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\begin{document}

\title{MERS-CoV in sheep, goats, and cattle, United Arab Emirates, 2019:
Virological and serological investigations reveal an accidental
spillover from dromedaries}



\author[1]{Pia Weidinger}%
\author[1]{Jolanta Kolodziejek}%
\author[1]{Jeremy Camp}%
\author[2]{tom loney}%
\author[3]{Dafalla Ahmed}%
\author[4]{Sathishkumar Ramaswamy }%
\author[2]{Ahmad Abou Tayoun}%
\author[5]{Victor Max Corman}%
\author[1]{Norbert Nowotny}%
\affil[1]{Veterinarmedizinische Universitat Wien}%
\affil[2]{Mohammed Bin Rashid University of Medicine and Health Sciences College of Medicine}%
\affil[3]{Al Ain City Municipality}%
\affil[4]{Al Jalila Children's Specialty Hospital}%
\affil[5]{Charite Universitatsmedizin Berlin}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The recent COVID-19 pandemic has demonstrated again the global threat
posed by emerging zoonotic coronaviruses. During the past two decades
alone, humans have experienced the emergence of several coronaviruses,
such as SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019. To
date, MERS-CoV has been detected in 27 countries, with a case fatality
ratio of approximately 34.5~\%. Similar to other coronaviruses, MERS-CoV
presumably originated from bats; however, the main reservoir and primary
source of human infections are dromedary camels. Other species within
the \emph{Camelidae} family, such as Bactrian camels, alpacas, and
llamas, seem to be susceptible to the infection as well, although to a
lesser extent. In contrast, susceptibility studies on sheep, goats,
cattle, pigs, chickens, and horses obtained divergent results. In the
present study, we tested nasal swabs and/or sera from 55 sheep, 45
goats, and 52 cattle, collected at the largest livestock market in the
United Arab Emirates, where dromedaries are also traded, for the
presence of MERS-CoV nucleic acid by RT-qPCR, and for specific
antibodies by immunofluorescence assay (IFA). All sera were negative for
MERS-CoV-reactive antibodies, but the nasal swab of one sheep (1.8~\%)
was positive for MERS-CoV nucleic acid. Next generation sequencing (NGS)
of the complete \emph{N} gene of the sheep-derived MERS-CoV revealed
\textgreater{}99 \% nucleotide identity to MERS-CoV sequences of five
dromedaries in nearby pens and to three reference sequences. The NGS
sequence of the sheep-derived MERS-CoV was confirmed by conventional
RT-PCR of a part of the \emph{N} gene and subsequent Sanger sequencing.
All MERS-CoV sequences clustered within clade B, lineage 5. In
conclusion, our study shows that non-camelid livestock, such as sheep,
goats, and cattle do not play a major role in MERS-CoV epidemiology. The
one sheep that tested positive most likely reflects an accidental viral
spillover event from infected dromedaries in nearby pens.%
\end{abstract}%



\sloppy


\textbf{MERS-CoV in sheep, goats, and cattle, United Arab Emirates,
2019: Virological and serological investigations reveal an accidental
spillover from dromedaries}

Short running title (less than 70 characters):

\textbf{Study on MERS-CoV in sheep, goats, and cattle, UAE, 2019}

Pia Weidinger\textsuperscript{1}, Jolanta
Kolodziejek\textsuperscript{1}, Jeremy V. Camp\textsuperscript{1,2}, Tom
Loney\textsuperscript{3}, Dafalla O. Kannan\textsuperscript{4},
Sathishkumar Ramaswamy\textsuperscript{5}, Ahmad Abou
Tayoun\textsuperscript{3,5}, Victor M. Corman\textsuperscript{6},
Norbert Nowotny\textsuperscript{1,3}

\textsuperscript{1}Viral Zoonoses, Emerging and Vector-Borne Infections
Group, Institute of Virology, University of Veterinary Medicine, Vienna,
Austria

\textsuperscript{2}Center for Virology, Medical University of Vienna,
Austria

\textsuperscript{3}College of Medicine, Mohammed Bin Rashid University
of Medicine and Health Sciences, Dubai, United Arab Emirates

\textsuperscript{4}Al Ain City Municipality, Al Ain, United Arab
Emirates

\textsuperscript{5}Al Jalila Genomics Center, Al Jalila Children's
Specialty Hospital, Dubai, United Arab Emirates

