189 Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 Accepted: 15.04.2021 | Available on line: 31.12.2021 1Department of Veterinary Medicine, University of Nigeria Nsukka, Enugu State, Nigeria. 2Istituto Zooprofilattico Sperimentale della Sicilia ‘A. Mirri’, Palermo, Italy. 3Department of Animal Health, Federal College of Animal Health and Production Technology, National Veterinary Research Institute Vom, Plateau State, Nigeria. 4Transboundary Animal Disease Laboratory, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa. 5Applied Biotechnology Division, National Veterinary Research Institute Vom, Plateau State, Nigeria. 6Department of Veterinary Pathology and Microbiology, University of Nigeria Nsukka, Enugu State, Nigeria. *Corresponding author at: Istituto Zooprofilattico Sperimentale della Sicilia ‘A.Mirri’, Palermo, Italy. E-mail: dottoremira@gmail.com. Ijeoma Chekwube Chukwudi1, Francesco Mira2*, Kenneth Ikejiofor Ogbu3, Refiloe Petunia Malesa4, Giuseppa Purpari2, Pam Dachung Luka5, Emmanuel Ikenna Ugochukwu1, Livio Edward Heath4, Annalisa Guercio2 and Kennedy Foinkfu Chah6 Keywords Genetic characterization, Goat, Lineage II, Lineage IV, Nigeria, Peste-des-petit- ruminants virus, Sheep, Small ruminant morbillivirus. Summary Peste des petits ruminants virus (PPRV) is the aetiologic agent of Peste des petits ruminants (PPR), an important viral disease of sheep and goats. PPR is endemic in Nigeria and leads to social and economic losses. The objective of this study was to determine the prevalence of PPR infection and genetically characterize PPRV strains obtained from sheep and goats in three States of Southeast Nigeria. A total of 285 nasal swab samples collected in 2017-2018 were processed for PPRV genome detection by RT-PCR. Sixty-five (22.81%) of the samples were positive for PPRV. Sequence and phylogenetic analyses revealed that the PPRV belonged to lineages II (11/38, 28.9%) and IV (27/38, 71.1%). The N gene fragment sequence showed a 99.77%-100% and 99.98%-100% identity among the strains of lineages II and IV, respectively. Fourteen amino acid substitutions, previously unreported in PPRV strains from Nigeria, were recorded. This study confirms the circulation of PPRV lineages II and IV in Southeast Nigeria, the dominance of strains belonging to lineage IV in recent years, and their close genetic relationship with those previously reported in other parts of Nigeria and neighboring countries. Peste des petits ruminants in Nigeria: identification and variations of lineage II and lineage IV in sheep and goats spread and has been reported in several countries across African, the Near and Middle East, Asia and, more recently, Europe (Parida et  al. 2015, Donduashvili et al. 2018). PPR is caused by Peste des petits ruminants virus (PPRV ), taxonomically classified by the International Committee on Taxonomy of Viruses (ICTV ) as specie small ruminant morbillivirus (SRMV ) (Amarasinghe et  al. 2019), a member of the Morbillivirus genus, belonging to the family Paramyxoviridae, subfamily Orthoparamyxovirinae (Gibbs et al. 1979). PPRV is a single-stranded negative-sense RNA virus, whose genome consists of approximately 15-16 Kb in size, encoding for six structural proteins, namely Introduction Peste des petits ruminants (PPR) is a transboundary viral disease of small ruminants, whose clinical signs are characterized by pneumonia, ocular and nasal discharges, conjunctivitis, necrotic stomatitis and diarrhoea (Zahur et  al. 2011, Gibbs et  al. 1979). The disease associated with high morbidity (100%) and mortality (up to 90%), particularly among naïve populations of sheep and goats, might cause severe social and economic losses amongst rural households/small ruminant farmers (Abu Elzein et al. 1990). Following its first report in Côte d'Ivoire (Gargadennec and Lalanne 1942), the disease has 190 Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 PPRV in Southeast Nigeria Chukwudi et al. Institute (OVI), South Africa, for processing, analysis, and sequencing. RNA extraction and RT‑PCR Sterile phosphate-buffered saline (PBS) (500 µl) was added into each tube containing the swab sample. This was centrifuged at 10,000 x g for 