Transboundary movements of foot-and-mouth disease from India to Sri Lanka: A common pattern is shared by serotypes O and C.

Foot-and-mouth disease (FMD) affects the livestock industry in a transboundary manner. It is essential to understand the FMD phylodynamics to assist in the disease-eradication. FMD critically affects the Sri Lankan cattle industry causing substantial economic losses. Even though many studies have covered the serotyping and genotyping of FMD virus (FMDV) in Sri Lanka, there is a significant knowledge gap exists in understanding the FMDV phylodynamics in the country. In the present study, the VP1 genomic region of FMD viral isolates belonging to serotype C from Sri Lanka and other South Asian countries were sequenced. All the published VPI sequences of serotype C and most of the published VP1 sequences for lineage ME-SA/Ind-2001d of serotype O from Sri Lanka, India, and other South Asian countries were retrieved. The datasets of serotype C and serotype O were separately analyzed using Bayesian, maximum likelihood, and phylogenetic networking methods to infer the transboundary movements and evolutionary aspects of the FMDV incursions in Sri Lanka. A model-based approach was used to detect any possible recombination events of FMDV incursions. Our results revealed that the invasions of the topotype ASIA of serotype C and the lineage ME-SA/Ind-2001d have a similar pattern of transboundary movement and evolution. The haplotype networks and phylogenies developed in the present study confirmed that FMDV incursions in Sri Lanka mainly originate from the Indian subcontinent, remain quiet after migration, and then cause outbreaks in a subsequent year. Since there are no recombination events detected among the different viral strains across serotypes and topotypes, we can assume that the incursions tend to show the independent evolution compared to the ancestral viral populations. Thus, we highlight the need for thorough surveillance of cattle/ruminants and associated product-movement into Sri Lanka from other regions to prevent the transboundary movement of FMDV.

Introduction

Foot-and-mouth disease (FMD) is a highly contagious viral infection that shows significant transboundary movements [1, 2, 3]. Foot-and-mouth disease virus (FMDV) (genus Aphthovirus, family Picornaviridae) is a small positive-sense RNA virus [4, 5] that affects cloven-hooved ruminants, including cattle [6, 7, 8]. FMDV imposes a massive threat to the livestock industry, causing substantial economic losses [9, 10, 11, 12]. The understanding of the viral phylodynamics is one of the necessities in planning the measures for disease eradication. The knowledge of the demographic histories and tracking the transboundary movements of viral populations using genetic features provide detailed insights into the viral phylodynamics [13].

The VP1 genomic region of the FMDV is one of the common markers used in phylodynamic assessments [5]. The FMDV capsid proteins are encoded by four regions, including VP1 [3, 14, 15]. The protein coded by VP1 is a vital component in host cell attachment and entry [16] and used in characterizing FMDV [17]. Due to the association of type-specific mutations, the VP1 genomic region is commonly used in typing and subtyping of FMDV isolates [18, 19]. The viruses that are undergoing adaptive selection shapes the genetic features enabling the effective host-pathogen interactions [20]. Thus we can use the VP1 genomic region to track the transboundary movements and evolutionary aspects of FMDV.

Many studies suggest that the transboundary FMDV incursions play a dominant role in disease movement [1, 7, 18, 21, 22, 23, 24, 25]. Numerous FMD outbreaks of serotypes O, A and Asia 1 have originated within South Asia [26, 27, 28, 29, 21]. The heterogeneous distribution of FMD within India and the distribution of livestock and their products to neighboring countries lead to transmission of FMDV into the Middle East, South East Asia, and East Asia [30, 31, 32]. FMD has also spread from South Asia to North Africa on several occasions [2]. The characteristic patterns of the epizootic outbreak within a country is visible when introducing FMDV [33, 34, 35, 36]. Commonly, novel incursions often tend to replace old FMDV lineages within a country [37]. In most cases, the spread of FMDV within the country occurs by seasonal outbreaks or severe epidemics in subsequent years after spending quite periods [22]. The lineage expansion events due to recombination or specific mutations can give rise to pandemic situations if the recurrent lineage exists for an extended period [38].

