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\DOI{10.5802/crbiol.157}
\datereceived{2024-05-15}
\dateaccepted{2024-06-18}
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\title{The dangerous biology of pathogenic germs}

\alttitle{La biologie dangereuse des germes pathog\`{e}nes}

\author{\firstname{Patrick} \lastname{Berche}\CDRorcid{0000-0002-8941-1564}}
\address{Emeritus Professor at the Universit\'{e} Paris Cit\'{e}, 
Member of the Acad\'{e}mie Nationale de M\'{e}decine,
France}
\email{patrick.berche@gmail.com}

\dedicatory{\textit{In memoriam} of Professeur Henri Korn} 

\keywords{\kwd{Gain-of-function}
\kwd{Synthetic biology}
\kwd{Biological weapons}
\kwd{Biosecurity}}

\altkeywords{\kwd{Gain de fonction}
\kwd{Biologie synth\'{e}tique}
\kwd{Arme biologique}
\kwd{Bios\'ecurit\'e}}

\begin{abstract} 
The convergence of biotechnologies with other disciplines, including
computer science and Artificial Intelligence (AI), may make it possible
to carry out dangerous genetic manipulations on pathogenic germs, as
the gain-of-function experiments exacerbating virulence, as those
carried out on myxoviruses and coronaviruses. Moreover, it is now
possible to chemically synthesise any microorganism from \textit{in
silico} sequences, including the most dangerous viruses (poxviruses,
Ebola, etc.), whose sequences are accessible. It might even be possible
to use AI to design new germs that could be used as biological weapons.
\end{abstract}

\begin{altabstract} 
La convergence des biotechnologies avec de nombreuses disciplines,
notamment l'informatique et l'Intelligence Artificielle (IA), peut
permettre des manipulations g\'{e}n\'{e}tiques dangereuses sur les
germes pathog\`{e}nes. Il peut s'agir d'exp\'{e}riences de gain de
fonction exacerbant la virulence, telles que r\'{e}alis\'{e}es sur les
\textit{Myxovirus} et les coronavirus. On peut aussi synth\'{e}tiser
n'importe quel microorganisme \`{a} partir des s\'{e}quences \textit{in
silico}, y compris les virus les plus dangereux (poxvirus, Ebola, etc.)
dont les s\'{e}quences sont accessibles. On pourrait m\^{e}me concevoir
par IA des germes nouveaux qui pourraient \^{e}tre des armes
biologiques.
\end{altabstract}
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\xsection{}
\noindent
We are witnessing an unprecedented revolution in the biological
sciences, marked by the convergence of biology with many other
disciplines, as chemistry, physics, biotechnology, nanotechnology,
mathematics, computer science and artificial intelligence, etc. The
major technological impacts have been the rapid sequencing of DNA
(1977), enabling the creation of data banks, the discovery of reverse
transcriptase (1970), to copy RNA into complementary DNA (cDNA), and
the chemical synthesis of DNA from \textit{in silico} sequences.

It is now possible to modify or even create biological entities (genes,
proteins, microorganisms, viruses, etc.) with new properties, mainly
used for medical applications. Experiments inactivating certain genes
of pathogenic germs through mutations or deletions allow the
identification of those involved in virulence and contagiousness. Thus,
it is possible to link the phenotype to the genotype of pathogens,
which is useful for preparing vaccines and anti-infectious drugs.
Conversely, virulence can be exacerbated by modifying or introducing
certain genes involved in pathogenicity into other\unskip\break 
microorganisms,
which is facilitated by CRISP-Cas9 technology~\cite{1}. These
experiments are known as gain-of-function (GoF) or Dual-use research of
concern (DURC). Although the majority of these experiments are designed
for useful purposes, some pose problems of dual research and biosafety,
with possible accidental escapes from the laboratory, especially when
dealing with potentially pandemic pathogens (PPP) viruses, such as
influenza viruses, coronaviruses or poxviruses.

Thanks to DNA sequencing, hundreds of thousands of complete pathogen
sequences are available in databases. For example, 11,694~complete
viral genome sequences have been deposited in the Viral Genome 
Resource~\cite{2}, including all known pathogenic viruses.

