint hampering the export of livestock and livestock products to the Middle East and African countries; the Egyptian trade ban of 2005, in which Ethiopia lost more than US$14 million [21] Phylogenetic analysis of the VP1 region of FMD viruses has been used to study the genetic relationships between different FMD virus isolates, geographical distribution of lineages and genotypes, the establishment of geographically and genetically linked topotypes and tracing the source of virus during outbreaks [9]. Topotypes are geographically clustered viruses in a single genetic lineage and sharing >85% (O, A, C, and Asia 1) or >80% (SAT 1, SAT 2, and SAT 3) nucleotide similarity in the VP1-coding region [22]. VP1 nucleotide sequences are used for reconstructing the evolutionary history of organisms and molecular evolutionary studies [9].
Although animals are protected against FMD by the Government through vaccination in Ethiopia, lack of vaccination strategies and presence of free animal movement without certification are the main factors that could increase the spread of FMD. Moreover, lack of veterinary infrastructure to handle outbreaks on large scale greatly contributes to the increasing occurrence of the disease [12]. The detailed research work on serotyping and molecular phylogeny of FMD in the present study area is needed to determining the circulating serotype(s), establishing the geographic relatedness between isolates and molecular evolution of the virus for planning a proper control and preventive measure in the country.
Therefore, the objective of the present study was to undertake phylogenetic analysis on virus circulating in the study area and to determine the amino acid sequence similarity/variability between the VP1 protein of the field isolate and the vaccine strain isolate (O/ETH/38/2005 with the Accession number FJ798108) [23,24] currently in use for vaccine production in Ethiopia.

Virus isolation
Out of 21 samples collected 13 representative samples from each outbreak were subjected to virus isolation using Baby Hamster Kidney (BHK-21) monolayer cell culture.
From 13 samples cultured, cytopathic effect (CPE) was observed in 10 (76.9%) samples (Table 1), while virus did not grow on the rest of the three samples even when blind passaged three times. The CPE observed was fast destruction of mono-layer cells and the infected cell appeared as singly and round in shape. Additionally, the CPE was characterized by complete destruction of the cell and cell detachment which was mostly seen within 48 hrs of inoculation.

Serotype identification
All the 21 samples were subjected to serotype differentiation using antigen detection sandwich ELISA and only serotype O was detected (Table 1), indicating that this serotype was the predominant in the study area. The same serotype was found in both Shewarobit and South Wollo areas of Amhara regional state.   This may be probably because these were oro-pharyngeal samples collected from animals that had previous history of the disease and at early stage of recovery during sample collection. This suggested that the chances of virus isolation from tissue samples taken from old lesion (in recovery stage) and oro-pharyngeal fluid samples were less due to lower concentration of the virus. In agreement with the present findigs, OIE [10] also recommended that the preferred sample for virus isolation is epithelial tissue from unruptured or freshly ruptured vesicles. In isolated virus, the CPEs observed were characterized by single appearance, rounding, detachment as well as destruction of monolayer cell. This result is in line with previous reports [26,27,15] in which, FMDV growth on BHK-21 cell, characterized by fast destruction of monolayer cells and infected cells appeared singly and round in shape.
The serotype identification in the present study area revealed that FMDV serotype O was the only identified and predominant serotype, which is in agreement with the reports made earlier in different regions of Ethiopia [12,15,26,27,28,29]. Sequence coding for the FMDV VP1 protein has been used extensively to investigate the relationship between different isolates of the virus worldwide [30]. On the basis of VP1 gene sequence comparison, existence of 8 serotype O topotypes has been confirmed within samples collected around the World. Out of the 8 topotypes, only 2 were found in Africa, with one each in East and West Africa [22]. FMD viruses with > 95% of nucleotide similarity in the VP1 sequence were considered as closely related [30], while those virus serotypes (O, A, C) with > 85% nucleotide similarity were clustered in a single genetic lineage [12]. It is also assumed that FMD viruses that differ between 2-7% from each other are generally believed to originate from the same epizootic [31]. [34] also reported, closely related viruses could either be from the same outbreak or from viruses temporally closely related.
In the present study, the deduced nucleotide and amino acid sequences were aligned and known to be an immunodominant region of VP1 gene [24]. In an other report, the amino acid site from 141-160 was found to be the imunodominant region of serotype O of FMDV [4]. The existence of even small genetic variation in the immunodominant regions can alter the antigenic specificity of FMD viruses [9]. Thus, in the present study, the existence of amino acid variation especially at immunodominant region indicates the need for invitro vaccine matching studies at frequent intervals to establish the level of protection conferred by the strains available in the vaccine against the currently circulating viruses.
These changes have implications in vaccine strain selection as these changes occurred in G-H loop as compared with the vaccine strain in use in Ethiopia.

