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\begin{document}

\title{Genomic diversity of mpox virus in Paris area (France) during the 2022
outbreak}



\author[1]{Géraldine Piorkowski}%
\author[2]{Jade Ghosn}%
\author[2]{Romain Coppée}%
\author[2]{Morgane Mailhe}%
\author[2]{Valentine M. Ferré}%
\author[2]{Nadhira Houhou-Fidouh}%
\author[2]{Yazdan Yazdanpanah}%
\author[2]{Quentin  Le Hingrat}%
\author[3]{Hervé Raoul}%
\author[2]{Charlotte Charpentier }%
\author[2]{Diane Descamps}%
\author[1]{Xavier de Lamballerie}%
\affil[1]{Aix-Marseille Universite}%
\affil[2]{Hopital Bichat - Claude-Bernard}%
\affil[3]{Agence Nationale de Recherches sur le SIDA et les Hepatites Virales}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
In May 2022, several countries reported mpox cases from patients without
history of traveling to endemic areas. France was one of the most
affected European countries. In this study, we described the clinical
characteristics of mpox cases in France, and studied the genetic
diversity of the virus. Patients diagnosed with mpox infection (qPCR
ct\textless{}28) between 21 \textsuperscript{th} May and 4
\textsuperscript{th} July 2022 and between 16 \textsuperscript{th}
August and 10 \textsuperscript{th} September 2022 were included to this
study. Twelve amplicons corresponding to the most polymorphic regions of
the mpox genome and covering \textasciitilde{}30,000 nucleotides were
generated and sequenced using the S5 XL Ion Torrent technology to
evaluate the genetic diversity of mpox sequences. One hundred and
fourty-eight patients were diagnosed with mpox-infection. 95\% were men,
5\% transgender (M-to-F), 50\% were taking HIV pre-exposure prophylaxis,
and 25\% were HIV seropositive. One hundred and sixty-two samples were
sequenced and compared to GenBank sequences. Thirty-two distinct
mutational patterns were identified. Overall, low genetic diversity of
mpox sequences was found compared with pre-epidemic Western-African
sequences, with 32 distinct mutational patterns. Our results provide a
first glance at the mutational landscape of early mpox 2022 circulating
strains in Paris (France).%
\end{abstract}%



\sloppy


\textbf{Genomic diversity of mpox virus in Paris area (France) during
the 2022 outbreak}

G\selectlanguage{ngerman}éraldine Piorkowski\textsuperscript{1}, Jade Ghosn\textsuperscript{2},
Romain Coppée\textsuperscript{3}, Morgane Mailhé\textsuperscript{2},
Valentine Marie Ferré\textsuperscript{3}, Nadhira
Houhou-Fidouh\textsuperscript{3}, Yazdan Yazdanpanah\textsuperscript{2,
4}, Quentin Le Hingrat\textsuperscript{3}, Hervé
Raoul\textsuperscript{4}, Charlotte Charpentier\textsuperscript{3},
Diane Descamps\textsuperscript{3 *} and Xavier de
Lamballerie\textsuperscript{1 *}

\textsuperscript{1} Unité des Virus Émergents (UVE : Aix-Marseille
Université -IRD 190-Inserm 1207), Marseille, France.

\textsuperscript{2} Université Paris Cité, Inserm, UMR1137, IAME,
Service des Maladies Infectieuses et Tropicales, AP-HP, Hôpital
Bichat-Claude Bernard, Paris, France.

\textsuperscript{3} Université Paris Cité, Inserm, UMR1137, IAME,
Service de Virologie, AP-HP, Hôpital Bichat-Claude Bernard, Paris,
France.

\textsuperscript{4} Agence nationale de Recherche sur le SIDA et les
hépatites virales, maladies Infectieuses émergentes, France.

\textbf{Corresponding author:} Xavier de Lamballerie
(xavier.de-lamballerie@univ-amu.fr)

*: equally contributed

\textbf{Running title:} Genomic analysis of Paris mpox outbreak



\textbf{Abstract}

In May 2022, several countries reported mpox cases from patients without
history of traveling to endemic areas. France was one of the most
affected European countries. In this study, we described the clinical
characteristics of mpox cases in France, and studied the genetic
diversity of the virus.

Patients diagnosed with mpox infection (qPCR ct\textless{}28) between
21\textsuperscript{th} May and 4\textsuperscript{th} July 2022 and
between 16\textsuperscript{th} August and
10\textsuperscript{th}September 2022 were included to this study. Twelve
amplicons corresponding to the most polymorphic regions of the mpox
genome and covering \textasciitilde{}30,000 nucleotides were generated
and sequenced using the S5 XL Ion Torrent technology to evaluate the
genetic diversity of mpox sequences.