FMDV exists in Sri Lanka since the 19th century [39]. FMD pandemics in Sri Lanka generally appear in every four to six years [40], although the evolutionary dynamics of the patterns of appearance remain ambiguous. To date, only FMDV serotypes O and C have caused outbreaks in Sri Lanka. The FMDV samples from the first outbreak (in 1950) were serotyped and identified as serotype O by a Danish Laboratory. The systemic surveys conducted from 1962 to 1967 and 1977 to 1981 revealed the presence of serotype O in Sri Lanka [41, 39]. Abeyratne et al. (2018) characterized the outbreaks during 1990–2014 [37]. The FMDV infections were reported during 1997–2014, where the numbers were higher in 1997, 1999, 2003, and 2014 [42]. Abeyratne et al. (2018) identified an endemic viral lineage (Srl-97) that caused three outbreak situations in 1997, 1999, and 2000 [38]. In 2014, a massive outbreak was recorded due to the Ind-2001d lineage, sweeping through all the provinces resulting in 58,645 infected cattle and 1,265 deaths [38, 42]. According to the analyses of VP1 and complete genome sequences, the outbreaks of Ind-2001d in Sri Lanka, which occurred in 2013 and 2014, were different from each other. However, FMDV lineages of the 2013 and 2014 outbreaks were closely related to the contemporary Indian FMDV indicating two independent-introductions [43].

FMDV serotype C was first identified in Sri Lanka in 1954 by serotyping of two samples at the Animal Virus Research Institute, Pirbright, England: however, these viral isolates are no longer available. Serotype C was thought to be accidentally introduced from India in 1970, which caused a massive outbreak in Sri Lanka till 1975 [44]. Despite the several attempts on serotyping and genotyping of the FMDV in Sri Lanka, none of the studies have carried out to understand the transboundary movements and the demographic histories of viral populations considering their genetic features.

Cattle farming is one of the most critical sources of living in the rural areas of Sri Lanka. The development of the cattle industry is a high priority in economic development [45]. FMDV is one of the significant obstacles in developing the cattle industry in Sri Lanka. One of the most effective ways of FMDV eradication in Sri Lanka is the prevention of the transmission of incursions into the country. Moreover, it is essential to understand the FMDV phylodynamics for disease management. Thus the objective of the present study was to find the transboundary movements and evolutionary dynamics of FMDV using genetic data of incursions belonging to O and C, the only serotypes reported in Sri Lanka to date.

Materials and methods

Sampling, RT-PCR and sequencing

The VP1 genomic regions of the viral samples collected during the outbreaks caused by FMDV serotype C in Sri Lanka, India, Bangladesh, Nepal, Bhutan, Saudi Arabia, Kuwait and, Tajikistan (former USSR) were sequenced. The vesicular epithelial tissue samples or vesicular fluids were collected from infected cattle. The recommended procedures and the ethical guidelines were followed, as described in Kitching and Donaldson, (1987), for the collection and transportation of the samples [46]. The sampling procedure from the infected cattle was approved by the ethics review committee of the Pirbright Institute under the Animal Scientific Procedures Act (ASPA). The field samples of the present study were submitted to the World Reference Laboratory for FMD (WRLFMD; The Pirbright Institute, UK) during the outbreaks. The total RNA was extracted using the RNeasy kit (Qiagen, Crawley, West Sussex, UK) according to the manufacturer's protocols. The RNA was eluted in nuclease-free water and stored at -20°C. Soon after RNA extraction, RT-PCR was performed by using the forward primers: C-1C536F 5′ TAC AGG GAT GGG TCT GTG TGT ACC 3′ and C-1C616F (5′ AAA GAC TTT GAG CTC CGG CTA CC 3′), each with the reverse primer: EUR-2B52R 5′ GAC ATG TCC TCC TGC ATC TGG TTG AT 3′ to amplify the VP1 genomic region employing the kits and protocols described in Knowles et al. (2016) [9] for RT-PCR. To confirm the product length of the amplicon, the agarose gel electrophoresis was carried out using the protocols described in Knowles et al. (2016) [9]. The DNA sequencing was carried out using BigDye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies) following the manufactures instructions. Each product was sequenced using the forward primer C-1C616F and the reverse primers, C-1D535R (5′ ARA GYT CIG CIC GYT TCA T 3′) and NK72 (5′ GAA GGG CCC AGG GTT GGA CTC 3′). Sequencing was performed using an ABI 3730 DNA analyzer following the instructions provided in Knowles et al. (2016) [9]. The sequences generated during the present study (n = 27) were submitted to GenBank under the accession numbers MK390941 to MK390966, KY091301, and KY091302.