3-5  minutes (+  4  °C) and supernatants (swab extract) was decanted into a sterile tube. Viral RNA was extracted from 140 µl of swab homogenate using the commercial viral RNA kit, QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. Presence of PPRV RNA was evaluated using the forward primer NP3 and reverse primer NP4 amplifying a 351-bp fragment of the nucleocapsid (N) gene (Couacy-Hymann et al. 2002). Reverse transcription-polymerase chain reaction was carried out in 25 µl volume reaction mix using the Qiagen® OneStep Ahead RT-PCR Kit (Qiagen) consisting of 10 µl of 2.5x OneStep Ahead RT-PCR Master mix, 1 µl of 25x OneStep Ahead RT-Mix, 1.5 µl of 10 pmol (each) of forward and reverse primers, 5 µl of 5x Q solution, 3.8 µl dH 2 O, 0.2  µl of 125x OneStep Ahead Master Mix Tracer and 2 µl of template RNA. Aliquot of molecular grade water was used as the negative control while the positive control consisted of an established positive sample (a clone of the live-attenuated PPRV vaccine derived from the Nigeria/75/1 strain, grown on VERO cell, and imported from The Pirbright Institute, Pirbright, United Kingdom). Amplification was conducted under the following thermal conditions: 50 °C for 10 min to activate the reverse transcriptases, 95 °C for 5 min to activate the DNA polymerases followed by 40 cycles of 95 °C for 10 sec, 55°C for 10 sec, 72  °C for 10 sec and a final extension of 72  °C for 2 min. Each amplicon (5 µl) nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin (H) and large (L), and two nonstructural (V and C) proteins (Bailey et al. 2005). Although PPRV has only one serotype, PPRV isolates are genetically grouped into four distinct lineages (I, II, III and IV), based on sequence analysis of the fusion (F), nucleocapsid (N) or haemagglutinin (H) protein genes (Forsyth and Barrett 1995, Dhar et al. 2002, Couacy-Hymann et al. 2002, Kumar et  al. 2014). Lineage I, which was circulating in West African countries in 1988-1994 (Kwiatek et  al. 2007), has not been identified at molecular level in Africa since 2001 (Tounkara et  al. 2018). Lineage II has been identified in Central and Western Africa; lineage III in Eastern Africa and southern part of the Middle East while lineage IV circulates across the Middle East, including Israel and Turkey, part of Asian continent and Northern, Western, Central and Eastern Africa (Kwiatek et al. 2007, Banyard et al. 2010, Munir et al. 2013, Cosseddu et al. 2013, Parida et  al. 2015, Mantip et  al. 2016, Woma et  al. 2016, Baazizi et al. 2017). Lineages III and IV co-circulation in the United Arab Emirates and Qatar (Kwiatek et al. 2007, Libeau et al. 2014) has been reported. Because of these previous reports, it has become necessary to provide further epidemiological and genetic data on the spread of PPRV among sheep and goats in Nigeria. This study was therefore conducted to determine the prevalence of PPRV infection and genetic characteristics of PPRV strains circulating in South-East Nigeria. Materials and methods Samples collection A multistage sampling method was performed with three hierarchical stages and the size of the sample used in this study was estimated using the formula according to Thrusfield (Thrusfield 2005), as described by Chukwudi and colleagues (Chukwudi et al. 2020). Nasal swabs were collected within a period of seven months (from December 2017 to June 2018) from 285 randomly selected sheep (n =  172) and goats (n = 113) at local markets, households, abattoirs and veterinary clinics in three randomly selected states: Anambra, Ebonyi and Enugu (latitudes 7°07’N and 3°90’N and longitudes 6°51’E and 8°30’E), located in South-East Nigeria (Figure 1). The animals had no history of vaccination and the sick animals showed one or a combination of the following clinical signs: nasal and ocular discharges, coughing, diarrhoea, ulcerative stomatitis, weakness, emaciation (Supplementary Table). The samples were properly packaged in dry ice and sent to the Transboundary Animal Disease Laboratory, Agricultural Research Council (ARC) - Ondersteport Veterinary Research Figure 1. Map of Nigeria showing the three States sampled (marked with gray background). 191Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 Chukwudi et al. PPRV in Southeast Nigeria Results Detection of PPRV viral genome by RT‑PCR Sixty-five (22.81%) of the 285 nasal swab samples were positive for PPRV by RT‐PCR assay. The highest infection rate of 27.73% was obtained from Enugu State while 21.73% and 16.21% were recorded for Anambra and Ebonyi States, respectively. Infection rate was higher in sheep (23.26%) than in goats (22.12%) while more females (24.5%) than males (20.8%) tested positive for PPRV. Based on breed distribution, Uda, Yankass and West African Dwarf breeds of sheep had infection prevalence of 33.3%, 13.3% and 23.5%, respectively, while Red Sokoto and West African Dwarf breeds of goats had 25% and 20.8%, respectively. Each analysed parameter is presented in Table I. There were no significant associations (p > 0.05) between the infection prevalence and state/location, species, sex or breed of animals sampled. Based on the month of sampling, the highest infection prevalence (68.8%) was obtained in March while the least (5.6%) was obtained in June. There was a significant association (p < 0.05) between the infection prevalence and the months of sampling (Figure 2). Sequence analysis Among the 65 samples that tested positive for PPRV by RT-PCR, 38 good quality sequences (303  nts was analysed by electrophoresis at 120 volt/80 mA for 45 min on a 1.5% agarose gel stained with ethidium bromide, examined and photographed under ultraviolet light using a gel documentation system (BioRAd Gel DocTM xRT model no. Universal HoodII, BioRad, USA). Sequencing Amplicons were purified with the commercial kit Qiaquick® Gel Extraction kit (Qiagen), according to manufacturer’s instruction. The concentration and purity of the DNAs were quantified using spectrophotometer (NanoDrop ND-1000, PeQlab Biotech Gmbh), according to manufacturer’s protocol. Amplicon that had more than 20 ng DNA load were stored at -  20  °C and then submitted to Inqaba Biotech™ (Pretoria, South Africa) for direct Sanger sequencing in both forward and reverse directions with primer pair NP3/NP4. Sequences were viewed and reversed using Chromas ver 2.6.4 (Technelysium Pty Ltd, South Brisbane, Australia) and low-quality sequences were not included in the further analyses. Sequences were then assembled and analysed using BioEdit ver 7.2.5 software and submitted to the nBLAST program (https://blast.ncbi. nlm.nih.gov/Blast.cgi) to search related sequences in public domain databases. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number as presented in Supplementary Table. Phylogenetic analysis To elucidate the genetic relationships of the analysed PPRV strains, a phylogenetic tree based on the partial N gene sequences was constructed with the MEGA X software. The model selection was performed using the best-fit model of nucleotide substitution with MEGA X software. The phylogenetic tree was constructed using the maximum-likelihood method, according to the Kimura 2-parameter (K2) model with invariant sites (I) and bootstrap analysis with 1,000 replicates. Statistical analysis The infection prevalence (total positive/total sample) was calculated. Pearson’s Chi-squared test was used to determine if there were associations in the number of positive and negative test results within each variable (area/location, species, sex, breeds, and months of sampling). Significance was accepted at P < 0.05. Table I. Presence of the Pestes des petit ruminants virus in sheep and goats in South-East Nigeria according to state, species, breed and sex. State No. positive No. negative Total samples Enugu 33 86 119 Anambra 20 72 92 Ebonyi 12 62 74 Total 65 220 285 Species No. positive No. negative Total samples Sheep 40 132 172 Goat 25 88 113 Total 65 220 285 Breed No. positive No. negative Total samples Uda 4 8 12 Yankassa 2 13 15 WAD sheep 34 111 145 Red Sokoto 9 27 36 WAD goat 16 61 77 Total 65 220 285 Sex No. positive No. negative Total samples Male 27 103 130 Female 38 117 155 Total 65 220 285 192 Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 PPRV in Southeast Nigeria Chukwudi et al. N gene fragments, by excluding the primers sequences; sequences ranged from nucleotide 