Clinical Manifestation and Phylogenetic Analysis of Peste des Petits Ruminants in Local Iraqi Breed Sheep in Al-Diwaniyah Province

Peste des petits ruminants (PPR), a contagious virus that infects sheep and goats, damages livestock globally. This study examined the clinical features and phylogenetic analysis of the PPR virus in Iraqi breed sheep from Al-Diwaniyah province. A clinical trial of 610 sheep from different flocks found 150 oral lesions. Special primers for RT-PCR and Mega11 for phylogenetic analysis were used to study the PPR virus nucleoprotein (N) gene. The PPR infection rate was 44.6% in 4–12 month olds (n = 33/131) and 4.8% in 36–48 month olds (n = 3/75). A 608-bp PPR virus partial N gene sequence was found in 49.3% of samples by RT-PCR. In leucine, isoleucine, proline, glycine, alanine, glutamine, asparagine, threonine, serine, arginine, and lysine codons, 25 amino acid alterations were found. The protein codon 56 alanine-valine alteration was most significant. Moving from a smaller hydrophobic amino acid to one with a bigger side chain may reduce protein stability. Steric hindrance or protein shape change from Valine's extended side chain may impact folding, stability, functionality, and interactions with other molecules. Furthermore, phylogenetic analysis showed that the Nigerian strain (MN271586) was most similar to our Iraqi strain, with 100% identity and coverage. This study found the Peste des Petits Ruminants (PPR) virus in sheep flocks in Al-Diwaniyah Governorate, Iraq, which is genetically similar to neighboring countries. PPR virus strains must be monitored and genetically characterized since N gene alterations can affect infection and propagation.

Te International Committee on Virus Taxonomy identifes PPR as a member of the Paramyxoviridae family, subfamily Paramyxovirinae, genus Morbillivirus, and species caprinae.Te genome is a negative-sense, single-stranded RNA.Morbilliviruses, including rinderpest and measles, are closely related and immunologically cross-reactive.Te genome is a negative-sense, single-stranded RNA.Morbilliviruses, which include rinderpest and measles, are closely related and have immunological interactions.Te nucleoprotein (N) gene, which produces the nucleocapsid when combined with the RNA genome, has been meticulously sequenced, revealing four lineages that correspond to the virus's geographical origins [8].Te nucleocapsid (N) attaches to the phosphoprotein (P) and the large protein (L), both of which crosslink during viral replication; the matrix protein (M) attaches it to the envelope; and the envelope contains hemagglutinin (H), which aids the virus's adhesion to host cells.Te F fusion protein facilitates the infection of host cells.Neutralizing antibodies primarily regulate the orientation of the H protein [9].
Sheep and goats are the primary carriers of PPR, but cattle and pigs do not exhibit clinical signs or symptoms of the disease; therefore, they play an important role in virus transmission.PPR has been found in Laristan sheep, gazelles, Gumbok (Arabian, Arabian Mountain, Dorcas, Reem, Tompson), bufalo, impala, and Afghan goats [10].Wildlife may serve as reservoirs for small ruminant pests, although there is little evidence that they sustain or disseminate them [11].
PPR infection in sheep and goat herds is expected to cause annual economic losses of US$1.44 billion, placing food security at risk in multiple countries.As a result, international organizations aspire to eliminate the disease by 2030 [12].It is critical to act quickly to combat PPR in order to limit the large economic losses caused by the disease and reduce its spread, particularly in developing countries.
Clinical signs typically diagnose most PPR outbreaks, but it can be difcult to diferentiate them from foot-andmouth and bluetongue infections.As a result, PPRV diagnosis with antibodies is the most efective because infected animals have lifetime antibodies and a prolonged antibody response [13,14].RT-PCR is a highly sensitive and efective method for detecting morbilliviruses.Tis technique can extract genetic information from sequencing results in order to track the spread and migration of virus lineages [15].Furthermore, in order to enhance the disease management program, it is necessary to conduct regular surveillance of both domestic and wild animals [16].
Te following questions guided our study: how prevalent is PPR in Iraqi-bred sheep in Al-Diwaniyah province?What are the most notable clinical signs?Are clinical signs different in adjacent countries?What is the origin of the current stain in Iraq?What is a genetic variation?Does the mutation afect evaluation and phylogenetic analysis?And based on these questions, the aims of the present study were the evaluation of the clinical manifestations of Peste des Petits Ruminants (PPR) in Iraqi breed sheep in Al-Diwaniyah province, using PCR tests for confrmation of the PPR infection, investigating its molecular characteristics, and analyzing the phylogenetic relationships of PPR virus strains circulating in Iraqi breed sheep.