Phylogenetic analysis

Two separate VP1 sequence datasets were constructed for the serotypes O and C. The dataset for serotype O contained most of the previously published FMD/O/ME-SA/Ind-2001 viral isolates from Sri Lanka and India (S1 Table). For the serotype C dataset, all the sequenced data generated in the present study and all the previously published sequences of FMD/C from India and Sri Lanka (S1 Table) were incorporated. For comparison, the sequence records from all the other topotypes of serotype C were added to make a robust phylogeny. Since the VP1 genomic region is a coding region, the datasets were manually aligned, and the gaps were added without disrupting the reading frame. The DNA sequence alignment was converted to an amino acid alignment to check for any stop codons in the middle using MEGA 7 [47]. According to the reading frame, the ambiguous ends of the analyzed sequences were trimmed.

The phylogenies for the two datasets were constructed separately following the same procedure. First, coding alignment was used to choose the best nucleotide substitution model that fits the dataset. To achieve a well-fitting model, the dataset was analyzed using multiple methods; namely, Akaike Informative Criteria (AIC) [48], Corrected Akaike Information Criteria (AICc) [49], Bayesian Information Criteria (BIC) [50] and Decision Theory (DT) [51]. The best model was selected using the J model test software [52] in CIPRES Science Gateway [53]. A tree search was implemented in maximum likelihood (ML) criteria in RAxML [54] using the rapid bootstrap algorithm [55] and GTR+GAMMA nucleotide substitution model [56]. The run was iterated for 1000 times and all the bipartitions were used to draw a single topology. Then the bootstrap values were imprinted from bipartition results to the best tree given by RAxML tree search.

Moreover, a Bayesian tree search was carried out in MrBayes [57]. The evolutionary model parameters were incorporated and two hot and cold chains of MCMC were run for 10 million generations. The analysis was set to discard the initial 10% of the trees as the burn-in. From all the trees probed during tree search, the 50% majority-rule consensus tree was taken as the final tree output. To check the robustness of the chain performance and to probe the trees from a stationary distribution, the Effective Sample Size (ESS) was tested using TRACER [58]. All the constructed trees were modified using FigTree [59] for better visualization.

Construction of haplotype network

The Sri Lankan and Indian FMDV isolates were used to determine the haplotype relationships. The input nexus file was modified to assign the trait labels with respective geographic origins. A Minimum spanning network, Median-joining network [60] and Templeton, Crandall, and Sing (TCS) [61] were constructed separately for both datasets in PopART [62]. The haplotype network with the highest clarity was used to represent the haplotype relationships.

Detection of possible recombination events

Although a haplotype network can detect particular genetic events, the identification of specific recombination events is difficult. Since the FMDV is an RNA virus, it could undergo recombination to produce novel lineages. Potential recombination events within VP1 were checked using four independent datasets: i) serotype C sequences from India and Sri Lanka (n = 15); ii) serotype C sequences from Sri Lanka and sequences of O/ME-SA/Srl-97 (n = 24); iii) serotype C sequences from Sri Lanka and sequences of O/ME-SA/Ind-2001d from Sri Lanka (n = 22); and iv) O/ME-SA/Ind-2001d sequences from Sri Lanka and India (n = 150) (S1 Table). The Genetic Algorithm for Recombination Detection (GARD) approach [63] was implemented in Datamonkey 2.0 platform [64]. By separately inserting each dataset, the possible recombination events were checked in different serotype/lineage combinations assessed.

Results

The TrN+I+G model was obtained as the best fit to describe the dataset of FMD/O and TPM1uf+I+G as the best model for FMD/ C dataset (S2 Table). All four criteria used (AIC, BIC, AICc, and DT) yielded the same models separately for two datasets indicating the higher fitness of the models.

The phylogenies created using ML and Bayesian frameworks had almost similar topologies for the two datasets. However, the Bayesian trees had higher resolution than ML trees. The MCMC chains had maximally converged and gave >200 ESS values for all the priors checked. Thus only the Bayesian trees are presented. In the serotype C phylogeny, all the Sri Lankan FMDV isolates that we sequenced and acquired from GenBank fell into a monophyletic group (high bs and pp values) (Fig 1). The south Asian FMDV C strains we sequenced including most of the Indian isolates formed a sister group with the Sri Lankan clade in the Bayesian tree of serotype C. The Bayesian tree constructed for FMDV O/Ind-2001 isolates contained a monophyletic group with Sri Lankan FMDV isolates with higher node support (Fig 2). The GARD analysis did not detect any recombination event within any of the four datasets tested. However, DNA and amino acid sequences analysis detected phylogenetically informative SNPs between Sri Lankan and Indian FMDV.

Fig 1

The Bayesian 50% majority rule consensus tree drawn for the VP1 sequences of the serotype C.