\textsuperscript{6}Institute of Virology, Charit\selectlanguage{ngerman}é-Universitätsmedizin
Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, and
German Centre for Infection Research (DZIF), Partner Site Charité,
Berlin, Germany

*Corresponding author: Norbert Nowotny, Viral Zoonoses, Emerging and
Vector\selectlanguage{english}-Borne Infections Group, Institute of Virology, University of
Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria.
Phone: +431250772403; Fax: +431250772390; Email:
Norbert.Nowotny@vetmeduni.ac.at

\emph{ORCID IDs}

Pia Weidinger https://orcid.org/0000-0003-4028-4151

Jolanta Kolodziejek https://orcid.org/0000-0001-5736-3644

Jeremy V. Camp https://orcid.org/0000-0002-9040-5786

Tom Loney https://orcid.org/0000-0003-1687-6587

Dafalla O. Kannan https://orcid.org/0000-0001-5819-0028

Sathishkumar Ramaswamy https://orcid.org/0000-0002-7467-6658

Ahmad Abou Tayoun https://orcid.org/0000-0002-9134-1673

Victor M. Corman https://orcid.org/0000-0002-3605-0136

Norbert Nowotny https://orcid.org/0000-0002-3548-571X

\textbf{Ethics Statement}

The authors confirm that the ethical policies of the journal, as noted
on the journal's author guidelines page, have been adhered to. No
ethical approval was required for sampling of the involved animals.

\textbf{Summary}

The recent COVID-19 pandemic has demonstrated again the global threat
posed by emerging zoonotic coronaviruses. During the past two decades
alone, humans have experienced the emergence of several coronaviruses,
such as SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019. To
date, MERS-CoV has been detected in 27 countries, with a case fatality
ratio of approximately 34.5~\%. Similar to other coronaviruses, MERS-CoV
presumably originated from bats; however, the main reservoir and primary
source of human infections are dromedary camels. Other species within
the \emph{Camelidae} family, such as Bactrian camels, alpacas, and
llamas, seem to be susceptible to the infection as well, although to a
lesser extent. In contrast, susceptibility studies on sheep, goats,
cattle, pigs, chickens, and horses obtained divergent results. In the
present study, we tested nasal swabs and/or sera from 55 sheep, 45
goats, and 52 cattle, collected at the largest livestock market in the
United Arab Emirates, where dromedaries are also traded, for the
presence of MERS-CoV nucleic acid by RT-qPCR, and for specific
antibodies by immunofluorescence assay (IFA). All sera were negative for
MERS-CoV-reactive antibodies, but the nasal swab of one sheep (1.8~\%)
was positive for MERS-CoV nucleic acid. Next generation sequencing (NGS)
of the complete \emph{N} gene of the sheep-derived MERS-CoV revealed
\textgreater{}99 \% nucleotide identity to MERS-CoV sequences of five
dromedaries in nearby pens and to three reference sequences. The NGS
sequence of the sheep-derived MERS-CoV was confirmed by conventional
RT-PCR of a part of the \emph{N} gene and subsequent Sanger sequencing.
All MERS-CoV sequences clustered within clade B, lineage 5. In
conclusion, our study shows that non-camelid livestock, such as sheep,
goats, and cattle do not play a major role in MERS-CoV epidemiology. The
one sheep that tested positive most likely reflects an accidental viral
spillover event from infected dromedaries in nearby pens.

\textbf{Keywords}

Sheep, goat, cattle, Middle East respiratory syndrome, Middle East
respiratory syndrome coronavirus, MERS-CoV