1205 to 1507 of the N gene sequence acc. no. KR828813) were obtained by Sanger sequencing. Based on the amino acid (aa) residues, the 38 sequences clustered into lineage II (11/38, 28.9%) and IV (27/38, 71.1%). The N gene fragment showed 99.77%-100% and 99.98%-100% nt identities among the strains of the lineages II and IV, respectively. When compared with the reference strain NGKW2012-MSLN (acc. no. KR828814), a number of amino acid substitutions previously reported among Nigerian PPRV strains belonging to lineage II (Mantip et al. 2016, Woma et al. 2016), were also found among the 11 strains in this study. However, two of these strains varied from previous lineage  II strains from Nigeria by additional substitutions IV and PA at Table II. Amino acid changes of PPRV Lineage II strains collected in Nigeria. Strain/Isolate id. Acc. no. Year Amino acid residue 403 424 425 435 442 451 456 459 469 473 477 482 NGKW2012-MSLNb KR828814 2012 E I G K I I P P L P V G N67-127-GYM31KD-Oyob KJ124767 2011 D T D R I T S P P Q L S Vaccine Nigeria/75/1b KY628761 1975 D T D R I T S P P Q L S NGEN_038 CA 2018 MT038903 2018 D T D R I T S P P Q L S NGEN_228 OV 2018 MT038904 2018 D T D R I T S A P Q L S NGEN_003 OV 2017 MT038905 2017 D T D R V T S P P Q L S Otherc Reference 2010-2013 E/D I/T G/D K/R I I/T P/S P L P V G/S bReference strain; cOther previous PPRV (Peste des petit ruminants virus) sequences from Nigeria (Mantip et al. 2016, Woma et al. 2016); Bolded are amino acid changes reported for the first time among all the PPRV sequences collected in Nigeria In fe ct io n p re va le n ce 0 Dec Jan Feb Mar Apr May Jun 10 20 30 40 50 60 70 80 Months Pearson chi square value, df 32.571a, 6 P Value 0.00 Figure 2. Trend of PPR virus infection prevalence in small ruminants in South-East Nigeria. Table III. Amino acid changes of PPRV Lineage IV strains collected in Nigeria. Strain/Isolate id. Acc. no. Year Amino acid residue 415 423 429 432 434 437 442 443 454 462 470 472 478 483 NGYO2013-2162d KR828813 2013 A R S P T G I S T E E M S Q PPRV/Cameroon/ CMRS6/2017d MH447983 2017 A K S P T G I P T E E T S Q SRMV/Niger/12/2017d MK673131 2017 - K L P T G I P T E E T S Q Nig12:IM21d KF479428 2012 A K S P T G I P T E E T S Q Nig12:TR10d KF479417 2012 A K S P T G I S T E E M S Q NGAN_150 CA 2018 MN271590 2018 A K S P T G I P T E E T S Q NGAN_084 OV 2018 MN271599 2018 A K S P A G I P T E E T S Q NGEN_137 OV 2018 MN271587 2018 A K S P T G I P A E E T S Q NGEN_255 OV 2018 MN271601 2018 A K S P T E I P T E E T P Q NGAN_217 OV 2018 MN271594 2018 A K S P T G I P T E E T S K NGEN_146 CA 2018 MN271593 2018 A K S P T G T S T E D M S Q NGAN_087 OV 2018 MT038906 2018 A K S P T G T S T K D M S Q NGAN_156 CA 2018 MN271592 2018 V K L L T G I P T E E T S Q Othere Reference 2010-2013 A/T R/K S P T/I G/E I S/P T/P E E M/T S/L Q dReference strain; eOther previous PPRV (Peste des petit ruminants virus) sequences from Nigeria (Mantip et al. 2016, Woma et al. 2016); Bolded are amino acid changes reported for the first time among all the PPRV sequences collected in Nigeria 193Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 Chukwudi et al. PPRV in Southeast Nigeria Figure 3. Unrooted maximum‐likelihood tree based on 59 partial N gene sequences of peste des petits ruminants virus strains (bootstrap 1,000 replicates; bootstrap values greater than 65 are shown). Black squares () and black dots () markings indicate PPRV strains of lineage II and IV, respectively, analysed in this study. Each sequence is indicated with strain/isolate name, country and year of collection, and accession number. clustered with lineage II were 99% related to Nigeria 75/1 vaccine strain and all were obtained from Enugu State. Discussion Peste des petits ruminants is a transboundary viral disease of small ruminants, associated with high morbidity and mortality rates, leading to social and economic losses amongst rural households/small ruminant farmers (Abu-Elzein et al. 1990). Because of the dramatic economic consequences it may cause, particularly in developing Countries, the Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health (OIE) supported a campaign for global eradication of PPR (https://www.oie.int/eng/ppr2015/background. html - http://www.fao.org/ag/againfo/programmes/ en/empres/news_271014.html). positions 442 and 459, respectively (Table II). When compared with PPRV sequences previously analysed and available in public domain databases, the N gene sequences of the analysed lineage II strains showed the highest nt identities (99.00%-100%) with the vaccine strain 75/1, collected in 1975 in Nigeria (acc.  no. DQ840160 and KY628761), and with the strain N67-127-GYM31KD-Oyo, collected in 2011 in Oyo State (Nigeria) (acc. no. KJ124767, Woma et al. 2016). A number of amino acid substitutions were equally observed among the sequences of the 27 PPRV strains belonging to lineage IV. Based on the deduced amino acid profiles, the strains were assigned into eight haplotypes or groups. Group  1 (n  =  2; strain  no.  9,  53) which consisted of strains with amino acid profiles similar to those of reference strains (acc.  no. MH447983 and KF479428) or those previously reported in Nigeria (Mantip et  al. 2016, Woma et  al. 2016) while Groups 2 (n  =  5; strain no. 3, 19, 20, 48, 49), 3 (n = 2; strain no. 46, 180), 4  (n  =  1; strain  no.  55) and 5 (n = 4; strain no. 12, 14, 16, 52) consisted of strains with single amino acid substitutions T434A, T454A, S478P and Q483K, respectively. Amino acid profiles of the representative of each of the eight haplotypes are shown in Table III. The amino acid changes recorded for the seven haplotypes (groups  2-8) have not been reported in PPRV strains from Nigeria. Sequences clustered in haplotypes 1, 3, 4 and 8 showed 99.33%-97.35% nt identities with the strain PPRV/Cameroon/ CMRS6/2017 (acc.  no. MH447983), collected in Cameroon in June 2017, and 99.21%-97.63% with the isolate SRMV/Niger/12/2017 (acc. no. MK673131), collected in Niger in January 2017. While sequences clustered in haplotypes 2, 5 and 6, 7 showed 99.66% and 99.34%-98.01% nt identities with the strain Nig12:IM21 and with the strain Nig12:TR10 (acc. no. KF479428 and KF479417, Woma et al. 2016), collected in Nigeria in 2012, respectively. Phylogenetic analysis The N gene nucleotide sequence of the PPRV strain representing each of the haplotypes was used for phylogenetic analysis. A phylogenetic tree constructed based on the N gene sequences of the representative PPRV strains in our study and other PPRV viruses representing the four lineages is shown in Figure 3. The PPRV strains obtained in this study clustered with the PPRV strains of the lineage II (11 strains) and IV (27 strains). The 27 PPRV strains that clustered with the Asian lineage IV were collected from all the States sampled (Enugu, Anambra, and Ebonyi). These strains were 76%-85% related to PPRV isolates from Imo, Anambra, Plateau, Taraba and Ondo States of Nigeria and isolates from Cameroon and Niger. The 11 PPRV strains that 194 Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 PPRV in Southeast Nigeria Chukwudi et al. Genetic characterization of the circulating PPRV strains has largely been based on the comparison of partial or complete N or, less frequently, F gene sequences. According to the sequence analysis, PPRV strains could be clustered in four different lineages (I-IV). Due to the divergence in the N gene, the analysis of this gene highlights the relationship of the circulating strains more clearly (Kwiatek et al. 2011). Woma and colleagues (Woma et  al. 2016) and Mantip and colleagues (Mantip et al. 2016) first described the spreading of the lineage IV in the years 2010 to 2013 in Nigeria, where the lineage II was, for a long time, the prevalent genetic type. In the present study both lineages II and IV were detected in samples collected from sheep and goats in South-East Nigeria, with lineage IV being the dominant genotype. Thus, this study further confirms the wide circulation in Nigeria of PPR viruses belonging to the Asian lineage IV. This study allowed to depict the prevalence of the lineages and their genetic features in selected areas. Mantip and colleagues (Mantip et al. 2016) obtained negative results from the samples collected in Enugu State while Woma and colleagues (Woma et al. 2016) obtained only two positive samples from Anambra State, both