One hundred and fourty-eight patients were diagnosed with
mpox-infection. 95\% were men, 5\% transgender (M-to-F), 50\% were
taking HIV pre-exposure prophylaxis, and 25\% were HIV seropositive. One
hundred and sixty-two samples were sequenced and compared to GenBank
sequences. Thirty-two distinct mutational patterns were identified.
Overall, low genetic diversity of mpox sequences was found compared with
pre-epidemic Western-African sequences, with 32 distinct mutational
patterns.

Our results provide a first glance at the mutational landscape of early
mpox 2022 circulating strains in Paris (France).

\textbf{Keywords:} mpox, genetic diversity, sequencing, 2022 outbreak,
phylogenomics, France, co-circulation

Mpox (formerly monkeypox) is a zoonotic disease caused by an
\emph{Orthopoxvirus} . The first human case was described in 1970 in the
Democratic Republic of Congo. Since then, endemic circulation has been
reported in West and Central Africa\textsuperscript{1}. An increase in
mpox infections may have occurred following the worldwide decline in
\emph{Orthopoxvirus} herd immunity, after the cessation of smallpox
vaccination when smallpox was declared eradicated in 1980
\textsuperscript{2}. It had been foreseen that mpox was likely to emerge
as the most important \emph{Orthopoxvirus}infection in humans
\textsuperscript{3}.

Sporadic mpox clusters and cases have occurred outside of Africa. In
2018, a first human-to-human mpox transmission was reported in the
United Kingdom, in a nosocomial context \textsuperscript{4} and in a
household cluster \textsuperscript{5}; in May 2019, one case occurred in
a man who traveled from Nigeria to Singapore\textsuperscript{6}; and in
May 2021, a family returned to the United Kingdom after traveling to
Nigeria, and three family members became infected with the mpox
virus\textsuperscript{7}.

On 7\textsuperscript{th} May 2022, a human mpox infection was diagnosed
in an individual who traveled from Nigeria to the United Kingdom. This
case was the beginning of an unprecedent outbreak that spread across
several non-endemic countries worldwide including Europe and North
America. Countries reported mpox cases with no epidemiological link to
Africa, mostly among men-who-have-sex-with-men (MSM)\textsuperscript{8}.
Between May 2022 to January 2023, it is estimated that 80,000
individuals contracted mpox, resulting in more than 100 deaths according
to the Centers for Disease Control and Prevention (CDC).

Historically, mpox virus has been divided into two major clades
(separating West from Central African cases). The current mpox outbreak
outside Africa showed the existence of a novel lineage (clade 3). Since
its identification, some mpox lineages have been characterized (such as
A.1 and B.1), suggesting that evolutionary events may have occurred
throughout the outbreak. The lineage B.1, associated with most cases
during the 2022 outbreak, forms a divergent branch originating from the
A.1 lineage associated with the exportation of mpox in 2018-2019 from
Nigeria.

In this study, we aimed to better understand both clincal and
epidemiological characteristics of mpox cases observed in the Paris area
(France). We also studied the genomic diversity of the mpox genome,
allowing to differentiate cases belonging to the outbreak (B.1 lineage)
from imported lineages (clades 1 or 2, or non-B.1 lineage).

\textbf{Material and methods}

\textbf{Ethic statement}

The Research Ethics Committee of the North Parisian Academic Hospital
Group approved the study. All the participants included in the study
gave consent for the use of their anonymized medical data.

\textbf{Sample collection}

The study was conducted in Bichat-Claude Bernard university hospital,
one of the mpox referral centers in the Paris Metropolitan area
(France), between 21\textsuperscript{th} May and
4\textsuperscript{th}July 2022 and between 16\textsuperscript{th} August
and 10\textsuperscript{th} September 2022. Patients were diagnosed with
mpox infection by quantitative polymerase chain reaction (qPCR). Only
patients associated with a cycle threshold \textless{} 28 were included
for genomic analyses. Samples consisted of skin, pharyngeal or anal
swabs.