\textbf{Introduction}

Since the 1930s, members of the family \emph{Coronaviridae} have been
identified in humans and many different animal species, such as cats,
dogs, horses, pigs, cattle, mice, and fowl . In the last 20 years, three
zoonotic coronaviruses have emerged into the human
population:\emph{Severe acute respiratory syndrome-related coronavirus}
(SARS-CoV) in 2003, \emph{Middle East respiratory syndrome-related
coronavirus}(MERS-CoV) in 2012, and most recently, SARS-CoV-2 in 2019,
which led to the so far most devastating pandemic of the
21\textsuperscript{st}century . Since 2012, MERS-CoV has been detected
in 27 countries, with 84.0~\% of cases found in Saudi Arabia, and a case
fatality ratio of approximately 34.5~\% (WHO, 2019). Similar to other
coronaviruses, MERS-CoV presumably originated from bats; however, the
main reservoir and primary source of human infections are dromedaries
(\emph{Camelus dromedarius} ) . Although dromedaries usually remain
asymptomatic, or develop only mild symptoms due to MERS-CoV infection,
it has been shown that they can shed considerable amount of virus .
Studies on Bactrian camels (\emph{Camelus bactrianus} ), hybrid camels,
alpacas (\emph{Vicugna pacos} ), llamas (\emph{Lama glama} ), and even
pigs (\emph{Sus scrofa} ), have revealed that these animals are also
susceptible to MERS-CoV infection (Adney et al., 2019; Lau et al., 2020;
Reusken et al., 2016; Vergara-Alert et al., 2017). In contrast, studies
on sheep, goats, cattle, horses, and chickens have indicated that,
although sheep and goats may produce antibodies to MERS-CoV, none of
these species effectively shed the virus (Adney et al., 2016; Hemida et
al., 2013; Reusken, Ababneh et al., 2013; Vergara-Alert et al., 2017). A
study about domestic mammals in contact with infected dromedaries found
MERS-CoV nucleic acid in nasal swabs not only from sheep and goats, but
also from a cow and donkeys (Kandeil et al., 2019). To provide more
information about the potential exposure and infection of other
livestock, we performed a survey for MERS-CoV at a livestock market in
the United Arab Emirates (UAE), as live animal markets are known to be
common sources of virus spillover.

\textbf{Materials and Methods}

\emph{Sampling}

Sampling was performed at the largest national livestock market in the
UAE, the Al Ain central market in the emirate of Abu Dhabi, in October
2019. The market is divided into three main areas, with mostly mixed
sheep and goat pens located between camel and cattle stalls (Fig. 1).
The distance between camel pens and the closest pens holding sheep and
goats is approximately 20~m.

In total, we sampled 152 animals, which appeared healthy at the time of
sampling. Nasal swabs and blood samples were taken from 55 sheep (41
female and 14 male) with an age range from three months to four years
(mean of 19 months), and 45 goats (31 female and 14 male) from three to
40 months old (mean of 12 months). In addition, we collected blood
samples from 52 cattle (43 female and nine male) from one to six years
(mean of 3.8 years). Nasal swabs were collected in tubes containing a
virus inactivation solution (DNA/RNA Shield, ZymoResearch, Irvine, CA,
USA). Serum was separated from each blood sample by centrifugation. All
samples were then stored at -80degC at the laboratory of the College of
Medicine, Mohammed Bin Rashid University of Medicine and Health
Sciences, Dubai, UAE, before shipment on dry ice to the University of
Veterinary Medicine, Vienna, Austria, for virological and serological
investigations.

\emph{MERS-CoV nucleic acid screening}

Nasal swabs of sheep (n=55) and goats (n= 45), as well as serum samples
of all animals (n=152) were screened for MERS-CoV nucleic acid by
reverse transcription real-time (RT-q)PCR. For this, 200~\selectlanguage{greek}µ\selectlanguage{english}l of each
sample were subjected to automated nucleic acid extraction employing
QIAamp Viral RNA Mini Kit on QIAcube HT (both Qiagen, Hilden, Germany).
MERS-CoV-specific RT-qPCR was performed using qScript XLT One Step
RT-qPCR ToughMix (QuantaBio, MA, USA) and previously described primers
and probe in the open reading frame (\emph{ORF} ) \emph{1a} gene .
RT-qPCR-positive samples were additionally tested by conventional
RT-PCRs (OneStep RT-PCR Kit, Qiagen, Hilden, Germany) employing two
previously published primer pairs within the nucleocapsid (\emph{N} )
and\emph{ORF1b} genomic regions of MERS-CoV . Amplicons from the RT-PCRs
were subjected to Sanger sequencing (Eurofins Genomics, Ebersberg,
Germany). In addition, next generation sequencing (NGS) was performed on
putative positive samples and five selected MERS-CoV-positive samples
from dromedaries in nearby pens. For this, adapter sequences were
trimmed using bcl2fastq2 conversion software v2.20.0, and high-quality
sequencing reads were aligned to three reference MERS-CoV genome
sequences (GenBank acc. numbers KF192507, MG757604 and MK462253) using
BWA v0.7.17. Consensus sequences were generated for the \emph{N} gene
using GATK v3.8--1--0 and BCFtools v1.3.1. Multiple sequence alignment
of the \emph{N} gene of MERS-CoV (1.2 kb) derived from one sheep and
five dromedary camels was performed using MAFFT v7.455.