Viral RNA Extraction
. By using a sterile scalpel, the oral lesions were collected gently from infected sheep.Ten, the collected oral lesion samples were placed in a sterile microcentrifuge tube containing 200 μl of Trizol solution.Moreover, a sterile pestle or homogenizer was used to disrupt the oral lesion samples in the Trizol solution.Tis step helps release the viral RNA from the cells and tissues into the Trizol solution.Ten, the samples in Trizol solution were incubated at room temperature for a few minutes to allow for complete dissociation of nucleoprotein complexes.After incubation, the samples were stored at an appropriate temperature according to the Trizol manufacturer's instructions.
Follow the instructions of the Maxime ™ RT-PCR Pre- Mix (iNtRON Biotechnology, Korea), 200 μl of suspension was loaded in 1.5 ml tubes, and 1 ml of AccuZol reagent was added.Ten, 200 μL of chloroform was added and shacked and incubated on ice for 5 minutes.Tese contents were centrifuged at 12000 rpm for 15 minutes at 4 °C, and a fresh 1.5 ml tube was used to transfer the aqueous phase.Equal volume of isopropyl alcohol was added.After that, the contents of the tube were mixed well by turning it 4-5 times and then put in freeze −20 °C for 10 min.Next, the supernatant was carefully removed after centrifuging at 12000 rpm for 10 min at 4 °C.One milliliter of 80% ethanol was added, and the mixture was thoroughly vortexed.Te supernatant carefully removed after centrifuging at 12000 rpm for 5 minutes at 40 °C.Te aqueous phase was removed, and the RNA pellet was left to dry for fve minutes.Subsequently, the extracted RNA was then dissolved in diethyl pyrocarbonate (DEPC) water by running the solution through a pipette tip a few times and allowing it to rest in the water path at 55-60 °C for 10 minutes.Te RNA was stored at −70 °C as well as PCR will be done.Te concentration and purity of extracted RNA were measured using a nanodrop spectrophotometer at absorbance 260/280 nm at ratio 1.8 as pure RNA [17,18].

Reverse Transcription PCR (RT-PCR)
2.3.1.cDNA Synthesis Preparation.Te complementary DNA synthesis master mix (with a total volume of 10 μL) was prepared using 9 μL of total RNA template and 1 μL of Handom Hexamer primer (20 pmol) with M-MLV Reverse Transcriptase, following the instructions provided by iNtRON company, Korea.Te components of the RT mix, including the mentioned ones, were added to the HiSenScriptTM RH(−) RT PreMixKit kit strip tubes, which contained all the necessary elements for cDNA synthesis.After this, the strip tubes underwent centrifugation at 3000 rpm for three minutes in an Exispin vortex centrifuge and were then incubated in a BioRad-USA thermocycler following a 2-step protocol: (step 1) cDNA synthesis (RT step) at 50 °C for 1 hour, and (step 2) heat inactivation at 95 °C for 5 minutes.

Preparing the PCR Master Mix.
A 20 μl total volume of the AccuPower ® PreMix for PCR was utilized for the PCR reaction, following the manufacturer's instructions.Tis involved adding 5 μl of cDNA template (5-50 ng), 1 μl (10 pmol) each of the forward and reverse primer, and 13 μl of PCR water.Tese PCR master mix components, along with other necessary components like Taq DNA polymerase, dNTPs, Tris-HCl pH: 9.0, KCl, MgCl2, stabilizer, and 2 Veterinary Medicine International tracking dye, were added to the standard PCR PreMix Kit.Te PCR tubes were then spun in an Exispin vortex centrifuge at 3000 rpm for three minutes before being placed in a PCR thermocycler.Te RT-PCR primers (F: CCTTCTGGCCAAGGCTGTTA and R: CAGAGCTGA CCTTTCCTGCA) designed to amplify a 608 bp segment of the nucleoprotein (N) gene for PPR detection were provided by Macrogen Company, Korea.Te conventional PCR thermocycler settings used are listed in Table 1.
Finally, the PCR products were visualized using a UV transilluminator and evaluated through agarose gel electrophoresis.

DNA Sequencing Method.
Te positive results of the morbillivirus N gene protein RT-PCR were sent to Macrogen Technology in Korea for DNA sequencing using the AB DNA sequencing system.Te DNA sequencing analysis involved multiple alignment assessment using Clustal W alignment analysis and NCBI-BLAST to identify homologous sequences.Mutations and amino acid variations were analyzed using the bioEdit tool.Subsequently, MEGA software version 11 was used to perform phylogenetic analysis based on the sequences obtained from the current study and those available in GenBank.

