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\begin{document}

\title{Genetic characterization and Temporal dynamics of Orf virus in Small
Ruminants from Republic of Niger and parts of Northern Nigeria sold in
Livestock Market in Abuja}



\author[1]{Babatunde Motayo}%
\author[2]{Anyebe Bernard ONOJA}%
\author[3]{Oluwaseyi AJAGBE}%
\author[4]{Andrew Musa ADAMU}%
\author[5]{Cornelius Arome OMATOLA}%
\author[6]{Ismaila Shittu}%
\author[7]{Adenike AJAGBE}%
\affil[1]{Federal Medical Centre Abeokuta}%
\affil[2]{University of Ibadan College of Medicine}%
\affil[3]{University of Abuja}%
\affil[4]{James Cook University Australian Institute of Tropical Health and Medicine}%
\affil[5]{Kogi State University Faculty of Natural Sciences}%
\affil[6]{National Veterinary Research Institute}%
\affil[7]{University of Maiduguri}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background}: Orf virus (ORFV) is an important zoonotic parapox
virus, with 100\% morbidity. It affects mainly domesticated ruminants
such as sheep and goats. This study determined the molecular
epidemiology of ORFV local as well as imported sheep and goats in
Nigeria and evolution of ORFV in Africa. \textbf{Methods}: A total of 30
small ruminants with orf were sampled in a livestock market Abuja
Nigeria.Skin scabs from pathological tissues were collected and
processed for viral genomic DNA. PCR and Sanger sequencing of B2L gene
of ORFV. Phylogenetic analysis, phylogeography, and Bayesian skygird
reconstruction (BSK), including mutational changes were performed on B2L
gene sequences. \textbf{Results}: An ORFV positivity rate of 67\% was
determined from samples. Animals \textless{}2 years had the highest
prevalence of 76.5\%.Hundred percent attack rate was observed among the
Uda and WAD breeds, followed by Niger 71.4\%, Kano Brown 66.7\%, Yankasa
25\% and Balami 0\% breeds. ORFV from Africa breeds clustered into 2
major lineages, Asian and African, with an evolutionary rate of 7.45 \selectlanguage{ngerman}×
10 \textsuperscript{-4}, 95\% HPD (3.46 × 10 \textsuperscript{-4} to
1.17 × 10 \textsuperscript{-3}) substitutions/ site/year. Viral
population demography showed a constant population growth with a slight
rise in viral population growth towards year 2020. \textbf{Conclusion}:
We report molecular evolution of ORFV in Africa and identified gap in
molecular data; we recommend regional molecular surveillance of ORFV and
other zoonotic trans-boundary diseases in global health prevention and
control effort.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

Orf virus (ORFV) is a member of parapoxvirus genus which belongs to the
family~\emph{Poxviridae} . Ithas a linear double-stranded DNA which is
about 135 kbp in size. Orfis (also known as contagious ecthyma) is an
infectious pustular dermatitis which primarily affects sheep, goats and
wild ruminants\textsuperscript{\textbf{1}} . It is characterized by
proliferative skin lesions on the lips, muzzle, ears, eyelids, and found
around the mouth and nostrils of lambs\textsuperscript{\textbf{2}} .
Transmission within the herd is by direct contact between animals. The
morbidity may reach 100\%, but mortality is usually less than 1\%. It is
zoonotic and humans can become infected through direct contact with sick
animals. In humans, it has predilection for the hands where it appears
as rash-shaped papule and can progressively extend to form
pustules\textsuperscript{\textbf{3}} . Sheeppox and goatpox are
important trans-boundary diseases that are notifiable to the Office
Internationale des Epizooties\textsuperscript{\textbf{4}} . Mortality
and morbidity in young animals can be high\textsuperscript{\textbf{5,6}}
. Exotic breeds of sheep and goats are more susceptible than indigenous
breeds that exhibit some level of natural
immunity\textsuperscript{\textbf{7}} while in na\selectlanguage{ngerman}ïve animals, mortality
can reach100\%\textsuperscript{\textbf{8}} .The disease is endemic in
North African countries of Morocco, Tunisia, Algeria and Libya because
of porous land borders coupled with legal and illegal trade of animals.
It is widespread in Central Africa\textsuperscript{\textbf{9}} . It has
been reported in Southern Europe, the Middle East, Central Asia, India
and China\textsuperscript{\textbf{10}} . In West Africa, pastoral
farming is widely practiced because of unrestricted trans-border
movement leading to importation of ORFV which infects sheep and goats.
Nigeria has the largest small ruminant population in Africa with about
73.8 million goats and 42.1 million sheep located mainly in the Northern
part of the country\textsuperscript{\textbf{11}} . Clinical and
serological evidence of Orf has been reported in
Nigeria\textsuperscript{\textbf{12}} , with few reports of molecular
identification. Since there is increased livestock trade and cross
border activities between Nigeria and neighboring countries, it is
important to determine the evolutionary diversity of ORFV in Nigeria.
Also, the zoonotic nature of ORFV underpins the public health importance
of this study. We determined the prevalence, and molecular epidemiology
of ORFV in sheep and goats imported into Nigeria's capital Abuja from
neighboring countries, the temporal dynamics and evolution of ORFV
across Africa.

