Emergence of Lineage IV Peste des Petits Ruminants Virus in Ethiopia: Complete Genome Sequence of an Ethiopian Isolate 2010

to the epidemiology of PPRV with a focus on the emergence of different of


Introduction
Different morbilliviruses are the cause of numerous clinically devastating diseases including measles in humans, canine distemper in dogs, rinderpest in large ruminants and peste des petits ruminants (PPR) in small ruminants and camelids. With the eradication of rinderpest focus has shifted onto PPR and it is increasingly regarded as an important viral disease of domestic ruminants that is a significant burden to the agricultural sectors in endemic areas (Munir, 2013). Currently, PPRV is endemic in Africa, the Middle East and across much of Asia (Banyard et al., 2010). The detection of PPRV or antibodies that denote natural infection has been reported from almost all African countries with the exception of vast territories across Southern Africa . For some time, it has been clear that at least one lineage of PPR virus, lineage III, is present within East Africa having been reported from Ethiopia, Tanzania, Uganda and Sudan. Interestingly, lineage IV PPRV has also been reported in Sudan (Banyard et al., 2010;Kwiatek et al., 2011), in Egypt (Sharawi and Abd El-Rahim, 2010) and in Eritrea (Cosseddu et al., 2013). However, circulation of lineage IV PPRV in Ethiopia is not known.
Ethiopia has over 62 million PPR-susceptible livestock (sheep and goats) and has the third largest agricultural sector across Africa. Ethiopia also ranks eighth in the world in terms of small ruminant populations, playing an economically important role in the livelihood of resource-poor farmers. PPR was first suspected in Ethiopia in 1977 following clinical observations consistent with infection with PPRV (Pegram and Tereke, 1981) and was later diagnosed as the causative agent of disease in goats in the country (Roeder et al., 1994). The virus detected in 1994, alongside a further isolate reported in 1996 was genetically determined to cluster within lineage III (Kwiatek et al., 2007;Banyard et al., 2010). Although PPR is endemic in Ethiopia, the circulation of lineage IV PPRV has not previously been reported. Therefore, the main aim of this study was to identify whether lineage IV virus is circulating in Ethiopia. Further, as there is no PPRV full genome sequence available from East African regions, we have sequenced the full genome of the Ethiopian virus and have made comparisons with existing available PPRV genetic data. The isolate reported here (KJ867541; Ethiopia/2010) is the first report of lineage IV in Ethiopia and is the first complete PPRV genome to be characterized from an outbreak in East Africa. Further, we have performed a full phylogenetic assessment of African PPRV isolates using both full genome sequences and partial genes (N and F) sequences available in the GenBank database.

Materials and Methods
Outbreak and virus A PPR outbreak was reported at the National Veterinary Institute (NVI) at Addis Ababa, Ethiopia following the procurement of 50 male goats aged 6-8 months from Debre Zeit market showing no signs of ill health. Within 2 weeks of animal purchase, the goats developed clinical disease consistent with PPRV infection. Numerous clinical samples were collected at post-mortem for confirmatory diagnosis. The RNA for this PPRV was recovered from an intestinal tissue collected on 13th of August 2010 and sent to The Pirbright Institute and as such the virus had not been subjected to selective pressures that may cause cell culture adaptation.

Complete genome sequencing
Viral RNA was extracted using Qiagen Viral RNA mini kit (Hilden, Germany), and the SuperScriptTM III One-Step RT-PCR System with Platinum â Taq High Fidelity poly-merase (Life Technologies, Paisley, UK) was used for the reverse transcription of viral RNA into cDNA and further amplification in a single steps. PPRV specific primers were used to generate twelve overlapping amplicons covering the entire genome of the isolate. Amplicons were gel purified and sequenced using an ABI-3730 automated sequencer (Applied-Biosystems, Foster City, CA, USA) as described previously (Muniraju et al., 2013). The genome termini were determined using 3 0 /5 0 RACE (Li et al., 2010). A total of 525 sequences were assembled into overlapping contigs that represented the full genome (Lasergene v. 10.1, Madison, WI, USA), with an average of 12.6-fold coverage at each nucleotide position.