The minimum spanning network drawn for Sri Lankan and Indian isolates are given next to the phylogenetic tree. The tip labels in the tree indicate the haplotypic origin of the sequences (Red: Sri Lanka; Yellow: India; Blue: Bhutan; Orange: Nepal; Green: Bangladesh; Purple: Middle East). Between each haplotype, the distance is given as number of mutations. The scale of the tree is given as substitution per site. In the phylogenetic tree, the node supports are indicated in each node (gray dots: PP>90; white dotes with black outline: bs>70; black dots: PP>90 and bs>70).

Fig 2

A clade of the 50% majority rule consensus tree drawn in the Bayesian framework for FMD/O isolates.

We have inserted the median-joining network drawn for Sri Lanka of Indian FMD/O/Ind-2001 strains next to the phylogenetic tree. The tip labels indicate the haplotype origin of the sequences (Red: Sri Lanka; Yellow: India). In the phylogenetic tree, the node supports are indicated in each node (gray dots: PP>90; white dotes with black outline: bs>70; black dots: PP>90 and bs>70). The complete phylogenetic tree is given in S1 Fig. Between each haplotype, the distance is given as number of mutations. The scale of the tree is given as substitution per site.

Haplotype diversity

The construction of the phylogenetic network provides insights into the relationships between different haplogroups and their movements (i.e., gene flow). Since the virus is more prone to events such as recombination and rapid evolution, it is essential to display the broad picture using the network topologies. The minimum spanning network represents the relationships between Indian and Sri Lankan serotype C viral isolates more clearly than other networks. Due to the availability of a limited number of samples and the fact that FMDV serotype C has not been detected since 2004, the complete haplotypic diversity and the epidemiological patterns in South Asia could not be deduced. However, the gene flow pattern and the direction of the transmission could be recognized via a constructed haplotype network. In the present study, all the published FMD/C sequences in the South Asian region were assessed. For comparison, the FMD/C sequences from the Middle East were used. With these sequences, 38 haplotypes were obtained out of the 40 sequences analyzed. Only shared haplotypes were observed within Sri Lanka and India. We detected 167 parsimony information sites, and the haplotype network had Tajima’s D value of 7.759. The D>0 indicates the presence of rare alleles at low frequencies. Thus it is possible to have independent evolutions in Sri Lankan and Indian FMDV populations. We obtained a star topology for the haplotype network separating Sri Lankan and Indian groups with no shared haplotypes. A unique haplotype sampled in India (C/IND/6/71) was the closest nested haplotype with Sri Lankan haplogroup, indicating the possible introduction event from India to Sri Lanka.

The median-joining network with ɛ = 0 was the best-describing haplotype network with higher clarity for FMD/O/Ind-2001 dataset. With 150 sequences used, we obtained 107 haplotypes, and Sri Lankan and Indian strains did not share common haplotypes. The Sri Lankan haplotypes nested within one haplogroup having a star topology. We observed four more nested haplogroups having the same topology. The Tajima’s D value was below 0 (D = -1. 164) for the median-joining network indicating a recent event of population expansion. The distance between haplogroups suggested that the expansion events might be due to recent bottleneck effects or significant changes in the VP1 genomic region.

Discussion

The VP1 genomic region is one of the widely employed markers in phylogeographic and evolutionary studies of FMDV [5, 19, 65, 6]. Based on VP1 sequences, FMD/C viruses fall into three topotypes; viz. Europe/South America, Asia, and Africa (Fig 1). In the present study, we looked at the evolutionary and phylogeographic perspectives of the VP1 genomic region of FMDV that caused outbreaks in Sri Lanka and India. We checked the transboundary movements and underlined evolutionary aspects of the viral outbreaks of Sri Lanka that caused by FMDV serotypes O and C.

In addition to serotype O, which caused several outbreaks in Sri Lanka, three studies reported the existence of the serotype C within the country [8, 40, 44]. For the first time, the present study reveals the dynamics of serotype C virus prevalent in Sri Lanka. All the Sri Lankan viral isolates of FMD/C fell into a single cluster, and they, along with all the South Asian and the Middle East viral strains, fell into the ASIA topotype. However, one of the Indian isolates (C/IND/6/71, collected on 22/09/1970) clustered with the Sri Lankan viruses. According to the nucleotide sequence of the VP1 protein-coding region, there were unique nucleotide substitutions shared by both Sri Lankan isolates and C/IND/6/71. We identified three such nucleotide substitutions at the positions of 255, 408, and 486 (A255G, C408T, T486C) located within the coding region of DE loop, GH loop and β sheet, respectively. Furthermore, deduced amino acid analysis showed the substitution of T151A situated within the GH loop in IND/6/71, which is also shared by Sri Lankan viral isolates (S3 Table). The other Indian viral strains did not share the specific nucleotide substitutions of the Sri Lankan isolates and C/IND/6/71.