\textbf{Nucleic acid extraction and mpox q-PCR}

After a heat inactivation step (12 minutes at 70°C), nucleic acids
(500\selectlanguage{greek}µ\selectlanguage{english}L of samples) were extracted using MagNA Pure LC 2.0 Instrument
(Roche, Meylan, France) and eluted in 100\selectlanguage{greek}µ\selectlanguage{english}L.~ Mpox virus specific
real-time PCR assay (qPCR) validated by both Centers for Disease Control
\& Prevention (CDC)\textsuperscript{9} and~ French Orthopoxvirus
national reference center\textsuperscript{10} was
performed\textsuperscript{11}. An exogenous internal extraction and
amplification control (IEAC) was added to each sample before extraction
(Simplexa Extraction and Amplification Control Set, DiaSorin, Saluggia,
Italy)\textsuperscript{12}. Negative controls were added to each
extraction batch and a positive control (DNA extracted from a MXPV cell
culture given by the French Orthopoxvirus national reference center) was
tested in each PCR run.

\textbf{Design of primers and PCR protocol}

Primers were designed after alignment of 311 sequences from clades 2 and
3 (published up to 29\textsuperscript{th} June 2022) and sequences
published by Isidro and colleagues\textsuperscript{13}, using MAFFT 7
online software\textsuperscript{14}. Twelve amplicons
(\textasciitilde{}30,000 bp) were realized to detect 26/35 of the
characteristic mutations which differentiated 2022 outbreak clade 3 and
pre-epidemic clade 3 2018\_UK\_P2 (GenBank:
MT903344.1)\textsuperscript{13}. The nine uncovered mutations were all
synonymous or non-coding.

Amplification was realised using 3\selectlanguage{greek}µ\selectlanguage{english}L of RNA, 500nM of each primer
(\textbf{Supplementary Table S1} ) and the Master Mix for PCR Platinum
SuperFi~II kit (Thermo Fisher) (12.5\selectlanguage{greek}µ\selectlanguage{english}L of 2X reaction mix) in a 25\selectlanguage{greek}µ\selectlanguage{english}L
final volume. The thermal profile used was 2 min at 98\selectlanguage{ngerman}°C, 40 cycles
consisting of denaturation at 98°C, 10 s; hybridisation at 56°C, 10 s;
and elongation 68°C, 2 min 30 s ending with final elongation 68°C 5 min.
PCR products were pooled in equimolar proportions after purification
using Nucleofast PCR Plate kit (Macherey Nagel).

\textbf{Sequencing of mpox fragments}

After Qubit quantification using~Qubit® dsDNA HS Assay Kit and Qubit 4.0
fluorometer (Thermo Fisher)~amplicons were fragmented (Bioruptor,
Diagenaode) into fragments of 250 bp long. Libraries were built adding
barcode, for sample identification, and~primers~to fragmented DNA with
Ion Plus Fragment Library Kit using AB Library Builder System (Thermo
Fisher Scientific).~~To pool equimolarly the barcoded samples, a
quantification step by real time PCR using Ion Library TaqMan\selectlanguage{english}
Quantitation Kit (Thermo Fisher) was realized. An emulsion PCR of the
pools and loading on 530 Chip were performed~using the automated~Ion
Chef instrument (Thermo Fisher).~Sequencing was performed using the S5
XL Ion torrent technology (Thermo Fisher) following manufacturer's
instructions. Consensus sequence was obtained after trimming of reads
(reads with quality score \textless{} 0.99, and length \textless{} 100
bp were removed and the 30 first and 30 last nucleotides were removed
from the reads) and mapping on the MT903344.1 mpox reference sequence
using CLC genomics workbench software v.21.0.5 (Qiagen). Parameters for
reference-based assembly consisted of match score = 1, mismatch cost =
2, length fraction = 0.5, similarity fraction = 0.8, insertion cost = 3,
and deletion cost = 3. A~\emph{de novo} ~contig was also produced to
ensure that the consensus sequence was not affected by the reference
sequence.

\textbf{Phylogenetic analysis and genetic diversity}

A phylogenetic tree was produced with MEGA 6
software\textsuperscript{15} using the maximum likelihood method
(options: Tamura-Nei 93 + G model, 1,000 replicates). For ease of
representation, only one representative sequence was for each pattern of
mutation. The sequences produced were compared to pre-epidemic and
epidemic sequences retrieved on GenBank (\textbf{Supplementary Methods
S1).}

\textbf{Results}

\textbf{Patient characteristics}

A total of 148 patients were positive to mpox by qPCR (ct \textless{}
28). Available medical records showed that 95\% were men and 5\%
transgender (M-to-F). Ninety percent of patients were MSM, of whom 50\%
were taking HIV pre-exposure prophylaxis, and 25\% lived with HIV. A
quarter of the patients had travelled within 3 weeks prior to mpox
diagnosis (\textgreater{}85\% to Europe, none to Africa). Only, 5\% of
the patients were hospitalized, mostly for severe pharyngeal/tonsils.