Sequence analysis
The complete genome sequence of PPRV Ethiopia 2010 (GenBank accession no. KJ867541) generated in this study was compared and phylogenetically analysed with other eight PPRV complete genomes available in the NCBI database. Similarly, the partial N and F gene sequences extracted from the PPRV Ethiopia 2010 in this study were compared and phylogenetically analysed with other available PPRV partial N/F gene sequences from African isolates.
The PPRV partial N gene sequence data of 255 nucleotides (genome position 1360-1614) or partial F gene sequence data of 322 nucleotide (genome position 5779-6100) sequences that have a detailed history of collection date and place were retrieved from the NCBI database (including submission to July 2014). These sequences were aligned using the ClustalW algorithm incorporated in Bioedit software v7.2.0 (Hall, 1999) and edited to remove unreliable sequences/regions. Further, the identical sequences originating from the same geographical location, host and year were excluded to avoid redundancy in phylogenetic analysis. The partial N gene data set contained 83 sequences sampled in Africa over a period of 46 years , and the partial F gene data set contained 38 sequences sampled in Africa over a period of 40 years . Phylogenetic analyses were performed using MEGA6 (Tamura et al., 2013). The neighbour-joining tree was computed using Kimura 2-parameter model. The test of phylogeny was carried out through bootstrap method with 1000 number of replications and the gaps/missing data treated with pairwise deletion.