The Sri Lankan serotype C sequences were nested into one haplogroup with geographically unique haplotypes. The most closely nested haplotypes for Sri Lankan haplogroup were the Indian haplotypes. The haplotype network had a star topology, and a unique haplotype (C/IND/7/76) was nested as the core haplotype (Fig 1). This pattern reveals the diversification of FMD in South Asia could have started in India. Also, the incursions of FMDV/C in Sri Lanka must have originated from India. The haplotypic relationships indicate that all the FMD incursions in South Asia originated from India and dispersed throughout the region.

In the present study, we did not find any possible recombination of Sri Lankan FMDV. Thus it is possible that the Sri Lankan cluster could have originated from India, where the rapid evolution of Sri Lankan viral isolates formed a genetically distinct form compared to Indian strains through independent evolution. The Sri Lankan viral strains separate into two clades, in which one clade represents the samples collected during 1971–1978, whereas the other clade represents the viral isolates collected during 1984 (Fig 1). These two events could be the lineage expansion events from independently evolving viral strains in Sri Lanka. Our haplotype network also shows that there was no introduction event. Furthermore, the comparison of the nucleotide sequence revealed three nucleotide substitutions at the positions of 33, 39, and 69 (T33C, T39C and A69G) located within the N-terminus. Two nucleotide substitutions at the locations of 409 and 450 (G409A, A450G) located within the coding region of GH loop shared among all Sri Lankan viral isolates found during the 1971–1978 outbreak but not shared with the viral strains collected in 1984 (S3 Table) supporting the fact that the separation could be due to lineage expansion.

Although there have been multiple FMD epidemic situations in Sri Lanka, only a few attempts have been made to characterize the viruses genetically. The sequence data are only available for outbreaks from 1990–2014. Abeyratne et al. (2018) [38] revealed most of the outbreak situations occurred before 2013 due to O/ME-SA/Srl-97 lineage, which is endemic to Sri Lanka. The study showed that Srl-97 endemic lineage is directly monophyletic to “Pak-98” lineage. Therefore, due to lack of data, the linear transboundary movements of the common ancestor of Srl-97 and Pak-98 cannot be deciphered. Moreover, our GARD analysis did not show any recombination event between FMD/C viral isolates and Srl-97 lineage. Thus it is possible that the Srl-97 lineage underwent independent evolution within Sri Lanka to acquire a unique set of mutations to circulate within the country.

Our analysis of O/ME-SA/Ind-2001 viral isolates of Sri Lanka and India shows a congruent pattern of diversification to serotype C viral isolates. Although the separation in the phylogeny of different groups of strains is low, the median-joining network displayed the grouping of viral strains (Fig 2). The haplotype network shows the diversification patterns of the Ind-2001d sub-lineage. The nested haplogroup of Ind-2001d isolates collected in Sri Lanka was closely related (only four mutations observed) to the haplogroup nested with Ind-2001d isolates collected in India during 2013. Remarkably two isolates collected in Sri Lanka in late 2013 (O/SRL/1/2013 and O/SRL/2/2013) also clustered with this Indian haplogroup. It is likely that O/SRL/1/2013 and O/SRL/2/2013 are closely related to FMD viruses that moved into Sri Lanka from India (Fig 2) [43]. The comparison of nucleotide sequence analysis of the VP1 coding region of viral isolates also supported the proposed hypothesis. We found nucleotide substitution at the position of 78 (G78A) located within the coding region of N-terminus and three nucleotide substitutions at 471st, 480th and 492nd positions (G471A, T480C, T492C) within the GH loop shared in Indian viral isolates and O/SRL/1/2013 and O/SRL/2/2013 (S4 Table).

Similarly, O/IND26(54)/2014 formed a unique haplotype that clustered with the Sri Lankan haplogroup suggesting possible limited spread back to India from Sri Lanka. However, it is evident that viruses introduced into Sri Lanka in 2013 and 2014 evolved independently from the ancestral populations (Indian virus) and became enzootic in Sri Lanka. The star topology of the Sri Lankan haplogroup, as well as Indian haplogroup, inferred a rapid expansion of two viral populations. Since the core haplotypes of two disease epidemics are well separated, we can assume that the viruses evolve independently from each other after the incursions occurred.