Statistical Analysis.
In the current study, we performed statistical tests to construct the tables and determine the link between clinical indications of PPR and its incidence across age cohorts.We calculated incidence rates and clinical indications using percentages (%).Te authors used percentages to determine fock PPR and clinical sign frequency.Furthermore, we also used the t-test, correlation test (r), and p value (P < 0.05) for statistical analysis.We employed the t-test to show signifcant diferences in means across unrelated groups and to determine the statistical signifcance of infection rates by age group.Furthermore, t-test has been used to assess incidence rates at 6-14 and 30-40 months.Te correlation test coefcient (r) measured the strength and direction of the relationship between two variables.Te authors used the correlation coefcient (r) to study clinical symptoms and infection rates.

Clinical Signs and Observations.
During clinical examination, suspected sheep with PPR showed symptoms such as high fever (above 40 °C), diarrhea, oral ulcers, respiratory distress, serous discharge from the eyes and nostrils which later became purulent, as well as anorexia and depression.Te most common and notable clinical sign was the presence of oral lesions.Tere were no signifcant diferences in the infection rate between males 52.95% (323/610) and females 47.05% (287/610).On the contrary, age was signifcant because there were 33/131 cases (25.2%) infected in the age 4-12 months, 16/91 cases (17.58%) infected in the age 24-36 months, and 22/326 cases (6.75%) infected in the age 12-24 months, as illustrated in a, b, and c in Table (2).Besides, it was not signifcant in age 36-48 months (d).

Correlation between Clinical Signs of PPR with Age Group.
Based on Table 2, which shows PPR infection rates by age, Table 3 estimates the association between clinical symptoms of PPR and incidence and proportions for each age category.Four age groups exist: 4-12 months, 12-24 months, 24-36 months, and 36-48 months.Table 2 shows a signifcant decrease in popularity percentages between the 4-12-month group and the 36-48-month group, with resistance rates dropping to 4.8%.Various age groups display distinct signs of lymph node dysfunction.For instance, the age group of 6-14 months demonstrates elevated temperatures compared to the other age groups.Based on the data presented in Table 2, the symptoms of "oral ulcers" and "fever" rank as the most prevalent.All of the age groups depicted in the table exhibit these features.
As indicated by Table 3, between the ages of 4-12 months, there was a signifcant positive correlation (r � 0.93) between age and the incidence of mouth ulcers.High t-test results (10.87) and a very low p value (p < 0.0016) support this association and show a statistically signifcant relationship.Tere was a weak correlation (r � −0.03) between age and the incidence of mouth ulcers in the 12-24month age group.A p value larger than >0.05 and a low ttest correlation coefcient (−0.40), which showed no discernible relationship, support this.Tere is a signifcant inverse relationship (r � −0.91) between age and the incidence of mouth ulcers within the 24-36-month age range.A statistically signifcant link is indicated by this association's enormous t-test result (−10.72) and incredibly low p value (p < 0.001).According to this study, as animals in this age group age, the incidence of mouth ulcers appears to be declining dramatically.Similarly, there is a considerable negative correlation (r � −0.87) between age and the incidence of mouth ulcers between the ages of 36-48 months.A statistically signifcant link is indicated by the huge t-test value (−8.86) and the incredibly low p value (p < 0.001).Within the specifed age range, this investigation suggests a negative correlation between age and the incidence of mouth ulcers.1.
Te result of this study revealed that the partial N gene sequences of the detected isolates in this investigation were related to those of PPR-V.Tese sequences have been deposited in the GenBank database with the accession codes (MK408669.1).