Conclusions
In the present study, serotype O was identified and isolated on BHK-21 cell culture from the different outbreaks and found as the dominant serotype circulating in the study area.
The phylogenetic characterization of this serotype revealed that it was clustered with East

Study areas
The study was conducted from October 2017 to May 2018 in Shewarobit and South Wollo districts of Amhara regional states of Ethiopia. Shewarobit is a town in KewetWoreda having a tropical climate with an average temperature of 23.0 °C. South Wollo is one of ten Zones in the Amhara National Regional State of Ethiopia having an average temperature and rainfall of 19.5 °C and 1185.1 millimeters, respectively [35]. The study areas were selected due to the occurrence of FMD outbreaks in different times. The distribution and density of animal populations vary across agro-ecological zones and woredas even though the distribution follows the pattern of human population. In both study areas animals were mostly reared under extensive management systems although semi-intensive farming is practiced rarely in some kebeles of the area.

Study animals
The study was conducted in cattle that had experienced outbreaks of FMD and manifesting the clinical signs of the disease. The animals were owned by farmers in the study area and their consent were asked before starting sampling and sample was collected only from willingness owners. Cattle of all age groups, breed, and sexes reared under semi-intensive and extensive production and management systems were included in the study.

Study Design
Based on reports of active cases of FMD outbreaks, a cross-sectional study design was employed to collect samples. While active cases were reported, a field investigation was conducted at a particular site of outbreaks within the study districts. Animals with active lesions and those in a history of infection but having healing lesions were used to collect samples for diagnostic purposes and confirmation of the cases.  [36]. OP sampleswere collected by passing a probang cup into oro-pharyngeal area from previously FMD suspected cattle and asymptomatic cattle that were found in close proximity to the affected herds. The collected fluid samples were examined for its clarity and 2ml of it containing cellular material were poured into a sampling bottle containing equal amounts of transport media (composed of 0.08M phosphate buffer containing 0.01% bovine serum albumin, 0.002% phenol red, antibiotics), pH7.2-7.4 [36].

Sample collection
The samples were, then, labeled, placed in icebox and transported to National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta and stored at -80 o C until required for further laboratory investigation.

Virus isolation
Sample processing and virus isolations on BHK-21 cells were conducted following the procedure of OIE manual [37]. Briefly, the epithelium samples were first taken from the transport media and blotted dry on absorbent paper.  [38]. Finally, isolated samples were labeled and stored at -80 °C for further experiments.

Serotype identification
FMDV serotype was detected and identified from the suspension by using antigen detection sandwich ELISA (IZSLER, Brescia, Italy) according to the manufacturer protocol and the protocol described in the OIE Manual for Terrestrial Animals [37]. Briefly, first samples were diluted ½ in diluent buffer and 50μl/well of each sample was distributed to 8 wells of a column (a total of 80 wells of A-H rows (each row were coated with FMD serotype-specific monoclonal antibodies)). Then, 50μl of diluents per well were added in all wells including thepositive and negative control wells and plates were incubated at room temperature for 1hour. After incubation, all the fluid in each well was discarded and the plate was tap hard to remove all the residual fluid. Then 200μl of washing solution was added and incubated for 3min at room temperature, subsequently wells were emptied and the washing repeated twice (three washing cycles in total). Then all residual fluids were removed by tapping on clean absorbent paper and 50μl/well of conjugate A was added from row A to F and the same volume of conjugate B was added into row G and H. Plates were covered and incubated at room temperature for 1hour. After incubation 50μl of substrate per well was added to all wells and plates were covered and left at room temperature for 20 minutes in the dark. The reaction was stopped by adding 50μl/well of stop solution (sulfuric acid, H 2 SO 4 ). Immediately after stopping, reading the optical density (OD) of each well was taken at 450 nm wavelength using micro plate reader.

Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing
Ten virus isolates were sent to World Reference Laboratory for FMD (WRLFMD), The Pirbright Institute, Pirbright, United Kingdom, for sequencing the VP1 gene of the FMDV strain according to recommended international standard. All RNA extraction, RT-PCR, and sequencing were conducted in WRLFMD.

Data management and analysis
Optical density (OD) values of the samples generated from antigen detection ELISA were interpreted by subtracting the OD value of each negative control from the OD value measured for each test sample with the corresponding coated MAb. The results were interpreted as summarized in table 3 (Additional file 3).
The generated sequence data were aligned by multiple sequence alignments using the Clustal W algorithm as implemented in BioEdit and MEGA6 software packages to compare the VP1 of outbreaks sequences with other reference sequences retrieved from GenBank.
The aligned sequences were used to reconstruct phylogenetic tree using minimumevolution methods of analysis and maximum likelihood method imbedded in MEGA6 [39] and confidence levels were assessed by 1000 bootstrap replications. Serotypes were distinguished on the basis of nucleotide sequence differences of 30-50% and high bootstrap support (> 70%) while a divergence of 15% distinguishes topotypes [38]. Declarations analysis, interpreted the results, and prepared the manuscript. FD and BM assisted with refining the study design and critically reviewed the manuscript. DG, TB and DS assisted with sample collection and laboratory analysis. All authors read and approved the final manuscript.

Ethics approval and consent to participate
Ethical approval for this study was obtained from Addis Ababa University College of showing sign of the disease from willingness animal owner.

Consent for publication
Not applicable.