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\begin{document}

\title{SURVEY OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS IN RHIPICEPHALUS AND
DERMACENTOR SPECIES TICKS FROM SOUTH-EASTERN ROMANIA}



\author[1]{Bianca Brătuleanu}%
\author[1]{Delphine Chretien}%
\author[1]{Béatrice Regnault}%
\author[1]{Sarah Temmam}%
\author[1]{Philippe Perot}%
\author[2]{Adriana Anita}%
\author[2]{Dragos Anita}%
\author[2]{Gheorghe Savuta}%
\author[1]{Marc Eloit}%
\affil[1]{Institut Pasteur}%
\affil[2]{Universitatea de Stiinte Agricole si Medicina Veterinara Ion Ionescu de la Brad din Iasi}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Few previous studies suggested the circulation of Crimean Congo
hemorrhagic fever virus (CCHFV) in Romania, but were based on
serological techniques. The present work investigated the presence of
CCHFV RNA in Rhipicephalus sp. and Dermacentor sp. ticks, in
south-eastern Romania. In July 2019, 127 ticks were collected from the
environment and from domestic ruminants (sheep and goats) in five
different locations from Tulcea county. All tick samples were tested
using Real Time RT-PCR based on SYBR Green with the purpose to detect
the CCHFV. For this purpose, specific primer sets for each of the six
known CCHFV genotypes and one degenerate primer pair for the detection
of all genotypes were used. None of the samples was identified as
positive (prevalence \textless{}2,5\%, p=0.05), highlighting the nil or
the low prevalence of CCHFV RNA in Dermacentor sp. and Rhipicephalus sp.
ticks from Tulcea county, in 2019. Further studies based on the
serological surveillance of animals and more comprehensive ticks testing
by molecular biology methods if CCHFV circulates at very low level in
Romania.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Crimean-Congo hemorrhagic fever virus (CCHFV), a orthonairovirus of
the\emph{Nairoviridae} family is the causative agent of a severe human
hemorrhagic fever disease characterized by fever, weakness, myalgia, and
hemorrhagic signs (Bente et al., 2013). The disease, named Crimean
hemorrhagic fever was first observed in the Crimean Peninsula in 1944,
and the causative agent which was isolated in 1967, was found to be
identical to Congo virus isolated in 1956 from a febrile child in the
Belgian Congo hence the names Crimean and Congo are used in combination
(Casals, 1969). The mean of CCHF fatality rates in Africa (22.0\%) is
lower than Asia (33.5\%) and Europe (33.8\%) (Nasirian, 2020). The
natural vector and reservoir have been identified as~\emph{Hyalomma~sp.}
ticks, and the distribution of human cases closely mirrors vector
distribution CCHFV has been detected in more than 30 species of ticks,
including \emph{Dermacentor sp} . and\emph{Riphicephalus sp} . ticks
(Mehravaran et al., 2013). Studies conducted since then have
consistently found a relationship between various tick species
(\emph{Hyalomma, Rhipicephalus, Boophilus, Dermacentor} and
\emph{Ixodes} ) and the presence of CCHFV in ticks are believed to be
the principal means of viral transmission and persistence in nature
(Drosten, Kummerer, Schmitz, \& Gunther, 2003; Wolfel et al., 2007).
Nevertheless, the ability to transmit infection has been demonstrated
for ixodid ticks of several genera, and transovarial transmission of the
virus from adult females to the succeeding generation of larval ticks
has been shown to occur in a few members of the \emph{Hyalomma,
Dermacentor,} and \emph{Rhipicephalus} genera (Zeller, Cornet, Diop, \&
Camicas, 1997).