A phylogeographic study based on the transboundary movement of Malaysia and surrounding countries showed the patterns of FMD movement across the region [22]. The FMDV incursions occurred either annually with quick eradication or introduced virus caused outbreak situation in the subsequent year of the invasion. The latter pattern is much similar to the situation in Sri Lanka and India. From our phylogenetic analysis, it is visible that in serotype C, the disease could have introduced to Sri Lanka in 1971, where the outbreak situation persisted until 1984. During this period, a couple of lineage expansion events were observed, indicating two disease epidemic situations. The lineage expansion could be mainly due to the independently evolving nature of the introduced FMDV in Sri Lanka. Similarly, in serotype O, the virus could have been introduced to Sri Lanka in 2013, although the massive epizootic outbreak started in 2014 and continued throughout the year.

Conclusion

In this study, a molecular-systematic approach was used to decipher the transboundary movements and the evolutionary aspects of FMDV serotypes O and C into Sri Lanka. It is evident that the FMDV incursions in Sri Lanka were mainly originated from the Indian subcontinent and remained to cause outbreaks in a subsequent year. Moreover, the introduced FMDV strains tend to show independent evolutions from the ancestral populations, which may complicate the disease eradication. Thus we emphasize the need for policies and surveillance programs to stop the illegal cattle movement into Sri Lanka.

The Bayesian 50% majority rule consensus tree drawn for FMD VP1 sequence of FMDV O isolates from Sri Lanka and India.

The Black dots indicate the nodes with PP>90 and bs>70. The grey dots indicate the node with PP>90. The white dots with black outlines indicate the nodes with bs<70.

(TIF)

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The DNA sequences assessed in the present study.

(XLSX)

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The nucleotide substitution model parameters for serotype O and C datasets we used in this study.

(XLSX)

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The nucleotide and amino acid alignments of the serotype C VP1 sequences from Sri Lanka and India.

(XLSX)

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The nucleotide and amino acid alignments of the FMDV O/ME-SA/Ind-2001d VP1 sequences from Sri Lanka and India.

(XLSX)

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7 Nov 2019

PONE-D-19-15471

Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C

PLOS ONE

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Reviewer #1: The paper “Transboundary movements of foot-and Mouth disease from India to Sri Lankan: a common pattern is shared by serotypes O and C” present the sequencing data of foot-and Mouth disease virus VP1 region in order to evaluate the phylodynamics of serotypes O and C.

The data present in this study seems to be interesting in knowing the movement of FMD from the Indian subcontinent to Sri Lanka. Authors extended their study to understanding of genetic variability associated with FMDV VP 1 region to emphasize the effects of this virus on cattle and its impact on associated product movement into Sri Lanka.

During his study authors are mainly focused on retrieving literature data on FMDV and combined several new sequences of serotype C. Majority of those sequences are included Sri Lankan and Indian virus sequences. To infer thee evolutionary and transboundary aspects they used few major software and applications, while the model-based approach is used to detect possible recombination.

However, some issues related to the article should be solved.

Present study authors indicated that they have sequenced viral samples from serotype C. However they haven’t justified why they used only serotype C instead of using both serotypes O and C.

Describing of the sample selection procedure is unclear. Authors are stated that they have used already collected viral samples. But they were not clearly stated how the present study sample is selected. Original sample number and present study cohort selection should be clearly described in the method section. In the present study, authors are newly generated only t 27 sequences of serotype C. Hence clear understand of the sample numbers can justify the present study.

Authors need to expand the description of the structure & phylogenetic significance of VP1 region since that is the major interest of their study.

It will be informative that the authors can include the length of the PCR amplicons and the sequences they used to present study. Shorter the sequence length reduced the precision of the analysis.

It is understandable if the authors may clearly indicate the number of sequences belongs to each dataset that they used to detect possible recombination events (From line #’s 221 to 224)

Some of the references listed in the reference section are not aligned with the journal recommended “Vancouver” reference style

Minor issues

Reference style in some points of the introduction should be revised and corrected

Eg: Line # 106: Reference list should be rearranged

Improving written language style is recommended since some language parts of the article are unclear

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Reviewer #1: Yes: Dr Ruwandi Ranasinghe

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11 Nov 2019

November 11, 2019

Editor,

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Dear Editor,

Rebuttal Letter Containing Comments and Answers for the manuscript titled “Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C”

I wish to pay my sincere thanks for reviewing and taking necessary actions to review the earlier submitted manuscript titled “Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C”. I appreciate your valuable effort in this regard to point out areas to improve and strengths in our study which are helpful in improving the quality of the article. This article is of immense importance to the betterment of the Sri Lankan cattle industry. Therefore, with your valuable suggestions, we wish to provide convey our research findings to the FMD research community.