\section{Gain-of-function experiments}\label{sec1} 
Many pathogenic germs that have been colonising wild animal species for
a long time circulate silently in hosts that form viral reservoirs.
Highly adapted to each species, they recognise specific receptors that
enable them to colonise and penetrate host cells. When they encounter
new hosts, they are generally not infectious, as there is a species
barrier preventing any colonisation. Crossing this barrier is rarely
possible directly, as it depends on the frequency of interactions
between animal species and their genetic proximity, but also on the
plasticity of the microorganisms' genomes.

Pandemic germs are often RNA viruses with a high mutation rate. Their
main wild reservoirs are birds, bats and rodents. In these animals,
these viruses exist as quasispecies, which are swarms of several
genotypes. The emerging wild virus is the most efficiently
proliferating  genotype~\cite{3,4}. The transition to humans is a
sequential process, usually requiring progressive adaptations via
intermediate animal hosts in which adaptive mutations occur and 
accumulate~\cite{5}. These intermediate hosts have been identified for
avian viruses (pigs), SARS-CoV1 from 2002 (civets, raccoon dogs,
badgers), MERS-CoV (dromedaries) and Nipah virus  (horses)~\cite{6}.
Initial human contact with wild viruses is usually unsuccessful,
resulting at best in sporadic cases of non-contagious infection or
asymptomatic  seroconversion~\cite{7}. This was the case, for example,
with H5N1 avian influenza in  humans~\cite{8} or the SARS outbreak in
2002, when almost 80\% of the animals in Guangzhou markets showed
anti-SARS-CoV1  antibodies~\cite{9}.

Gain-of-function experiments can mimic this adaptation process of wild
viruses. There are several ways of increasing the virulence and
contagiousness of an infectious agent. It has long been known how to
create random mutations that progressively increase virulence through
passages in animals or cell cultures. One can also carry out genetic
manipulation to induce mutations, through directed mutagenesis or
insertion of genetic material. More recently, it was possible to create
chimeric mutants by recombining certain pathogenic viruses, for example
to modify their cellular tropism. Occasionally, an experiment can lead
to an unexpected gain-of -function. In 2001, Australian researchers
looking for ways to develop immunocontraceptive viral vaccines
generated an extremely virulent mouse poxvirus (ectromelia). By adding
the murine interleukin-4 (IL-4) gene, the recombinant virus caused
mortality in all lines of mice, even after  vaccination~\cite{10}. The
new virus completely suppressed NK and cytotoxic T responses. Bearing
in mind that the human smallpox virus is related to this virus and has
the same genetic organisation, there is every reason to fear that a
recipe for a formidable biological weapon was published. Was it
necessary to publish these results?

Faced with recurrent threats of pandemics, due to avian flu (H5N1,
H7N9), SARS (2002) and MERS (2012), researchers have tried to
understand the molecular mechanisms of the pathogenicity of these
viruses by studying the main virulence factors, such as the
haemagglutinin HA of myxoviruses and the Spike protein of
coronaviruses. The aim was to predict the emergence of a new virus,
potentially better adapted to humans, from mutations created in certain
putative virulence genes. Dangerous gain-of-function experiments were
thus carried out on influenza viruses and coronaviruses.