In south-eastern Romania, the presence of \emph{Hyalomma} was confirmed
in 2014 (Dumitrescu et al., 2014). The coincidence in distribution of
CCHF virus and \emph{Hyalomma} ticks implies that members of this genus
are important vectors of the virus (Shepherd, Swanepoel, Cornel, \&
Mathee, 1989). Arboviruses usually show low levels of genome diversity,
perhaps since they have to be adapted both to an arthropod vector and a
vertebrate host species, but CCHFV does not follow this rule and shows a
high nucleic acid diversity (Weaver, 2006). This virus is a
negative-sense, single-stranded RNA virus with a genome of approximative
19.2 kb in length. The genome contains three segments: small (S), medium
(M) and large (L). The L segment encodes the RNA-dependent RNA
polymerase, the M segment encodes the precursor of the two envelope
glycoproteins Gn and Gc, and the S segment encodes the nucleocapsid
protein (Aitichou, Saleh, McElroy, Schmaljohn, \& Ibrahim, 2005; Estrada
\& De Guzman, 2011; Honig, Osborne, \& Nichol, 2004). CCHFV is an
enveloped virus characterized by a degree of sequence diversity of 20\%,
22\%, and 31\% among the S-, L-, and M-segments of the virus genome
(Bente et al., 2013). M and L RNA segments of nairoviruses are larger
than other bunyaviruses (hantaviruses and phleboviruses) (Estrada-Pena
et al., 2007; Hoogstraal, 1979). As other members of the family
\emph{Bunyaviridae} , CCHFV glycoproteins target the Golgi apparatus,
where most viral assembly takes place. Recent studies have revealed a
new strain which shows unique S-segment phylogenies and which
constitutes a unique clade (provisionally identified as clade VII)
(Chinikar et al., 2010). Crimean-Congo hemorrhagic virus circulates in
nature in unnoticed enzootic tick--vertebrate--tick cycles. Asymptomatic
CCHFV infection has been reported in numerous vertebrate species and
appears to be pervasive in both wild and domestic animals. Asymptomatic
viremia last up to 7--15 days in several vertebrate animal species, and
CCHFV has been isolated from livestock and small mammals (horses,
donkeys, goats, cattle, sheep, and pigs (Ergonul, 2006).

In Romania there is still not sufficient data concerning CCHFV and the
virus has never been detected in ticks. Virus detection by real-time
quantitative reverse transcription polymerase chain reaction (RT-qPCR)
is essential to prove an actual circulation of CCHFV in a country and
can be used to identify animals and humans CCHFV infections, together
with viral detection in ticks. The predominant problem in CCHFV RT-qPCR
development has always been the high genetic diversity of the virus
(Deyde, Khristova, Rollin, Ksiazek, \& Nichol, 2006).

The aim of this study was to assess the prevalence of CCHFV
in\emph{Dermacentor sp} . and \emph{Rhipicephalus sp} . ticks collected
from Romania, more precisely in Tulcea county, where in previous studies
IgG antibodies anti-CCHFV were found in sheep and goats (Ceianu,
Panculescu-Gatej, Coudrier, \& Bouloy, 2012; R\selectlanguage{polish}ă\selectlanguage{english}ileanu, Anit\selectlanguage{polish}ă, \selectlanguage{english}Porea, \&
Savuta, 2015).

\textbf{MATERIAL AND METHODS}

Ticks were collected during July 2019 from different locations in
south-eastern Romania. The sampling consisted in environmental and
body-surface of domestic ruminants, from five sites across Tulcea
county. Tulcea county (latitude: 44.64\selectlanguage{ngerman}°S--45.42°N; longitude:
28.00°W--29.65°E) is situated in southeast Romania and forms the
northern part of Dobrogea province. Vegetation is represented by
submediterranean broadleaf forests and xerophytic pastures with shrubs.
The county is bordered by the river Danube (W and N), and the Black Sea
(E), and is neighboring Ukraine (N). Ground-feeding birds and small and
larger mammals, including livestock, are abundant in this area.