\textbf{Materials and method}

\textbf{Study design}

This is a prospective observational study which carried out in August
2019. A total of 30 small ruminants with contagious ecthyma were sampled
in a livestock market at the Federal Capital Territory (FCT) Abuja.
These ruminants were raised under extensive farming system in Kano (5),
Yobe (4), Katsina (5), Jigawa (1), Niger (1) and Niger Republic (14)
from where they were shipped to the FCT (Figure 1). Common clinical
signs include weakness, inappetence, lymphadenopathy, rough hair coat,
cough, scabby lesions on the commissures of the lips, mouth, muzzle,
skin, eyes, nose ears and head region.

\textbf{DNA Extraction}

Skin scabs from pathological tissues were centrifuged at 10,000 g for 10
min at +4 °C. The centrifuged scabs was used for viral genomic DNA
extraction using Blood-Animal-Plant DNA Preparation kit (Jena
Biosciences®, Germany). A total of 300 \selectlanguage{greek}µ\selectlanguage{english}l of lysis buffer was added to
200 \selectlanguage{greek}µ\selectlanguage{english}l of tissue homogenate and 2 \selectlanguage{greek}µ\selectlanguage{english}l RNAse A inhibitor was added and
vortexed for 30 seconds, then 8\selectlanguage{greek}µ\selectlanguage{english}l of proteinase K was added to digest
the tissue and placed on heating block at 60\textsuperscript{o}C for 30
minutes. This was cooled for 5mins and 300 \selectlanguage{greek}µ\selectlanguage{english}l binding buffer was added
and centrifuged at 10,000g for 5mins. The supernatant was decanted into
a spin column and washed twice with wash buffer. Fifty microliter of
viral DNA was eluted into a new eppendorf tube, labeled and kept at
-4\textsuperscript{o}C briefly before PCR.

\textbf{Molecular detection using conventional Polymerase Chain
Reaction}

Fragments containing the B2L genes were amplified by PCR in a 25 \selectlanguage{greek}μ\selectlanguage{english}l
reaction volume containing 20UM forward primer, 20 UM reverse primer, 2
mM dNTPs, 5x PCR Buffer (Applied Biosystem), 5X Taq Polymerase (Jena
Biosciences) and 5 \selectlanguage{greek}μ\selectlanguage{english}l template DNA. Cycling conditions were: initial
denaturation at 94\selectlanguage{ngerman}°C for 3 mins, followed by 35 cycles at 94°C for 50s,
52°C for 60s and 72°C for 90s, and final extension at 72°C for 7 min.
Primers ORFV-B2Lf-For 5'-GACCTTCCGCGCTTTAATTT-3 and ORFV-B2Lf-Rev
5'-CCCGCCTGCTAAAAGACT-3'. Each PCR run included a negative control
consisting of 5\selectlanguage{greek}μ\selectlanguage{english}l of PCR grade water instead of the DNA template.
Amplified PCR products were subjected to electrophoresis on 1.5\%
agarose gel stained with ethidium bromide and visualized using Biorad\selectlanguage{ngerman}®
transilluminator.

\textbf{Sequencing and phylogenetic analysis}

Amplified DNA fragments of 1,210bp were used forthe dideoxynucleotide
sequencing reactions. PCR-amplified products were analyzed on an ABI
Prism 3700 automated DNA sequencer (Applied Biosystems, Foster City,
California). Low quality sequences were trimmed with Chromas version
2.6.2 for windows licensed by Technelysium Pty Ltd 1998-2016
(www.technelysium.com.au). The sequences were combined with those
currently available from GenBank (as shown in Table 2). Pairwise and
multiple genomic alignments were done with Clustal
W\textsuperscript{\textbf{13}} alignment programs. The evolutionary
history was inferred by using the Maximum Likelihood (ML) method and
Kimura 2-parameter model\textsuperscript{\textbf{14}} . The percentage
of trees in which the associated taxa clustered together is shown next
to the branches. Initial tree(s) for the heuristic search were obtained
automatically by applying ML algorithm with a GTR+G model, and 1000
bootstrap resampling. The tree is drawn to scale, with branch lengths in
the same units as those of the evolutionary distances used to infer the
phylogenetic tree. This analysis involved 55 nucleotide sequences with
556 positions in the final dataset. Evolutionary analyses were conducted
in iqtree\textsuperscript{\textbf{15}} . Sequences were submitted to the
GenBank and accession numbers MW916908-MW916918 were assigned for 11
isolates in this present study. Information regarding the study
sequences can be accessed in supplementary table 1.