\subsection{Influenza viruses}\label{sec1.1} 
The influenza virus, \textit{Myxovirus influenza}, consists of
single-stranded RNA (12--15~kb) harboring eight genes carried by
separate segments, including the two virulence genes encoding
haemagglutinin HA and neuraminidase NA, expressed on the surface of the
virus envelope. When pigs are co-infected with several influenza
viruses, reassortments can occur, leading to the emergence of new
pandemic viruses. There are currently fears that a new influenza
pandemic could emerge from the H5N1 avian virus, which triggered
epizootics with human cases from 1996  onwards~\cite{11}. This virus
recognises avian sialic acids (SA) (SA $\rmalpha$-2.3 galactose),
but does not recognise human SA (SA $\rmalpha$-2.6 galactose)
present in the upper respiratory tract, which explains why it is not
contagious to humans. However, after deep inhalation in contact with
poultry, the virus can reach the human bronchioles and alveoli where
the avian receptor is present. This explains the high mortality rate
and low contagiousness of the virus to humans. A pandemic alert was
issued in Hong Kong in 2005, following clustered human cases without
any real  epidemic~\cite{12}. Between 2005 and 2024, there have been
888 cases of avian influenza in humans, including 463~deaths (52\% 
mortality)~\cite{13}. However, it is feared that a few mutations could
be enough to transform the H5N1~virus into a fearsome pandemic virus. In
2012, in an attempt to predict the risk of a new pandemic emerging, two
teams sought to identify the crucial mutations in the HA~gene that
would enable the H5N1~virus to become contagious to humans by
recognising human SA~receptors. Researchers in Madison replaced the
H1~haemagglutinin gene of an H1N1~strain with the H5~gene of an avian 
H5N1~strain~\cite{14}. Unlike the H1N1~strain, the new virus is not
contagious in ferrets. Random mutations in the H5~gene were created
\textit{in vitro} and then selected by adsorption onto turkey red blood
cells expressing human~SA. Thus, 370~mutants were obtained and
individually screened to select those capable of binding human~SA very
efficiently. Of the nine mutants thus isolated, all had mutations in
the receptor binding domain (RBD), in the 120--259 region of
haemagglutinin~H5. Four of which were contagious for the ferret
inoculated by nasal route (N186K, S227N, Q226L, G228S mutations). These
mutants cause mortality in 3--7 days after intra-tracheal and
intra-nasal inoculations (10$^{6}$ viruses). The transmissible
H5~reassortant virus preferentially recognized human-type receptors,
replicated efficiently in ferrets, caused lung lesions and weight loss,
but was not highly pathogenic and did not cause mortality. Another
team, in Rotterdam (Netherlands), used directed mutagenesis to
introduce two mutations in the haemagglutinin gene of an H5N1~strain
(Q222L and G224S, to allow it to bind to human SA), and a mutation in
the RNA polymerase gene (PB2) to make it active at 33~\textdegree C
(the temperature of the human respiratory tract) instead of
40~\textdegree C (E627K). This H5N1 mutant, which is not airborne
transmissible, was then nasally inoculated into ferrets to force
adaptation through repeated passages (10~in total).
Airborne-transmissible viruses were identified, all carrying two
additonal mutations in the hemaglutinine gene (H103Y and T156A). This
suggests that only 5~mutations (four in HA and one in PB2) may be
sufficient to confer airborne transmission between mammals to the H5N1
virus. This H5N1~mutant was inoculated into ferrets via 10~nasal
passages, resulting in a fatal and highly contagious airborne disease.
Researchers identified four~critical mutations in H5 (N182K, Q222L,
G224S, N154K), enabling adaptation to  mammals~\cite{15}.

Noting the frequency of co-infections of pigs with avian~H5N1 and
human~H1N1 viruses, a Chinese team reproduced a co-infection in cell
culture to generate emerging pandemic  strains~\cite{16}. They
generated all possible reassortants between two strains: a H5N1~strain
highly pathogenic for mice (carrying two mutations facilitating
replication and SA~binding in mammals) but unable to transmit
efficiently through respiratory droplets in guinea pigs, and a
H1N1~strain which is airborne transmissible in guinea pig but not
highly virulent in mice. Out of the 127~hybrid virus, 35~were more
pathogenic that the H5N1~strain. Among those, some were also capable of
very efficient respiratory droplet transmission between guinea pigs.
These dangerous experiments, published in 2012 and 2013 demonstrated
that a few mutations enabling better replication and better binding to
SA~receptor were sufficient to cross the species barrier. These
gain-of-function experiments led to a moratorium being imposed in the
United States from October~2014 to December~2017. In October~2022, a
major H5N1~epizootic occurred in mink farms in Spain, affecting almost
52,000~animals, with a mortality rising from 0.7\% at the start to
4.3\% after three weeks. Analysis of the viral genomes detected
mutations in the replicative apparatus and the HA~encoding genes that
differed from those previously predicted, as well as additional
mutations in other  genes~\cite{17}.