Questing ticks collection was performed by dragging. The surface covered
at each collection sites was \selectlanguage{english}~1 hectare and the flag was examined after
every 10 m of dragging, at the end of each segment. Between every 10 m
of dragging, every transect was separated by 20 m. Tick sampling in
Tulcea county was performed only once at each site (Table 1). Engorged
ticks were collected on domestic ruminants. The whole body of each
sampled animal was inspected for the presence of tick infestations by
palpation, mainly on the ears and along the nape of the neck, perineum,
scrotum or udder, and tail base. Ticks collected from each animal were
kept alive in separate vials and labeled. Live ticks were transported in
vials (within a closed secondary container) to the laboratory. Prior
handling, ticks were cooled by placing the vials in ice. Developmental
stages were identified under a stereomicroscope using standard
morphological identification keys. After the identification, each tick
was cut lengthwise, using sterile scalpels on an ice-cold surface Petri
dish, in a class II laminar flow hood. Half was used for additional
processing and the remainder was stored at \selectlanguage{english}-80degC. Ticks were transferred
to Institut Pasteur (Paris, France) and analysed by real time RT-qPCR.
For maintaining the integrity of RNA, ticks were introduced in RNA later
solution (Invitrogen, France), according to best security practices of
storing and transport.

\textbf{RNA extraction}

Extraction steps were conducted in a BSL-3 laboratory. The ticks were
pooled based on the geographical region before extraction, resulting a
final number of 13 pools (Table 2). The tick pools were homogenized in 2
mL reinforced tubes MK28-R from Precellys containing 1 ml of TRIzol
Reagent for cells.

Ticks were shaken two to three times for 30s using the Minilys (Bertin,
France). Between each shaking procedure the samples were placed on ice
for 1 minute. The homogenates were used for total RNA extraction. The
lysates were centrifuged at 12,000 g for 2 min at 4degC. RNA was purified
with a RNeasy mini kit (Qiagen, Hilden, Germany) according to the
manufacturer's instructions, including on-column DNAse treatment and
further analyzed using an Agilent Bioanalyzer. The RNA recovered in
nuclease-free water was stored at \selectlanguage{english}-80degC.

\textbf{Detection of CCHFV in \emph{Dermacentor sp}.
and\emph{Rhipicephalus sp}. ticks using Real Time RT-PCR based on SYBR
Green}

In the present study specific primer sets for each of the six known
CCHFV genotypes and one degenerate primer pair for the detection of all
genotypes, described by others were used (Sas et al., 2018). These
genotypes were assigned to different geographic areas: I -- West Africa,
II -- Central Africa, III -- South and West Africa, IV -- Asia and
Middle East, V- South and East Europe, VI -- Europe. The region targeted
by these primers was from nucleotide 1068 to 1248 for the S-sequence.
Viral RNA was transcribed to cDNA using SuperScript IV Reverse
Transcriptase (ref. 18090050, Invitrogen) reagent kit and a 6-mer random
primer (ref. N8080127, Invitrogen).

One \selectlanguage{greek}µ\selectlanguage{english}l of DNAc, 0.2 \selectlanguage{greek}µ\selectlanguage{english}l of each CCHF-deg primer, 0,2 \selectlanguage{greek}µ\selectlanguage{english}l of each
genotype-specific CCHF-primer, 10 \selectlanguage{greek}µ\selectlanguage{english}l SybrGReen reagent and 8.6 \selectlanguage{greek}µ\selectlanguage{english}l H2O
were used, in a total reaction volume of 20 \selectlanguage{greek}µ\selectlanguage{english}l. The real time RT-qPCR
was performed with Roche LightCycler~96 System (Roche Diagnostics,
Germany). The cycling conditions used as follows: 95\selectlanguage{ngerman}°c for 300 s
preincubation, followed by 45 cycles at 95°C for 10s (denaturation),
60°C for 10 s (annealing) 72°C for 30 s and 37°C for 30 s (cooling), the
annealing temperature being adjusted at 60°C depending on the primers
temperature.