Herewith I am sending the details of actions, answers and comments for each query indicated by the reviewers/editor.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Answer: The suggested changes were included in revised form of the article.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Answer: Thank you for the comment.

3. Have the authors made all data underlying the findings in their manuscript fully available?

medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Answer: Thank you for the comment.

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Answer: The article was edited to correct the language mistakes.

Review Comments to the Author

Reviewer #1

Comment 1: The data present in this study seems to be interesting in knowing the movement of FMD from the Indian subcontinent to Sri Lanka. Authors extended their study to understanding of genetic variability associated with FMDV VP 1 region to emphasize the effects of this virus on cattle and its impact on associated product movement into Sri Lanka. During his study authors are mainly focused on retrieving literature data on FMDV and combined several new sequences of serotype C. Majority of those sequences are included Sri Lankan and Indian virus sequences. To infer thee evolutionary and transboundary aspects they used few major software and applications, while the model-based approach is used to detect possible recombination.

Answer: Thanks for identifying the importance and strengths of this work.

Comment 2: However, some issues related to the article should be solved. Present study authors indicated that they have sequenced viral samples from serotype C. However, they haven’t justified why they used only serotype C instead of using both serotypes O and C.

Answer: Thank you very much for your comment. I believe a clarification is needed for this question. We wanted to cover FMDV incursions associated with both serotype O and serotype C since those are the only reported serotypes to carry out an FMD outbreak situation in Sri Lanka. (Introduction: Lines 121-122, 149 -150). The sequence data for FMDV Serotype O viral samples collected in Sri Lanka were publicly available and included in the analysis (Materials and methods: lines 180 - 181). Although FMDV epidemic situations are reported for Serotype C, the sequence data were not publicly available. Thus, we collected and sequenced the Serotype C data, in order to collectively analyze the viral phylodynamic for each incursion, as we wished to present a completed and more robust scientific story.

Comment 3: Describing of the sample selection procedure is unclear. Authors are stated that they have used already collected viral samples. But they were not clearly stated how the present study sample is selected. Original sample number and present study cohort selection should be clearly described in the method section.

Answer: Thanks for identifying this. We have added “We sequenced the viral samples that were collected during the outbreak situations caused by FMDV serotype C in Sri Lanka, India and, a few associated countries”.

Comment 4: In the present study, authors are newly generated only 27 sequences of serotype C. Hence a clear understand of the sample numbers can justify the present study.

Answer: We used sequences of all the relevant FMDV serotype C isolates submitted WRLFMD by the sample collectors. The number of samples for serotype C was turned out be 27. There were no other relevant sequences of serotype C available to have a larger sample size.

Comment 5: Authors need to expand the description of the structure & phylogenetic significance of VP1 region since that is the major interest of their study.

Answer: Thanks for the suggestion. However, I believe the Paragraph 2 of the Introduction justifies the usage of VP1 genomic region and its importance in FMDV phylogenetics.

Comment 6: It will be informative that the authors can include the length of the PCR amplicons and the sequences they used to present study. Shorter the sequence length reduced the precision of the analysis.

Answer: Thank for pointing this out and making the valuable suggestion. We have remodified the S1 Table including the sequence-lengths.

Comment 7: It is understandable if the authors may clearly indicate the number of sequences belongs to each dataset that they used to detect possible recombination events (From line #’s 221 to 224).

Answer: Thank you for valuable suggestion. The text in the particular section is corrected accordingly as given below.

“i) serotype C sequences from India and Sri Lanka (n= 15); ii) serotype C sequences from Sri Lanka and sequences of O/ME-SA/Srl-97 (n = 24) ; iii) serotype C sequences from Sri Lanka and sequences of O/ME-SA/Ind-2001d from Sri Lanka (n = 22); and iv) O/ME-SA/Ind-2001d sequences from Sri Lanka and India (n = 150)”.

Comment 8: Some of the references listed in the reference section are not aligned with the journal recommended “Vancouver” reference style.

Answer: Thanks for the comment and the references have been double checked to meet the journal style.

Minor issues

Comment: Reference style in some points of the introduction should be revised and corrected.

Eg: Line # 106: Reference list should be rearranged.

Answer: Thanks, corrected accordingly.