\emph{MERS-CoV antibody testing}

Altogether 148 serum samples from sheep (n=55), goats (n=45), and cattle
(n=48) were available in sufficient volumes to perform MERS-CoV antibody
testing. MERS-CoV spike protein-based immunofluorescence assay (IFA) was
done as described previously . Initial screening was performed with
sample dilutions of 1:40, retesting of putative positive (questionable)
samples was done with a dilution series of 1:10 -- 1:320. Detection was
facilitated using 1:200 dilutions of donkey-anti sheep, donkey-anti
goat, or goat-anti bovine secondary antibodies, all labelled with Alexa
Fluor 488 (Dianova, Hamburg, Germany).

\textbf{Results}

\emph{MERS-CoV nucleic acid and antibody prevalence}

The nasal swab of one sheep (1.8~\%) repeatedly tested positive for
MERS-CoV nucleic acid by RT-qPCR (Ct values 32.5-34.8; Table 1), and the
nasal swab of one goat tested borderline positive (Ct values 38.1-39.4;
Table 1). The positive sheep was a two-year-old female, presumably
locally raised Nuaimi breed. It was kept in a pen at the market used for
sheep only, which was the second closest pen to the camel area
(approximately 25~m away; Fig. 1, Supplementary Table 1). The borderline
positive goat, a one-year-old locally raised male Omani goat, was kept
in one of the pens directly adjacent to the camel pens, with only
approximately 20 m between them (Fig. 1, Supplementary Table 1).

The RT-qPCR-positive sheep sample was confirmed by two different
RT-PCRs. The obtained sequences {[}lengths without primer
sequences:\emph{N} -- 293~bp (GenBank acc. number: MZ558082),
\emph{ORF1b} -- 341~bp (GenBank acc. number: MZ558083){]} were
\textgreater{}99~\% identical to many MERS-CoV sequences belonging to
clade B, lineage 5 {[}according to the classification by , including
human and camel strains from the UAE, Saudi Arabia, South Korea, Jordan,
Qatar, and the United Kingdom. Unfortunately, we were not able to
uncover the complete MERS-CoV genomes using NGS; however we succeeded to
establish the complete \emph{N} gene sequences of the virus-positive
sheep and five selected MERS-CoV-positive dromedaries (Figure 2). The
sheep-derived\emph{N} gene sequences obtained by NGS and Sanger
sequencing were identical. By NGS, the average sequencing coverage of
the \emph{N} gene from the sheep (S90\_N) and five camel samples
(C53\_N, C5\_N, C6\_N, C28\_N, and C50\_N) was \textgreater{}6X across
all samples (Figure 2A). The percentage of sequence identity of the
\emph{N} gene of the same samples along with three reference MERS-CoV
sequences (GenBank acc. numbers KF192507, MG757604 and MK462253)
revealed that the sheep sample showed high nucleotide identity
(\textgreater{}99~\%) with all other MERS-CoV sequences (Figure 2B). In
detail, multiple sequence alignment revealed five positions of
nucleotide variations between the sheep-derived MERS-CoV sequence and
the camel-derived sequences and/or reference sequences (representative
positions 533 and 637 of the\emph{N} gene are shown in Figure 2C). The
GenBank accession numbers of the complete MERS-CoV \emph{N} gene
sequences are: sheep-derived (S90\_N): MZ558076; dromedary-derived:
C53\_N - MZ558080; C5\_N - MZ558077; C6\_N - MZ558078; C28\_N -
MZ558079; and C50\_N - MZ558081. Unfortunately, the borderline-positive
result of the goat sample could not be confirmed by either RT-PCR or
NGS.

The serological survey revealed that, although in the initial screening
with 1:40 dilutions 15 serum samples (eight sheep, four goats, three
cattle) appeared questionable, the presence of antibodies could not be
confirmed in any sample by retesting using a dilution series of 1:10 --
1:320 (Supplementary Tables 1 and 2).