For regulatory reasons regarding bioterrorism, it was not possible to
use a positive control. A negative control nuclease-free water was used
to rule out cross contamination of reagents and surfaces. Analysis of
the fluorescence data was conducted with the LightCycler~96 software.
Fifteen \selectlanguage{greek}µ\selectlanguage{english}l of PCR product was mixed with 3 \selectlanguage{greek}µ\selectlanguage{english}l of loading buffer and then
electrophoresed on 2\% agarose gel in Tris-acetate-EDTA buffer.

\textbf{Statistical analysis} : The upper range of prevalence was
calculated using VS Outbreak SurveillanceToolbox. The maximal prevalence
of viral infection in ticks was estimated assuming a sensitivity of
100\% and a confidence level of 95\%.

\textbf{RESULTS AND DISCUSSION}

During this study a total of 127 ticks were collected. Of these, 47 were
identified as \emph{Dermacentor sp} . questing ticks from the
environment and 80 were confirmed as \emph{Riphicephalus sp.} engorged
ticks, detached from adult goats and sheeps. All ticks were tested for
detection of the six CCHFV genotypes by real time qRT-PCR. For this
purpose, real time RT-qPCR based on SybrGreen with specific primer sets
for the detection of all the six known CCHFV genotypes (Sas et al.,
2018) in questing and engorged ticks were used. The detection of CCHFV
consisted in the amplification of a 180 bp region from S segment that is
coding for NP. CCHFV is classified as a risk group 4 pathogen, being
considered to have bioterrorism potential due its aerosol infectivity.
For regulatory reasons a positive control was not included in reaction.
By using the negative control, we have not encountered any
false-positive RT-PCR results due to contamination. In all collection
sites from Tulcea county, the tested samples using real time qRT-PCR
were identified as negative for the six genotypes of CCHFV. The results
are listed in table 3. As no positive result was recorded, this defines
a maximal prevalence \textless{}2.5\% (p=0.05). We consider this result
as robust because of the good sensitivity of the RT-qPCR used in this
study. Two copies/\selectlanguage{greek}µ\selectlanguage{english}l were detected for genotypes II (DR Congo), IV
(Afghanistan), V (Kosovo) and VI (Greece), while the limits of detection
for genotypes I and III were 200 copies/\selectlanguage{greek}µ\selectlanguage{english}l respectively (Sas et al.,
2018). During the same study the specificity was favorably evaluated
using ten viruses from the order \emph{Bunyavirales} as none of the
viruses was detected.

The present study highlight the nil or at most low prevalence of CCHFV
RNA in \emph{Dermacentor} \emph{sp.} and \emph{Rhipicephalus} \emph{sp}
. questing ticks and engorged ticks from south-eastern Romania, in 2019
from the same region (Tulcea county) where the presence of CCHFV
antibodies in animals has been detected previously (Ceianu et al., 2012;
R\selectlanguage{polish}ă\selectlanguage{english}ileanu et al., 2015).

Crimean Congo hemorrhagic fever has a widespread geographic
distribution, the disease being reported in many regions of Africa,
Middle East, Europe, and Asia, and outbreaks caused by CCHFV have been
recorded in several countries (Chinikar et al., 2010). A detailed review
about the epidemiology of CCHF in Asia, Europe and Africa was published
in 1979 by Hoogstraal (Hoogstraal, 1979).

The Balkan Peninsula is an endemic region for the disease, where Turkey
and Bulgaria are countries with the majority of the cases (Estrada-Pena
et al., 2007; Honig et al., 2004; Papa, Pappa, Panayotova, Papadopoulou,
\& Christova, 2016; Papa et al., 2013; Zeller et al., 1997). In
Bulgaria, country that is on the border with Romania, CCHFV has an
endemic evolution (Gergova \& Kamarinchev, 2013) which could suggest the
circulation of the virus in Romania, possibly by ticks carried on
migratory birds or through the international livestock trade. Migratory
birds' role in spreading Crimean-Congo hemorrhagic fever virus (CCHFV)
through attached ticks was demonstrated in Turkey (Leblebicioglu et al.,
2014). The geographic area of our present investigations comprises one
of the most active bird migration pathways in southeastern Europe, for
both spring and fall migrations.