DNA Sequencing and Phylogenetic Analysis.
Te NCBIblast-related studies using the DNA sequencing method to identify genetic relationships revealed that the sequence of N gene was matched with obtainable sequenced using Mega 11.Te sequencing obtained in this investigation was compared to 7 NCBI (strains/isolates) as mentioned in Table 4.After multiple sequence alignment, Mega 11 was used to create a phylogenetic tree (Figure 2) and percentage of homology between the N genes study sequence was estimated (Tables 5,  6, 7).
As demonstrated in Figure 2 and Table 5, the PPR virus detected in this study was highly related to those detected in Iran, Turkey, Erbil, and India in order which foretell the way disease was spread in and out of Iraq.Vaccination program should depend on the local strain rather than the imported vaccine because it will be more efective.Free movement of sheep and goat herds in the region is the most common cause beside the uncontrolled importation of animal product from Iran, Turkey, and India.Absence of the vaccine in the last decade is another important cause of the spread of the disease in the middle and the south of Iraq.
Te fndings of this study demonstrate that the Iraqi strain [MW682328] [19] exhibits 100% homology with Nigerian strain [MN271586], although it varies in composition when compared to Indian strains MT108445 and MT108445, turkey strain JQ51995, Iranian strains MH824414 and JX898862, and Pakistani strain KY967608.Moreover, the analysis of substitutions in the amino acid sequence of a protein encoded by the PPR virus genome, as presented in Tables 6 and 7, led to the classifcation of amino acids based on their structural and functional characteristics.
Te amino acid analysis revealed 25 substitutions in the N gene of Peste des Petits ruminants' virus (PPR) at diferent positions (Table 7).For instance, substitution of hydrophobic amino acids like leucine (Leu) and isoleucine (Ile) infuence the protein's interaction with hydrophobic regions, while proline (Pro) introduces structural kinks impacting fexibility.Additionally, small amino acids with small side chains like glycine (Gly) and alanine (Ala) may have similar roles in protein structure.Polar amino acids such as glutamine (Gln) and asparagine (Asn) afect protein function due to their ability to form hydrogen bonds, while threonine (Tr) and serine (Ser) infuence hydrogen bonding in protein function through their hydroxyl groups.Positively charged amino acids like histidine (His), arginine (Arg), and lysine (Lys) are potentially involved in interactions with negatively charged molecules or enzyme catalysis, whereas negatively charged amino acids like glutamic acid (Glu) have distinct roles due to their negative charge.
Furthermore, the study identifed a substitution between alanine (Ala) and valine (Val) at codon 56 of a protein sequence.Tis change from a smaller hydrophobic amino    Te Iraqi isolates were clustered in the same branch of the genetic tree with the isolates MN271586.1,KY967609.1,MH824414.1,and MTH108445.1.Also, the Iraqi isolates clustered with MN271586.1.Te genetic tree also showed that the other isolates strains LT629277.1,JX898862.1,and JQ519925 were found to be clustered in a separate branch of the genetic tree as in Figure 2.
Te results of nucleotide sequence alignments between the Nigeria isolate (MN271586.1)and the Iraqi isolate was (100%).In other hands, the results, as illustrated in

Discussion
Tis study found that sheep suspected of Peste des Petits ruminants (PPR) had high fever (above 40 °C), diarrhea, oral ulcers, respiratory discomfort, purulent serous discharge from the eyes and nose, anorexia, and depression [20] which showed clinical and pathological abnormalities that indicate PPR.PPRV loves lung tissue but looks like rinderpest.A fever (pyrexia), eye and nose drainage, necrotizing and erosive mouth ulcers, gastrointestinal infammation, diarrhea, and pneumonia are symptoms [21].According to [22], the main clinical lesions in PPR infection were oral lesions.Furthermore, the authors in [23] reported that the most oral mucosa layer was congestion, sloughed, and ulcerated in infected goats.
Males 52.95% (323/610) and females 47.05% (287/610) exhibited similar infection rates, possibly due to the increased proportion of females in the herds.Age also played a signifcance, with 33 (25.2%) incidences in sheep aged 4-12 months and 3 (4.8%) in those aged 3-4 years.Some owners and herders claimed that males were more susceptible to PPRV; however, the authors in [24] discovered no gender diference.PCR identifcation of the N gene resulted in 49.3% infection rates (74/150 samples).Based on an investigation by [25], the nucleoprotein (N) gene was found in 63.2% and 89.1% of the samples, respectively, using conventional and reverse transcription real-time quantitative PCR.By comparing sequences from a brief part of the fusion (F) or nucleoprotein (N) gene, we concur with [10] molecular epidemiology of Peste des Petits ruminants' virus (PPRV).Moreover, Omer Babashekh et al. [26] found F and N genes in all suspected PPR-infected sheep using PCR.
Te association between age and infection rate has several explanations: in younger animals with developing immune systems, mouth ulcers may be more common as immunological development continues.Second, vaccination status is important because younger animals may lack immunity due to inadequate vaccination or exposure, whereas older animals may have it through vaccination or natural exposure.Younger animals may be riskier or interact with sick people.At certain ages, specifc clinical symptoms increase as the illness progresses.Finally, larger and more diversifed populations are better able to estimate ageclinical sign correlations [27].
Tis study revealed a PPR virus that was closely linked to those reported in Iran, Turkey, Erbil, and India, showing its spread into and out of Iraq [25].Te study recommends using indigenous strains instead than imported vaccines for better efcacy.Free movement of sheep and goat herds and unrestrained imports of animal products from neighboring nations were said to spread the disease.
PPR N gene amino acid analysis revealed 25 changes.Tis substitution was based on structure-function similarities [28].Hydrophobic Leu and Ile may afect protein interactions [29].Protein chains kinked by proline changed shape and function [30].Short side chains of glycine (Gly) and alanine (Ala) likely altered protein structure similarly [31].Due to their polarity and hydrogen bond-forming ability, glutamine (Gln) and asparagine (Asn) may have had similar purposes.Hydroxyl group polarity of Tr and Ser implies hydrogen bonding [32].Histidine, arginine, and lysine may have responded similarly due to their positive charges.Negatively charged glutamic acid (Glu) likely worked.Valine (Val) replaced alanine (Ala) at codon 56, creating a larger hydrophobic amino acid with a broader side chain.Protein stability and shape afect molecular interactions and function [33].On the other hand, previous research identifed fewer PPR virus N gene amino acid alterations [34].
In addition, the NCBI GenBank received amino acid sequences of positive PPR strains from wild and domestic goats, accession codes LT629276-LT629277 and LT882721-LT882728.PPR virus N gene amino acid changes were found in 10 locations in other research [25].Ten positive strains from wild (B2, C1, L81, and N11) and domestic (P49, P56, S4, V67, V89, and V90) goats were also sent to NCBI GenBank for amino acid sequencing.Tese sequences have accession numbers LT629276-LT629277 and LT882721-LT882728.Te Iraqi strain [MW682328] was 100% comparable to the Nigerian strain [MN271586] but difered from the Indian strains MT108445 and MT108445, Turkish strain JQ51995, Iranian strains MH824414 and JX898862, and Pakistani strain KY967608 [25,26,35,36].Te Peste des Petits ruminants' (PPR) viral genome amino acid sequence alterations were used to classify amino acids by structure and function.Tese sequences were also similar to strains, particularly the Nigerian strain [26,37].PPR infection epidemiology in this study was linked to eight strains from adjacent Asian and African nations [38,39].