\textbf{Phylogeography and Bayesian Skyride analysis}

Phylogenetic trees were generated by Bayasian inference through Markov
chain Monte Carlo (MCMC) implemented in BEAST version 2.5. To
reconstruct the ancestral-state phylogeographic transmission across
countries, the discrete-trait model implemented in BEAST version
2.5\textsuperscript{\textbf{16}} . The coding genes were partitioned
into first+second and third codon positions and applied a separate
Hasegawa-Kishino-Yano (HKY+G) substitution model with gamma-distributed
rate heterogeneity among sites to each
partition\textsuperscript{\textbf{17}} .The relaxed clock with Gausian
Markov Random Field Skyride plot (GMRF) coalescent prior was selected
for the final analysis. The MCMC was set at 100, 000, 000 states
with10\% as burn in. Results were visualized using Tracer version 1.8.
(http://tree.bio.ed.ac.uk/software/tracer/) all effective sampling size
ESS values were \textgreater{}200 indicating sufficient sampling.
Bayesian Skyride analysis was carried out to visualize the epidemic
evolutionary history using Tracer v 1.8.
(http://tree.bio.ed.ac.uk/software/tracer/).

\textbf{Statistical analysis}

Statistical Packages for Social Sciences (SPSS) version 16.0 was used to
analyze the data. Prevalence was computed by dividing the number of
positive animals by the total animals tested and multiplied by 100.
Univariate logistic regression at 95\% confidence level was performed to
assess potential influence of demographic parameters on the occurrence
of Orf. Level of statistical significance was p value \selectlanguage{english}[?]0.05.

\textbf{Results}

Out of 30 samples screened for ORFV, 20 were positive giving a rate of
67\%. Animals \textless{}2 years had higher prevalence (76.5\%) of ORF
compared to older ages 2-3 years and \textgreater{}3 years with 50\% and
66.7\%, respectively. More females than males (80\% versus 64\%) were
infected with ORFV despite having five-fold more of the later in our
study. Logistic analysis showed rate of occurrence of Orf did not differ
significantly with animals' ages and sexes (P\textgreater{}0.05). Goat
with 71.4\% prevalence demonstrated higher susceptibility to ORFV
compared to sheep (55.6\%). Hundred percent attack rate was observed
among the Uda and WAD breeds followed by a decreasing susceptibility in
Niger (71.4\%), Kano Brown (66.7\%), Yankasa (25\%) and Balami (0\%)
breeds(Table 1). A differential distribution of Orf was observed in
relation to sources of livestock sampled from the market. Animals
sourced from Niger State (100\%) were more infected followed by Kano
State (80\%), Yobe State (75\%), Niger republic (71\%) and Katsina State
(40\%). Though, current study did not observe any significant influence
of source on the occurrence of ORFV infection (p\textgreater{}0.05).

The ML phylogenetic treeshowed a distinct diversification of the
isolates analyzed into 2 lineages, African and Asian lineage, with
majority of the Nigerian strains clusteringwithin the African lineage, a
few Nigerian stains also clustered within the Asian lineage along with
some South African strains and reference strains from China (Fig. 2).

Time scaled MCC phylogeny of the ORFV sequences indicates that the MRCA
of the African strains was around 2002. The African Viruses clustered
into 2 major clades with both clades containing West African (Nigerian)
and South African viruses (Fig. 3a). There was also a strain from
Botswana which clustered very closely with Nigerian isolates and shared
same TMRCA of around 2014 highest posterior density interval (HPD
2012-2017), while the TMRCA for the entire African sequences was around
2004 (HPD 2000-2006).. The evolutionary rate of the African ORFV B2L
genes was 7.45 x 10\textsuperscript{-4}, 95\% HPD (3.46 x
10\textsuperscript{-4} to 1.17 x 10\textsuperscript{-3}) substitutions/
site/year. The demographic history of the African ORFV B2L genes showed
a constant viral population expansion from 2005 to 2016, with a slight
rise in population expansion between 2018 to 2020 (Fig. 3b). Amino acid
alignment of the complete B2L protein coding sequence also showed some
non-synonymous mutations (Suppl. Figure 1) which are summarized in Table
2. The sampling dates and Genbank accession numbers of the study
sequences can be assessed in Suppl. Table 1.