Results and Discussion
The total genome size of Ethiopia/2010 was identical to that of previously published PPRV isolates, 15 948 nucleotides. As expected, the genome was organized as described for all other isolates of PPRV and included: the nucleocapsid (N), phospho (P/C/V), matrix (M), fusion (F), haemagglutinin (H) and the large polymerase (L) proteins. All untranslated regions including the genome and anti-genome promoter regions, gene start and stop sequences and intergenic trinucleotides were present as expected. Interestingly, this isolate clusters with lineage IV isolates of PPRV ( Fig. 1) whilst all previous isolates from Ethiopia (1994 and1996) have been phylogenetically typed as lineage III viruses Fig. 2 (Kwiatek et al., 2007;Banyard et al., 2010). We have *Lineages of PPRV isolates were named by following the partial N gene sequence phylogenetic analysis. recently reported a full genome sequence of lineage IV PPRV from Morocco (Muniraju et al., 2013) (Banyard et al., 2010;Kwiatek et al., 2011;Libeau et al., 2014). Lineage IV PPRV had also been recorded from Cameroon in 1997, the Central African Republic (CAR) in 2004 and in Nigeria in 2008 (Fig. 2). Multiple lineages have also been reported as being present on several other African countries such as Burkina Faso, Senegal and Nigeria (Table 1). In Ethiopia lineage III has been detected since 1994 along with lineage IV PPRV that has been record in 2010 in this study. As both lineage III (1971 isolate) and Lineage IV (2000 isolate) have been reported since 1971 and 2000, respectively in neighbouring Sudan, the presence of lineage IV virus in Ethiopia earlier than 2010 cannot be ruled out.
A neighbour-joining tree was constructed using 83 partial N gene sequences (255 nucleotide) from PPRV isolates reported across Africa (Fig. 2). The sequences available from 1968 to 2013 divided phylogenetically into four lineages. Lineage I was limited to Western Africa and has not been reported since 1994 (Batten et al., 2011). Lineage II is circulating in Western Africa (Nigeria, Ghana, Sierra Leone and Mali) having been reported sporadically in Nigeria, Ghana, Senegal, Sierra Leone, Mali andMauritania between 1975 and. Lineage III appears to be restricted to East Africa since initially being reported in Sudan in 1971, Ethiopia in 1994and most recently in Uganda (2007 and Tanzania (2010 and 2013, Fig. 2) (Banyard et al., 2010). Within Africa, lineage IV was first reported in Cameroon in HQ131950_Sudan_Atbara-NSUD05-3_Camel_2005 HQ131949_Sudan_Atbara-NSUD05-1_Camel_2005 HQ131951_Sudan_Atbara-NSUD05-2_Camel_2005 HQ131953_Sudan_Gedarif-KSUD08_Sheep_2008 HQ131955_Sudan_Bashagra-Gezira-BNSUD08_Sheep_2008 HQ131954_Sudan_Rabak-BNSUD09_Goat_2009 HQ131959_Sudan_EdDamar-NSUD08_Sheep_2008 As Lineage IV virus has been reported from East-, North-, West-and Central Africa, it may also be the cause of disease in other areas where recent outbreaks have been reported (Table 1) without genetic characterization. Further, it is clear that more than one lineage is circulating in some of the African countries, particularly in East Africa in Sudan (Lineage III and IV), Ethiopia (Lineage III and IV) and Uganda (Lineage III and IV) and in West Africa in Nigeria lineage II and IV (Table 1). Although Lineage I and II both were reported in Senegal and Burkina Faso, currently only lineage II appears to be present in Senegal. Lineage I PPRV (Ivory Coast/1989) has not been seen in Africa or elsewhere since 1994 (Batten et al., 2011). However, by analysing partial F gene sequence data, Luka et al. (2012) reported the circulation of Lineage I, II and IV in Uganda during outbreaks in 2007 and 2008. Using the existing data and that reported here a reanalysis of these data have shown that the sequences reported in 2007 and 2008 belong to lineage III and IV (Fig. 3). This also corroborates well with the one virus sequence reported in 2007 from Uganda as lineage III (Banyard et al., 2010). Two viruses (Ugn-LF1-07 HQ407501 and Ugn-LF3-07 HQ407502) reported as lineage II by Luka et al. (2012) grouped with viruses from lineage III in the present analysis. Similarly, three viruses (Ugn-14-08 HQ407497, Ugn-18-09 HQ407499 and Ugn-16-09 HQ407498) phylogenetically classified as belonging to lineage I by Luka et al. (2012) clustered with isolates from lineage IV in the present analysis. From the neighbour-joining tree of the available N gene sequence data for PPRV (Fig. 2), it appears that lineage IV PPRV has become the dominant strain circulating in many regions. From the phylogenetic analysis, lineage IV appears to differentiate into four clusters. Ethiopia 2010 (reported in this study) belongs to cluster one (C1) along with East and North African viruses from Sudan (2004Sudan ( -2009 (2000), and cluster four includes isolates from Cameroon (1997) and a single isolate from the Sudan (2000).
Comparing between the nucleotide sequence similarity of partial N gene sequence data of isolates from C1 viruses with Ethiopia 2010, 99.2% similarity was found with many viruses from Sudan and only one virus (2005) from Eritrea whereas 93.7-98.8% nucleotide similarity was seen for other C1 viruses (Table 2). From this analysis, it suggests that the Ethiopia 2010 outbreak may be due to the trans- boundary spread of PPRV between the neighbouring countries. This can occur readily in areas where land borders are ill defined, and the movement of animals between countries is unrestricted.
In conclusion, from the available gene sequences including the data generated in this study, only lineage III and IV are circulating in East Africa, lineage I (Ivory Coast/1998) appears restricted to western Africa and currently not in circulation, lineage II (Nigeria/ 1975) appears limited to western and central Africa with recent incursions into the western part of North Africa. Further, lineage IV PPRV appears to have extended its distribution following initial detection in Africa in 1997. Furthermore, in the absence of reports of the presence of other lineages it appears to have emerged as a dominant lineage of the virus, being detected in numerous regions where historically other lineages have predominated. However, limitations exist with our understanding of the epidemiology of PPRV. Further, genetic analyses are required to understand the epidemiology of the virus across Africa as although the eradication of rinderpest has led to a renewed interest in PPRV, the ability to genetically type viruses is still lacking in many regions where the virus remains endemic. As such where genetic data do exist, such information is likely to merely represent a snapshot of the true situation regarding this virus and its distribution across the developing world.