\textbf{Discussion}

Previous studies have demonstrated that, besides dromedaries, several
other members of the \emph{Camelidae} family are susceptible to MERS-CoV
infection, such as Bactrian camels, alpacas, and llamas , although human
contact infections have only been reported from dromedary camels .
However, the existing data on the susceptibility of non-camelids are
conflicting. Our results are in line with a series of publications
demonstrating that non-camelids do not play a major role in the
transmission cycle . For instance, in a seroepidemiological study in
Saudi Arabia (2010-2013), no antibodies were detected in sheep, goats,
cattle, or chickens, but a high prevalence was found in camels .
Similarly, after an experimental challenge of sheep, goats, and horses
by intranasal MERS-CoV inoculation, none of the animals effectively shed
the virus, and neutralizing antibodies were only found in goats .
Further regarding the family \emph{Equidae} , including horses, donkeys,
and mules, although viral replication has been observed in primary horse
kidney cells, no antibodies have been found in any of 1,053 equid sera
collected in the UAE and Spain . A study on livestock cell lines from
the Arabian Peninsula showed that MERS-CoV can replicate in goat,
alpaca, and dromedary cells, in contrast to sheep, cattle, rodent, and
insectivore cells . In a study experimentally challenging animals from
France and Spain, MERS-CoV nucleic acid in the respiratory tract as well
as seroconversion were detected only in llamas and pigs, but not in
sheep and horses . A serosurvey in a MERS-CoV-affected area in Jordan
identified MERS-CoV neutralising antibodies in the sera of all camels
but none in goats and cattle, while 4.8~\% of sheep sera reacted with
MERS-CoV antigen . In a monitoring study of several mammalian species in
direct contact with infected dromedaries in Egypt, Tunisia, and Senegal
(2015-2017), antibodies were identified in an astonishing 55.6~\% of
sheep from Senegal, 1.8~\% of sheep from Tunisia, and 0.9~\% of goats
from Egypt. MERS-CoV RNA was detected in nasal swabs from 1.2~\% sheep
and 4.1~\% goats from Egypt and Senegal, as well as 1.9~\% cattle and
7.1~\% donkeys from Egypt . In conclusion, it seems that several
non-camelid species may become infected with MERS-CoV during viral
spillover events when they are in direct contact with virus-shedding
dromedaries. Maybe this susceptibility is due to the close relatedness
of the \emph{Betacoronaviruses} (specifically their receptors) infecting
camels and especially bovids, but also humans, porcines, and equids .
However, whether these animals are implicated in the MERS-CoV
transmission cycle remains unknown, but seems to be highly unlikely.

The fact that none of the sheep, goats, and cattle in our survey tested
positive for antibodies indicates that none of them had previously
experienced a MERS-CoV infection, although with an age range of three
months to six years, all animals could have encountered the virus in the
past. Moreover, as the average time of stay at the market for goats is
usually 7-10 days, 10-14 days for sheep, and between 14 and 30 days for
cattle, there is also a not negligible risk of infection at the market.
However, the nasal swab of one sheep (1.8~\%) contained MERS-CoV nucleic
acid (Supplementary Table 1), suggesting either a current infection, or
a spillover event from nearby dromedaries, which appears to be more
likely since no antibodies were detected and the sheep was kept in a pen
in close proximity to the camel pens (approximately 25~m away; Fig. 1).
Of note, we additionally sampled 90 dromedaries at the same market and
at the same time as the non-camelids, of which 35.6~\% were positive for
MERS-CoV nucleic acid, and another 24.4~\% tested borderline positive
(Ct values 38.6-41.2). Moreover, 91.1~\% of the 90 camel sera were
MERS-CoV antibody-positive by ELISA, and 96.7~\% by IFA (data can be
provided upon request), further supporting the high prevalence of
MERS-CoV infections in dromedaries. This hypothesis is also supported by
our latest study, in which we screened 76 dromedaries during spring and
autumn 2019 at the same market, and found MERS-CoV nucleic acid in
57.9~\% of their nasal swabs .

The positive sheep from this study was a two-year-old female, presumably
locally raised Nuaimi breed. However, due to the fact that sex was not
equally distributed across the animals we sampled (115 females, 75.7~\%,
compared to 37 males, 24.3~\%), no conclusions can be drawn regarding
the susceptibility of the different sexes. The same is true for the
different breeds, with only one positive animal, we cannot infer any
breed-specific genetic predispositions to MERS-CoV infection.

In conclusion, our study shows that non-camelids such as sheep, goats,
and cattle do not play a major role in MERS-CoV epidemiology. The one
sheep that tested RNA-positive in our study most likely reflects a viral
spillover event from infected dromedaries in nearby pens. Furthermore,
serology revealed that the sheep did not develop an antibody response,
suggesting that the infection was very recent, or the sheep only carried
viral particles on its nasal mucosa without actually being infected.