\textbf{Discussion}

In this study, high prevalence of Orf occurred among animals of all ages
which reinforces the endemicity which may jeopardize the animal
productivity in the area. The greater incidence of ORF in ages
\textless{}2years indicates increased susceptibility due to lower
immunity in younger ages which has been corroborated in other
studies\textsuperscript{\textbf{18,19}} . The absence of statistical
significance despite higher prevalence of ORF among female animals
support reports from the eastern Sudan which posited that sex is not a
significant predictor of Orf\textsuperscript{\textbf{19}} . Higher rate
of susceptibility in goats compared to ovine species has been
reported\textsuperscript{\textbf{20}} . The naturally aggressive
behavior of the former compared to the latter predisposes them to
injury, thereby increases the susceptibility to ORFV transmitted via
direct contact. Most small livestock holders in Nigeria do not practice
dehorning as observed in this study and this may increase the risk of
deep wounds or injuries which serve as a predisposing factor for virus
penetration via the skin wounds. Current study observed 100\% morbidity
among the Uda and WAD breeds, similar to previous observation in Ibadan
(Onoja et al., unpublished data) and Jos\textsuperscript{\textbf{21}}
respectively. These findings suggest the influence of intrinsic genetic
factors in certain breed susceptibility to Orf which needs to be fully
investigated.

Over 70\% morbidity rates were observed among animals imported from
Niger republic and the ones sourced from Kano, Niger and Yobe States,
raising the concern of trans-boundary and national sporadic spread to
susceptible new herds which could be the starting point of contagious
ecthyma outbreaks often associated with production loss in livestock and
reduction in the market value\textsuperscript{\textbf{22}} . The high
morbidity observed in this study can be traceable to inadequate
hygienic\textsuperscript{\textbf{20}} and quarantine procedures as most
of the sick animals are not isolated before mixing up with previously
apparently healthy sheep and goats. Climatic change over the years has
made more ruminants susceptible to this hitherto low pathogenic but
highly contagious viral disease. In addition, Abuja been a commercial
centre and the nation's capital receives a high influx of both human and
livestock. With a burgeoning population, consumption of livestock is on
the increase consequently increasing importation of sheep and goat from
other African countries. It is noteworthy that all samples were
collected from live markets where sheep and goats are ready to be sold.
In the process of handling and bargain between the potential buyer and
seller, transmission and infection with contagious ecthyma may occur.
The highly contagious nature of contagious ecthyma affects production.
Animals refuse or reduce intake of food which leads to poor market value
consequently leading to low protein availability and loss to the
farmers.

The phylogenetic analysis revealed that majority of the isolated
Nigerian strains clustered within the African lineage, with a few stains
isolated from goats sold within the FCT, clustering within the Asian
lineage along with South African strains and reference strains isolated
from China (Figure 2). This observation suggests that the goats sampled
from Nigeria infected with the Asian Lineage, confirms Orf virus as a
transboudary animal disease. This confirms that some of the Nigeria
goats were breed outside the country in neighboring Countries such as
Niger republic or Chad and might have had contact with other ruminants
from North Africa or Arabia, where the Asian virus might have been
introduced. Another theory for this observation is that the Asian virus
might have been circulating within the African continent for decades
undetected because of poor surveillance and weak diagnostic
infrastructure. The MCC tree of African Orf B2L sequences was also
clustered into a similar topology with majority of the African sequences
falling in to the African Lineage and FCT, South African and Egyptian
strains falling within the Asian lineage (Figure 3a). This clustering of
sequences further buttresses our earlier observation that the sheep and
goats may have been infected with the same strain during transit. Since
majority of sheep and goats are from neighboring African countries
especially Chad and Niger republic, sharing borders with Northern
Nigeria, it is not unlikely that they came from a common source and were
transported together across the border to Nigeria. Moreover, the stress
of trans-border transportation makes the animals more susceptible to
contagious ecthyma virus. Unchecked and indiscriminate trans-border
activity of both humans and animals is a very important factor in the
dissemination of ORFV. The time to most recent common ancestor (TMRCA)
for the Nigeria African lineage was around 2014, highest posterior
density interval (HPD 2012-2017), while the TMRCA for the entire African
sequences was around 2004 (HPD 2000-2006). This is quite recent and
might not actually represent the true picture of when the virus was
introduced into circulation because of the paucity of sequence
information and regency of the data presented. However it shows the gap
in knowledge posed by insufficient molecular data on this virus in
Africa. In addition the calculated evolutionary rate of the African Orf
virus B2L genes was 7.45 x 10\textsuperscript{-4}, 95\% HPD (3.46 x
10\textsuperscript{-4} to 1.17 x 10\textsuperscript{-3}) substitutions/
site/year. This is higher than the calculated evolutionary mean rate of
1.32 x 10\textsuperscript{-6} derived for whole Orf gene sequences from
a recent study\textsuperscript{\textbf{23}} . This observation might be
due to the small genome size analyzed, as well as the limited number of
sequences, but reelects the relatively slower evolutionary rate for DNA
viruses in comparism to RNA viruses. The viral demographic history
portrays a steady population demography throughout the years past,
except for a slight increase towards the year 2020, around 2016 (Fig.
3b). This observation should be taken with caution as it may not
represent the true picture because of the lack of sequence data from
Africa before the year 2000, however the rise in viral population,
towards the more recent years shows an increase in detectable cases
reflected by more sequence information being made available in various
databases.