belonging to the lineage  IV. Genetically, the PPRV strains in this study had a close relationship with previous strains collected in other States of Nigeria and Cameroon. However, amino acid changes reported for the first time were found in strains belonging to both lineage II and lineage IV. Lack of recent further genetic analyses in Nigeria or neighboring countries makes it difficult to ascertain if these changes are due to the recent introduction of different but closely related strains from other territories or if they represent potential local adaptative genetic changes. The genetic close connection of the most prevalent haplotype of the lineage IV with PPRV strains collected in the same years in neighboring Cameroon and Niger suggests the potential introduction of the related strains from other countries. Indeed, in the phylogenetic tree depicted in Figure 3, the lineage IV viruses described in this study cluster in a sub-clade together with strains collected in Cameroon in 2017 (unpublished) and Niger in 2015/2017 (Souley et al. 2019). This close genetic relationship between PPRV strains from neighboring Countries (Nigeria, Niger, Cameroon) suggests a common origin, probably connected to the transboundary movements of animals due to the absence of natural barriers or the commercial routes of animal trade (Tounkara et  al. 2017, Souley et  al. 2019). The varying amino acid changes observed among the lineage IV strains in this study indicate the strains circulating in Nigeria are genetically diverse and reflect the geographical differences between the different parts of Africa (Dundon et al. 2020). Although PPR was first described in Nigeria in 1975 (Taylor and Abegunde 1979), it is only in the last decade that molecular analyses on PPRV strains circulating in Nigeria were provided. These studies, based on the N gene (Mantip et  al. 2016, Woma et al. 2016) or the F gene (Luka et al. 2011) sequence analyses, characterized strains collected from 2007 to 2013 in many but not all States of Nigeria, highlighting the circulation of the lineage II and, since 2010, of the lineage IV of PPRV (Mantip et  al. 2016, Woma et al. 2016). In the present study the molecular characteristics of PPRV strains circulating in Nigeria in 2017-2018 were determined and compared with those previuosly collected in Nigeria and other countries. The analysis was based on the N gene sequence of PPRV strains collected from States of Nigeria not included in previous studies (Ebonyi State) or with none or few tested positive samples (Anambra and Enugu States). PPRV was detected by RT-PCR in 65 (22.8%) of the 285 animals tested, confirming the current circulation of PPRV in South-East Nigeria (Anambra, Ebonyi and Enugu States). Different infection rates from clinical samples (9.7%, Mantip et al. 2016; 42%, Woma et  al. 2016; 51.52%, Luka et  al. 2016) were reported in previous studies conducted in other States of Nigeria. The differences in detection rates recorded may be related to the sample size, sampling method, season of the year, management practice and detection method (Couacy-Hymann et al. 2005, Devi et  al. 2016) as well as the clinical status of the animals at the period of sampling. The analysis of the results revealed that there were no significant associations between PPRV detection rate and location/states, species, sex and breeds of the animal sampled. These results suggest that PPRV affects equally both sheep and goats, males and females, and also all breeds of the animals studied. However, our findings differ with those of other investigators such as Anderson and colleagues (Anderson et  al. 1991) and Al-Majali and colleagues (Al-Majali et  al. 2008) in Northern Jordan. Additionally, Lefevre and Diallo (Lefevre and Diallo 1990) also reported that goats are more sensitive to PPRV. Previous studies reported the variability on susceptibility among domestic species but the PPRV spread is thought to be related also to the relationship of virus sequence and host species (Banyard et al. 2010). The highest PPR infection rate observed in March (68.8%) coincides with the harmattan period. Similar observation has been reported (Ezeibe et  al. 2008, Okoli 2003, Wosu 1994, Wosu et  al. 1990, Ezeokoli et al. 1986, Obi et al. 1983). This higher infection rate during the harmattan period has been attributed to the dry, cold and dusty weather, together with the poor nutrition as a result of inadequate pasture (Ezeibe et al. 2008, Wosu et al. 1990). 195Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 Chukwudi et al. PPRV in Southeast Nigeria of good laboratory practices, may contribute to clarify these hypotheses. Indeed, the partial N gene sequence was commonly used as an effective target for PPR detection and lineage identification (Kwiatek et  al. 2011, Tounkara et  al. 2019), aiming mainly to understand the epidemiology and transmission dynamics of PPR (Tounkara et  al. 2019), as in the present study. This study reports vast diversity in the amino acid data among the PPRV lineages IV and II circulating in South-East Nigeria,and confirms the predominance of the lineage IV in Nigeria. These data support the need for further molecular epidemiological investigation in order to better comprehend the spread of the PPRV strains and their transboundary circulation, to support the future control strategies. Acknowledgments The Authors would like to thank Dr. Webster Okonkwo and Dr. Adaeze Nwabueze of the Tropical Veterinary Centre Ekwulobia Anambra state, Dr. Sunday Ali and Mr. Ndubuisi of Ebonyi state, Dr. Ikechukwu Udeani of Veterinary Teaching Hospital University of Nigeria, Nsukka, for their assistance with sample collection. Grant Support This work was supported by the Tertiary Education Trust Fund (TETFund) of the Nigerian Government through University of Nigeria Nsukka TETFund (Needs Aassessment) Bench Space Intervention. Chukwudi I.C. is the only recipient of the grant. The Funder had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The high degree of sequence similarity in PPRV lineage II with the Nigeria 75/1 vaccine strain recorded in our study has also been reported by other authors in Nigeria (Mantip et  al. 2016, Woma et  al. 2016), Sierra Leone (Munir et  al. 2012), Tanzania (Misinzo et  al. 2015, Mahapatra et  al. 2015), China (Zhou et  al. 2018), India (unpublished, acc  no. DQ176750) and Iran (unpublished; acc.  no. KC534492), although Dundon and colleagues (Dundon et  al. 2020) posited that some of these previously reported cases of sequence similarity are most likely as a result of laboratory contamination during sample processing or due to circulating strains that are very similar to or variants of the vaccine strain Nigeria 75/1. Although homologous PPR vaccine available in Nigeria for immunization of small ruminant is of Nigeria PPRV 75/1 origin (Sen et  al. 2010), a recent study reported lack of awareness of PPR vaccination among small ruminant farmers and non-availability of PPR vaccine at veterinary establishments in Southeast Nigeria (Chukwudi et  al. 2020). There is not a documented evidence that animals tested in this study have been vaccinated against PPRV but the molecular analysis of the 11 lineage II strains collected from Enugu State in this study shows their close relationship with a vaccine strain. Therefore, albeit hypothesized, a clear origin of these viruses cannot be pointed out and necessarily this limitation deserves further evaluation or specific assays for discrimination between Nigeria PPRV 75/1-based vaccines and field strains of PPRV. 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Dia, Du 21/12/2017 2 3 AN OVI WAD M Nv Nd, Cou. Dia, Du, RHC, Wk, Us 25/01/2018 MN271599, NGAN_084 OV 2018 3 7 EN CAP WAD F Nv Nd, Od, Cou. Du, Ema, RHC, Wk 13/03/2018 4 8 EN CAP WAD F Nv Nd, Od 14/03/2018 5 9 AN CAP WAD F Nv Nd, Od, Dia, Us 19/03/2018 MN271590, NGAN_150 CA 2018 6 10 AN CAP WAD F Nv Nd, Od, Cou. Dia, Du, RHC, Wk, Se 26/03/2018 MN271592, NGAN_156 CA 2018 7 12 AN OVI WAD F Nv Nd, Dia, Cou 16/04/2018 MN271594, NGAN_217 OV 2018 8 13 AN OVI WAD F Nv Nd, Od, Cou. Du, Wk 16/04/2018 9 14 AN CAP WAD M Nv Nd, Cou. Du, Us, RHC 16/04/2018 10 16 EN OVI UDA F Nv Apparently healthy 22/12/2017 11 17 EN CAP RS F Unk Nd 09/01/2018 12 19 EN OVI WAD M Nv Severe mucoprulent Nd, Du 17/01/2018 13 20 AN OVI WAD F Nv Nd, Dia, Us, Cou. Du, RHC 25/01/2018 