In livestock the CCHFV does not cause clinical disease. However,
domestic animals are very important for the epidemiology of the virus.
Sheep have been recognized as CCHFV reservoirs in certain endemic
regions, and have been epidemiologically linked to human cases on
several occasions (Humolli, Dedushaj, Zupanac, \& Mucaj, 2010;
Mostafavi, Haghdoost, Khakifirouz, \& Chinikar, 2013). Not only are they
hosts for adult vectors, but they also may amplify the virus and infect
other ticks during their short-lived viremia, and thus may introduce the
virus into new areas via movement and importation of tick-infested and
virus-infected livestock (Gale et al., 2010).

So far, the presence of the CCHFV in ticks from Romania was not
confirmed and, consistently with our results, CCHFV human disease has
not been reported to date. There are few serological studies regarding
the circulation of CCHFV on Romanian territory. In 2012, Ceianu tested
471 sheep serum samples from different localities in Tulcea county and
obtained a prevalence of 27.8\% for IgG antibodies against CCHFV (Ceianu
et al., 2012). However, a more recent study conducted by Raileanu in
2015, reported an overall prevalence of 74\% IgG antibodies by testing
90 domestic ruminants (R\selectlanguage{polish}ă\selectlanguage{english}ileanu et al., 2015). In the study conducted by
Raileanu 74 \emph{Rhipicephalus sp} . ticks, collected from the positive
animals, were tested using rRT-PCR method obtaining negative results,
which suggest the seroconversion process occurred before the ticks were
attached and the transmission of the virus to vectors was not achieved
(R\selectlanguage{polish}ă\selectlanguage{english}ileanu et al., 2015). Many studies suggest that the viral RNA in
attached ticks does not directly indicate transmission to host species,
and vice versa: infected ticks have been found on seronegative animals
and uninfected ticks on seropositive animals (Zeller et al., 1997).

The results of this study together with previous published data may
suggest a silent tick-vertebrate-tick cycle of CCHFV in Romania.
Clinical cases occur when a range of factors related to tick, vertebrate
host, and human behavior align, increasing the level of viral
circulation, the interactions between humans and sources of infection.
Thus, detection of CCHFV antibodies in domestic animals has been
important in providing initial evidence of circulating virus and
localizing CCHFV and increased risk for human infection (Spengler,
Bergeron, \& Rollin, 2016).

There are many factors that can influence the circulation of CHHFV. For
example, abiotic variation by season and region is reported in CCHFV
evolution (Spengler et al., 2016). Longitudinal studies demonstrated
considerable variation when repeated sampling was performed in the same
location. These studies reported September as the optimum period for
detecting antibodies with a notable decrease in seroprevalence in the
winter--spring period (Gale et al., 2010). This could also partially
explain our negative results, because we collected the ticks only once,
in July 2019. Variation in seroprevalence is often associated with
competent vector distribution, host preference of competent tick
vectors, and tick load on a particular animal species. \emph{Hyalomma
sp.} ticks were not tested, which is a limit of our study. This study
can therefore be extended by exploring different tick species, areas and
seasons of sampling.

\textbf{CONCLUSIONS}

Our results highlight that in 2019, the presence of the CCHFV RNA was
not detected in \emph{Rhipicephalus sp} . and \emph{Dermacentor
sp.}ticks collected from five different sites in Tulcea county, even if
the CCHFV IgG antibodies were identified in animals from this area,
during researches undertaken in previous years.

Further studies should focus on increasing the number of collected ticks
and also in expanding the geographical area of research, correlated with
serological investigations in animals in order to accomplish a
longitudinal research for better understanding CCHFV epidemiology
throughout Romania.

\textbf{ACKNOWLEDGMENTS}

We wish to thank DVM Andreea Paula Cozma, DVM Anca Mihaela Dasc\selectlanguage{polish}ă\selectlanguage{english}lu, DVM
Ioana Alexandra R\selectlanguage{polish}ă\selectlanguage{romanian}țoi from the Public Health Department, ''Ion Ionescu
de la Brad'' University of Agricultural Sciences and Veterinary Medicine
Iasi, Romania for their contribution in this research.