Table 2 shows a summary of major amino acid mutations observed among our
B2L ORFV sequences in relation to the prototype vaccine strain
(Accession number AY278208.1). The mutations observed are concomitant
with that of a previously isolated strain reported in Sokoto State. The
phenotypic properties of these mutations have not been well described,
but some of them may affect the pathogenicity of the
isolates\textsuperscript{\textbf{24}} . Limitations of this study are
paucity of African ORFV genomic data, and the small sample size.

In conclusion, we report the genetic diversity and molecular evolution
of ORFV in Africa based on B2L sequences. Further, we highlighted the
epidemiology of ORFV among goats and sheep sold in Northern Nigeria. The
impact of trans-border movement of farm animals such as sheep and goats
on the transmission pattern of trans-boundary disease such as ORFV, and
have illuminated the information gap particularly genetic information of
this virus in Africa. We hereby recommend a regional approach to
molecular surveillance of ORFV and other zoonotic trans-boundary animal
diseases to help in prevention and control effort.

\textbf{Figure Legends}

\textbf{Fig. 1:} Map showing locations were samples were collected in
Nigeria and where animal were imported from Republic of Niger.

\textbf{Fig. 2:} Maximum likelihood phylogenetic tree of African ORFV
B2L genes including global reference sequences. Blue lines represent
study sequences, and other Nigerian isolates, red lines represent South
African sequences, green lines represent Egyptian isolates. Light blue
box represents the Asian lineage, light green box represents isolates
within the African lineage.

\textbf{Fig. 3a.} Bayesian MCC tree showing African isolates, countries
are represented by the color code the figure legend. Light blue box
represents isolates within the African lineage, light green box
represents isolates within the Asian lineage. \textbf{3b} . Population
demography of African ORFV B2L genes represented by a Bayesian skyride
plot showing effective viral population on the y axis and sampling time
on the x-axis.

\textbf{Author's Contributions}

OAB designed the study and carried out molecular identification;AO,
AAMcollected samples; OAB, AO, AAM, AA funded sequencing of the
amplified products. OCAperformed data analysis,MBperformed
bioinformatics analysis. IScleaned sequences, carried out the
phylogenetic analysis OAB, AO, AAM wrote the initial draft. All authors
reviewed and approved the final manuscript before submission.

\textbf{Funding}

This work was supported by grant award number D43TW010140 funded by
Fogarty International Centre of the National Institutes of Health to the
University of Ibadan Medical Education Partnership Initiative Junior
Faculty Project (UI-MEPI-J).

\textbf{Data Availability Statement}

The data that support the findings of this study are freely available in
GenBank, reference numbers MW916908-MW916918.

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\end{enumerate}

\textbf{Hosted file}

\verb`FIGURES_Orf figs.docx`  available at \url{https://authorea.com/users/577616/articles/619941-genetic-characterization-and-temporal-dynamics-of-orf-virus-in-small-ruminants-from-republic-of-niger-and-parts-of-northern-nigeria-sold-in-livestock-market-in-abuja}

\textbf{Hosted file}

\verb`Tables Orf pp.docx`  available at \url{https://authorea.com/users/577616/articles/619941-genetic-characterization-and-temporal-dynamics-of-orf-virus-in-small-ruminants-from-republic-of-niger-and-parts-of-northern-nigeria-sold-in-livestock-market-in-abuja}

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