Conclusion
Te current study indicates that the PPR symptoms in sheep included high fever, diarrhea, oral ulcers, respiratory pain, purulent serous discharge from the eyes and nose, anorexia, and sadness.Furthermore, the study found no signifcant diferences in infection rates between males and females.However, age showed a signifcant diference in relation to infection rates.Moreover, oral ulcers were more prominent in 4-12-month age groups, where a strong t-test (10.87) and low p value (p < 0.0016) have been calculated.While we estimated a decrease in the prevalence of oral ulcer lesions in 24-to 36-month olds.Tese fndings revealed that these animals' oral lesions exhibit an inverse relationship with age.A study of molecules and phylogeny showed that the PPR-V virus's N gene partial amplicon in Al-Qadisiyah sheep was 74/150 (49.3%) at 608 bp.Te PPR N gene amino acid analysis revealed 25 substitutions.Te analysis discovered a shift in protein codon 56 from Ala to Val. that may afect protein stability.Moreover, we recorded that the PPR strain of the local Iraqi sheep breed is identical 100% to the Nigerian strain and highly similar to the strains of neighboring countries.In conclusion, the present study indicated that PPR infection in the local Iraqi breed of sheep was genetically identical to that in neighboring countries.Since N gene mutations afect infection and dissemination, we must monitor and genetically defne PPR virus strains.

Table 1 :
PCR thermocycler program employed for PPR-virus diagnosis by PCR.

Table 2 :
Infection rates of PPR-V according to age.
3.3.PCR Analysis.Te results of the RT-PCR assay for detection partial amplicon of the viral N gene of PPR-V of infected sheep in the province of Al-Qadisiyah 74/150 (49.3%) at (608 bp) as shown in Figure

Table 3 :
Correlation between clinical manifestations of Peste des Petits ruminants and the occurrence rate across diferent age groups.
acid to a larger one with a bulkier side chain could significantly impact the protein's structure and stability.Te larger side chain of valine might introduce steric hindrance or alter the local conformation of the protein, potentially afecting its overall fold or stability and subsequently its function and interactions with other molecules.

Table 4 :
Accession numbers employed in this study to conduct the phylogenetic analysis.

Table 5 :
Identity and coverage percentage of PPR virus sequences from diferent hosts and sources.

Table 6 :
A total of 52 places variations found in multiple sequence alignment of feld PPR isolates from the current investigation (MW682328.1)and 8 GenBank nucleotide sequences of PPR samples.

Table 7 :
Amino acids substitution mutations recorded in the study isolate [MW682328] compared to 8 GenBank isolates of PPR virus samples.