\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 as this is a review article with no
original research data.

\textbf{CONFLICT OF INTERESTS}

We declare no competing interests.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of the study are available on request
from the corresponding author.

We declare no competing interests.

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Table 1. The distribution of tick collection sites in Tulcea county\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Locality & Number of ticks & Tick species\tabularnewline
\midrule
\endhead
Cataloi & 18 & \emph{Rhipicephalus sp.}\tabularnewline
Slava Rus\selectlanguage{polish}ă & 27 & \emph{Rhipicephalus sp.}\tabularnewline
Slava Cerchez\selectlanguage{polish}ă & 17 & \emph{Rhipicephalus sp.}\tabularnewline
Somova & 18 & \emph{Rhipicephalus sp.}\tabularnewline
Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc & 47 & \emph{Dermacentor sp.}\tabularnewline
TOTAL & \textbf{127} &\tabularnewline
\bottomrule
\end{longtable}

Table 2. Pooling protocol (grouping by sample place, species and site)\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Sample type & Site & Ticks number & Ticks genus & Sampling
origin\tabularnewline
\midrule
\endhead
1. Tick pool & Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc, Tulcea & 7 & \emph{Dermacentor sp.} &
Environment\tabularnewline
2. Tick pool & Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc, Tulcea & 10 & \emph{Dermacentor sp.} &
Environment\tabularnewline
3. Tick pool & Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc, Tulcea & 10 & \emph{Dermacentor sp.} &
Environment\tabularnewline
4. Tick pool & Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc, Tulcea & 5 & \emph{Dermacentor sp.} &
Environment\tabularnewline
5. Tick pool & Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc, Tulcea & 5 & \emph{Dermacentor sp.} &
Environment\tabularnewline
6. Tick pool & Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc, Tulcea & 10 & \emph{Dermacentor sp.} &
Environment\tabularnewline
7. Tick pool & Cataloi, Tulcea & 5 & \emph{Rhipicephalus sp.} & Adult
sheeps\tabularnewline
8. Tick pool & Cataloi, Tulcea & 15 & \emph{Rhipicephalus sp.} & Adult
goats\tabularnewline
9. Tick pool & Slava Rus\selectlanguage{polish}ă, \selectlanguage{english}Tulcea & 11 & \emph{Rhipicephalus sp.} &
Adult goats\tabularnewline
10. Tick pool & Slava Rus\selectlanguage{polish}ă, \selectlanguage{english}Tulcea & 16 & \emph{Rhipicephalus sp.} &
Adult sheeps\tabularnewline
11. Tick pool & Slava Cerchez\selectlanguage{polish}ă, \selectlanguage{english}Tulcea & 15 & \emph{Rhipicephalus sp.} &
Adult goats\tabularnewline
12. Tick pool & Somova, Tulcea & 5 & \emph{Rhipicephalus sp.} & Adult
sheeps\tabularnewline
13. Tick pool & Somova, Tulcea & 13 & \emph{Rhipicephalus sp.} & Adult
goats\tabularnewline
\bottomrule
\end{longtable}

Table 3. Results obtained using RT-qPCR for the presence of

CCHFV in ticks from Tulcea county\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Locality & Tick genus & No. of CCHFV positive ticks\tabularnewline
\midrule
\endhead
Cataloi & \emph{Riphicephalus sp.} & 0/18\tabularnewline
Slava Rus\selectlanguage{polish}ă & \emph{Riphicephalus sp.} & 0/27\tabularnewline
Slava Cerchez\selectlanguage{polish}ă & \emph{Riphicephalus sp.} & 0/17\tabularnewline
Somova & \emph{Riphicephalus sp.} & 0/18\tabularnewline
Sat Pesc\selectlanguage{polish}ă\selectlanguage{english}resc & \emph{Dermacentor sp.} & 0/47\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