Comment: Improving written language style is recommended since some language parts of the article are unclear.

Answer: Thank you for the suggestion. The relevant changes have been made.

Thank you very much for your consideration and I look forward to hearing back from you with the next step of the publication process.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

5 Dec 2019

PONE-D-19-15471R1

Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C

PLOS ONE

Dear Prof. Sooriyapathirana,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address the few minor comments listed below.

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We look forward to receiving your revised manuscript.

Kind regards,

Douglas Gladue, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

Please consider the following suggestions

1) Line # 157: Please indicates what are the "Nearby" Countries

2) Material and Method section: Better rephrase with Passive voice

3)Line # 182: Indicate which type of data set that you used

4)Line # 's 223- 226: Better indicate the individual sequence numbers for each country/Category

5)Line #: Duplication of "Viral"

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please consider the following suggestions

1) Line # 157: Please indicates what are the "Nearby" Countries

2) Material and Method section: Better rephrase with Passive voice

3)Line # 182:  Indicate which type of data set that you used

4)Line # 's 223- 226:  Better indicate the individual sequence numbers for each country/Category

5)Line #: Duplication of "Viral"

**********

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr Ruwandi Ranasinghe

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6 Dec 2019

Prof. S.D.S.S. Sooriyapathirana

Corresponding Author

December 6, 2019

Editor

PLoS ONE

Dear Editor,

Submission of the Revised Version of PONE-D-19-15471R1: Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C

We wish to pay our sincere thanks for reviewing the manuscript. Herewith we explain the answers and activities for the queries made by Editor and Reviewer.

Editor’s Comments (EC):

EC1: Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Answer: Thank you very much for the revisions and herewith we submit the revised version.

EC2: Please address the few minor comments listed below.

Please consider the following suggestions

1) Line # 157: Please indicates what are the "Nearby" Countries

Answer: The ‘Nearby’ countries were included separately in the sentence.

2) Material and Method section: Better rephrase with Passive voice

Answer: The Materials and Methods section was rephrased to passive voice.

3)Line # 182: Indicate which type of data set that you used

Answer: VP1 sequence datasets were used. The sentence was amended to make it clear.

4)Line # 's 223- 226: Better indicate the individual sequence numbers for each country/Category

Answer: We appreciate this comment. The no. of sequences for each country can be easily taken from S1 Table. If we prepare a summary, it would be like the Table given below. Therefore, under the editors permission we wish to keep the current form and guide the readers to S1 Table for sampling details.

Country No. of sequences

Argentina 12

Austria 1

Bangladesh 2

Belgium 3

Bhutan 2

Brazil 6

Denmark 1

Ethiopia 2

France 4

Germany 3

Greece 1

Hungary 1

India 108

Italy 1

Kenya 3

Kuwait 1

Nepal 5

Philipppines 1

Portugal 1

Saudi Arabia 2

Spain 2

Sri Lanka (Ceylon) 28

Switzerland 2

Uganda 1

United Kingdom 2

Uruguay 1

Tajikistan (USSR) 1

5)Line #: Duplication of “Viral”

Answer: We guess the line # has to be 242. The sentence was modified to accommodate the correction.

Reviewers' comments:

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Answer: Thank you!

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Answer: Thank you!

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Answer: Thank you!

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Answer: Thank you!

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Answer: The necessary corrections were made, and the Materials and Methods section was changed to passive voice.

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please consider the following suggestions

1) Line # 157: Please indicates what are the "Nearby" Countries

2) Material and Method section: Better rephrase with Passive voice

3)Line # 182: Indicate which type of data set that you used

4)Line # 's 223- 226: Better indicate the individual sequence numbers for each country/Category

5)Line #: Duplication of "Viral"

Answer: All these suggestions were addressed before.

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr Ruwandi Ranasinghe

Answer: Thank you! We would also prefer to publish peer review history according to the journal guidelines.

Again we wish to thank the Editor, Reviewer and PLoS ONE staff for your excellent service. I look forward to hearing back from you with the next step of publication.

Thank you!

Sincerely

Prof. S.D.S.S. Sooriyapathirana

Corresponding Author

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

13 Dec 2019

Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C

PONE-D-19-15471R2

Dear Dr. Sooriyapathirana,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Douglas Gladue, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

17 Dec 2019

PONE-D-19-15471R2

Transboundary movements of foot-and-mouth disease from India to Sri Lanka: a common pattern is shared by serotypes O and C

Dear Dr. Sooriyapathirana:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Douglas Gladue

Academic Editor

PLOS ONE