\textbf{Acknowledgements}

The authors thank the General Manager of Al Ain City Municipality, H.E.
Dr. Matar Mohammed Saif Al Nuaimi, and his team, including Dr. Babiker
Mohammed Osman, for supporting the study, as well as Hassan Mohammed
Alkaabi, Head of Veterinary Services, Hashim Ahmed Saeed and Md. Helal
Ahmed for assistance with sampling at the livestock market. We are
grateful for the help of Noushad Karuvantevida, Athiq Ahmed Wahab, and
Abubakkar Babuhan, from the Mohammed Bin Rashid University of Medicine
and Health Sciences, in facilitating the study.

This work was supported by research grants of the College of Medicine,
Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai,
United Arab Emirates (grant no. MBRU-CM-RG2018-14 to N.N., and grant no.
MBRU-CM-RG2019-13 to T.L.).

\textbf{Conflict of Interest Statement}

The authors declare no conflict of interest.

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\textbf{Table 1. Results of the MERS-CoV screening in sheep, goats, and
cattle.} Nasal swabs from sheep and goats, and sera from all animals
were tested for MERS-CoV nucleic acid by \emph{ORF1a} RT-qPCR. Serum
samples from all but four animals (\emph{i.e.} cattle sera with
insufficient volumes) were investigated for MERS-CoV-specific antibodies
by IFA.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Species} & \textbf{n (female/male)} & \textbf{Age range
(average)} & \textbf{Nucleic acid-positive} & \textbf{Nucleic
acid-positive} & \textbf{Antibody-positive}\tabularnewline
\midrule
\endhead
& & & \textbf{Nasal swabs} & \textbf{Serum samples} &\tabularnewline
Sheep & 55 (41/14) & 3-48 months (19 months) & 1 (1.8~\%) & 0 &
0\tabularnewline
Goat & 45 (31/14) & 3-40 months (12 months) & 1 borderline (2.2~\%) & 0
& 0\tabularnewline
Cattle & 52 (43/9) & 1-6 years (3.8 years) & 0 & 0 & 0\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure 1:} \textbf{Image of the Al Ain central market in Abu
Dhabi.} Camel pens are located on the left side, cattle stalls on the
right side, and sheep and goats are kept in between. The distance
between camel pens and the closest pens holding sheep and goats is
approximately 20~m (separated by a road). The red asterisk indicates
sheep pen S18, in which the MERS-CoV-positive sheep was sampled, the
green asterisk indicates the mixed pen S17, in which the borderline
positive goat was kept. Image retrieved from Google Maps.

\textbf{Figure 2: Next generation sequencing analysis of the
MERS-CoV\emph{N} gene of sheep and camels.} A) Sequencing coverage of
the MERS-CoV \emph{N} gene derived from one sheep (S90\_N) and five
camels (C53\_N, C5\_N, C6\_N, C28\_N, and C50\_N). The average coverage
of the\emph{N} gene across all samples was \textgreater{}6X. X axis =
\emph{N}gene of MERS-CoV (1.2~kb) and Y axis = sequencing coverage; red
line = sheep-derived MERS-CoV, blue lines = dromedary-derived MERS-CoV.
B) Percentage of \emph{N} gene sequence identity of the same samples
along with three reference MERS-CoV sequences (GenBank acc. numbers
KF192507, MG757604 and MK462253). The sample collected from the sheep
showed high nucleotide identity (\textgreater{}99~\%) with all
camel-derived MERS-CoV sequences as well as the reference sequences. C)
Multiple sequence alignment of the \emph{N} gene of the same MERS-CoV
samples (1.2 kb). Two short representative regions with nucleotide
sequence variations are shown: positions 533 and 637 of the \emph{N}
gene (highlighted by a red vertical box); conserved positions are
indicated by asterisks (*). Multiple sequence alignment was performed
using MAFFT v7.455.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/MERS-CoV-in-sheep,-goats,-and-cattle,-UAE,-2019-Figure-1/MERS-CoV-in-sheep,-goats,-and-cattle,-UAE,-2019-Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/MERS-CoV-in-sheep-goats-and-cattle-UAE-2019-Figure-2/MERS-CoV-in-sheep-goats-and-cattle-UAE-2019-Figure-2}

\end{center}
\end{figure}

\selectlanguage{english}
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