\textbf{Genetic variability of mpox sequences}

A total of 162 sequences (SRA, BioProject: PRJNA944928) were produced
from 148 patients (some patients had two samples). Among them, 32
different mutational patterns were distinguished -- defined in
comparison to the ON602722.2 reference sequence. Pattern \#1 was
identical to ON602722.2, while patterns \#2 to \#32 harbored at least
one mutation (\textbf{Supplementary Table} \textbf{S2} ). Pattern \#1
included most of the sequences (n=120, 74.1\%), and pattern \#9
encompassed 10 sequences (6.2\%). All other mutational patterns included
only one or two sequences. Fourteen patterns were already reported in
the GenBank database. The most common ones are patterns \#9 and 13
observed in 6.16\% and 1\% of the GenBank sequences, respectively
(\textbf{Supplementary Table S2} ). Others were observed in less than
1\% of GenBank sequences. Interestingly, all of the sequences produced
in this study included a 17-nucleotide deletion (position 150,621,
intergenic). This deletion was a succession of 17-nucleotides repeated
sequence and was present in only 30\% of the 5,661 GenBank mpox
sequences (15\textsuperscript{th} March 2023), and 32\% of 5,306
described during the 2022 outbreak. One sequence included an unreported
4 nt-deletion, pattern \#8. One sequence included an unreported 4
nt-deletion (position 14,509, ORF OPG25). Overall, 51\% of mutations
were non-synonymous, 37\% synonymous, and 12\% non-coding. A majority of
transitions (17 G-to-A; 17 C-to-T) was observed, confirming a highly
specific mutational typology.

Of note, a comparison of pre-epidemic strain MT903344.1 and the French
MPXV\_FRA\_2022\_TLS67 outbreak strain (ON602722.2) identified more
mutations (58 substitutions and 27 indels along the genome)
(\textbf{Supplementary Table S3} ).

\textbf{Phylogenetic reconstruction}

The 162 mpox sequences of this study were compared with representative
sequences of clades. Phylogenetic reconstruction was realized using
maximum Likelihood method and, Tamura-Nei 93 + G model, 1,000
replicates\textbf{; (Supplementary Method S1)} . All patterns from this
study, were closely related to other 2022 epidemic strains
(ON745225\_Spain\_2022, ON637938\_Germany\_2022; \textbf{Figure 1} ).
However, for one patient returning from Asia, pattern \#32 was
identified, closely related to clade 3 pre-epidemic and USA-2022\_FL001
(ON674051) strains. This suggests a possible low-level circulation of
non-epidemic mpox virus in Western countries, probably underdiagnosed
and detected here due to attention generated by the current outbreak.

\textbf{Discussion}

To conclude, this rapid parsimonious mpox sequencing strategy in 148
mpox-infected patients provides a unique insight into the genomic
variability of an epidemic DNA virus. The existence among the mpox
strains identified in France of a mutational pattern specific to the
2022 epidemic was confirmed, and showed the overall low genomic
variability of circulating strains. The fact that the majority of the
substitutions were transitions (G\textgreater{}A and C\textgreater{}T)
suggests a role of the human APOBEC3 enzyme editing in mpox sequences,
known to target G and C in specific nucleotide contexts for other
viruses as an innate immune response \textsuperscript{16}.

This study also highlights that, although rare, mpox can circulate in
Europe far from epidemics and should be included in differential
diagnosis of other sexually transmitted infections in particular in
MSMs.

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\textbf{Figure 1.} Phylogeny of mpox based on the analysis of selected
30,000 nucleotides from 80 sequences. The tree was resolved by maximum
likelihood using the TN93 + G model of evolution. Bootstrap values are
indicated at nodes. The blue circle in front of an identifier indicates
the sequences resulting of this study.

This work was presented at the Conference of retroviruses and
Opportunistic Infections (19\textsuperscript{th} to 22th February 2023),
abstract ndeg: 0236

Funding: This work was supported by ANRS MIE, Project APP PRI 22275,
Emergen-2021

ACKNOWLEDGMENTS This work was supported by the Agence Nationale de la
Recherche sur le SIDA et les Maladies Infectieuses Emergentes (ANRS
MIE), Medical Virology and pharmacology Network\selectlanguage{english}
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