14 26 EN CAP WAD M Nv Nd, Cou. Dia, Du, RHC, Ema, Wk, Se 12/03/2018 MN271593, NGEN_146 CA 2018 15 27 EN CAP WAD F Nv Nd, Cou. Du, RHC, Ema 19/03/2018 16 28 AN CAP WAD F Nv Nd, Od, Cou. Du, RHC, Ema 26/03/2018 17 29 AN CAP WAD F Nv Nd, Cou. Dia, Us, Du, RHC, Ema 26/03/2018 18 32 EB OVI WAD F Nv Nd, Od, Cou. Du, RHC 11/04/2018 19 33 EN OVI WAD M Nv Mucopurulent Nd, Us, Cou, Du 21/04/2018 20 35 EB OVI WAD M Nv Mucopurulent Nd, Us, Cou, Du 23/05/2018 21 46 EN OVI WAD M Nv Apparently healthy 17/02/2018 MN271587, NGEN_137 OV 2018 22 48 EB OVI WAD F Nv Mucopurulent Nd, Us, Cou, Du 06/04/2018 23 49 EB OVI WAD F Nv Mucopurulent Nd, Du 11/04/2018 24 52 AN OVI WAD M Nv Nd, Dia, Cou 16/04/2018 25 53 EN OVI WAD M Nv Mucopurulent Nd, Cou, Du 21/04/2018 26 55 EN OVI WAD M Nv Mucopurulent Nd, Du 28/05/2018 MN271601, NGEN_255 OV 2018 27 60 AN OVI WAD F Nv Nd RHC, Du 25/01/2018 MT038906, NGAN_087 OV 2018 28 61 EN CAP RS F Unk Apparently healthy 09/01/2018 MT038903, NGEN_038 CA 2018 29 64 AN OVI WAD M Nv Apparently healthy 30/05/2018 30 66 EN OVI WAD M Nv Nd 21/04/2018 31 70 EN CAP RS M Unk Nd, Du Wk 17/01/2018 32 71 AN OVI WAD M Nv Mucopurulent Nd, Cou 25/01/2018 33 75 EN CAP WAD M Nv Mucopurulent Nd, Dia, Od, Cou. Du, RHC, Us, Ema 17/01/2018 34 85 AN OVI YAN M Nv Nd, Cou 30/05/2018 continued S/NO = Sample number; ST/NO = Strain number; State of collection: EN = Enugu, AN = Anambra, EB = Ebonyi; Specie of collection: OVI = Ovine, CAP = Caprine; Breed: WAD = West African Dwarf, RS = Red Sokoto, YAN = Yankassa; Sex: M = Male, F = Female; Vaccination status: Nv = Not vaccinated, Unk = Unknown; Anamnesis: Nd = Nasal discharge, Od = Ocular discharge, Cou = Coughing, Ema = Emaciated, RHC = Rough hair coat, Wk = Weak, Du = Dull, Dia = Diarrhea, Se = Sunken eyeball, Us = Ulcerative stomatitis. 199Veterinaria Italiana 2021, 57 (3), 189-199. doi: 10.12834/VetIt.2390.14842.1 Chukwudi et al. PPRV in Southeast Nigeria Supplementary Table. Details of tested positive samples. —cont’d Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 57; Issue: 3; Year: 2021; doi: 10.12834/VetIt.2390.14842.1 S/NO ST/NO State Species Breed Sex Vaccination status Anamnesis Date of collection Accession number and sequence ID 35 90 AN CAP WAD M Nv Nd, Du, Cou 16/04/2018 36 93 EN OVI WAD F Nv Nd, Cou, Du 23/01/2018 37 97 EN CAP RS F Unk Nd, RHC, Du, 21/12/2017 38 108 EN OVI WAD F Nv Nd, RHC, Du 23/01/2018 39 116 EN OVI WAD M Nv Nd, Du 28/05/2018 40 118 EN OVI WAD M Nv Nd 21/04/2018 MT038904, NGEN_228 OV 2018 41 122 AN CAP WAD F Nv Nd, Cou, Du 26/03/2018 42 123 AN CAP WAD F Nv Nd Du, Cou 26/03/2018 43 127 EN CAP RS F Unk Apparently healthy 28/12/2017 44 131 EN OVI UDA F Unk Nd 09/01/2018 45 132 EB OVI WAD F Nv Nd, Cou, Du 23/05/2018 46 137 EB OVI WAD M Nv Nd Du, Cou 23/05/2018 47 141 EB OVI WAD M Nv Nd, Cou 05/06/2018 48 143 EN CAP RS F Unk Nd 09/01/2018 49 144 EB OVI WAD F Nv Nd 11/04/2018 50 149 AN OVI WAD F Nv Nd, Du, Wk 30/05/2018 51 150 AN OVI WAD M Nv Nd, Cou, RHC 30/05/2018 52 159 EN OVI WAD M Nv Nd, Cou, Du 17/01/2018 53 160 EN CAP WAD F Nv Mucopurulent Nd, Cou 22/05/2018 54 161 EN OVI YAN M Unk Apparently healthy 17/02/2018 55 165 AN OVI WAD M Nv Apparently healthy 30/05/2018 56 168 EN CAP WAD F Nv Nd, Du 13/03/2018 57 169 EN CAP RS F Unk Apparently healthy 28/12/2017 58 170 EN OVI UDA F Nv Apparently healthy 20/12/2017 MT038905, NGEN_003 OV 2017 59 172 EB OVI WAD F Nv Nd 06/04/2018 60 174 EB CAP WAD F Nv Apparently healthy 06/04/2018 61 175 EB OVI WAD M Nv Apparently healthy 06/04/2018 62 177 EN CAP RS F Unk Apparently healthy 28/12//2017 63 178 EN CAP RS F Unk Apparently healthy 22/12/1017 64 179 EN OVI WAD M Nv Du 21/04/2018 65 180 EB OVI WAD F Nv Apparently healthy 17/02/2018 S/NO = Sample number; ST/NO = Strain number; State of collection: EN = Enugu, AN = Anambra, EB = Ebonyi; Specie of collection: OVI = Ovine, CAP = Caprine; Breed: WAD = West African Dwarf, RS = Red Sokoto, YAN = Yankassa; Sex: M = Male, F = Female; Vaccination status: Nv = Not vaccinated, Unk = Unknown; Anamnesis: Nd = Nasal discharge, Od = Ocular discharge, Cou = Coughing, Ema = Emaciated, RHC = Rough hair coat, Wk = Weak, Du = Dull, Dia = Diarrhea, Se = Sunken eyeball, Us = Ulcerative stomatitis.