\subsection{Coronaviruses}\label{sec1.2} 
Coronaviruses consist of a single-stranded RNA of 26--32~kb with an
envelope bristling with spicules made up of the S (Spike) protein. The
genome of the 2002 SARS (Severe Acute Respiratory Syndrome) virus
contains eleven genes. There are seven pathogenic coronaviruses in
humans. Four (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1) cause mild
epidemic respiratory infections in children (rhinitis, etc.). Three
others are responsible for severe respiratory infections: SARS-CoV1 in
2002--2003 (8346~cases, 646~deaths, mortality~7.8\%); MERS-CoV from
MERS (Middle East Respiratory Syndrome), which appeared in 2012 and
remains rampant in the Middle East (${>}$2000~cases from 2012--2024,
35\%mortality); SARS-CoV2, responsible for the Covid-19 pandemic,
emerging in December~2019 and evolving in iterative waves to 
date~\cite{18} (${<}$14~million deaths, 0.6\% mortality). The
viruses~SARS-CoV1 and SARS-CoV2 recognise ACE2 (Angiotensin-Converting
Enzyme~2) receptors, while MERS-CoV recognises the DPP4 (human
dipeptidyl-peptidase~4) receptor. Bats are the wild reservoirs for the
three coronaviruses that are pathogenic for humans. Intermediate hosts
have been found for SARS-CoV1 (palm civet, raccoon 
dog, badger) and for MERS-CoV (dromedary).
For \mbox{SARS-CoV2,} no intermediate host has been
identified after three years of the pandemic. Its origin remains
controversial~\cite{19}.

In Wuhan, the emergence of SARS in 2002 stimulated major research into
coronaviruses and, from 2004 onwards, a collection of samples taken
from bats captured in caves in southern China and south-east Asia. To
date, a stock of almost 15,000~samples (blood, saliva, urine, faeces,
etc.) has been built up, making it possible to identify more than
200~SARS-like coronaviruses by RT-PCR and sequencing, of which around a
hundred sequences have been published. The reservoir of the SARS-CoV1
virus was identified in 2005 in \textit{Rhinolophus spp}~\cite{20}. In
2013, Chinese researchers discovered several coronaviruses in bat
samples with \textit{spike} gene sequences very similar to those of
SARS-CoV1. These viruses can bind and multiply in HeLa cells expressing
the ACE2 receptors from humans, civets or \textit{Rhinolophus
affinis}~\cite{21}. In a cave in Yunnan Province, the Chinese
researchers discovered in 2017 eight unknown bat viruses closely
related to SARS-CoV1, including two with a functional Spike and six
with RBD deletions. The Spike of one virus (Rs4874) is identical
(99.9\%) to that of SARS-CoV1~\cite{22}. 

In 2008, an American team carried out a GoF experiment on a wild-type
strain of SARS-like coronavirus (Bat-SCoV). They produced a cDNA that
was transfected into cultured cells, obtaining a replicative virus
poorly penetrating human cells expressing ACE2. The RBD of Bat-SCoV
Spike was then replaced by that of SARS-CoV1. The new virus then
penetrated and replicated easily in primate and mouse cells~\cite{23}.
In 2015, GoF experiments were carried out by Chinese and American teams
using the MA15~\cite{24}, a SARS-CoV1 virus adapted by passages on
BALB/c mice~\cite{25}. This MA15 mutant is unable to penetrate human
cells. The addition to the MA15 virus of the \textit{spike} gene of a
wild-type SARS-like coronavirus (SHC014-CoV), produces a new virus
capable of recognising the human ACE2 receptor. This virus was serially
transmitted \textit{in vitro} on human respiratory epithelium cells,
where it reached titres close to the epidemic strain of SARS-CoV1.
\textit{In vivo} passages in human ACE2 transgenic mice, previously
described~\cite{26}, showed significant replication in the lungs. The
chimeric virus was then fully synthesised, confirming its high
replication and virulence \textit{in vivo}. It was no longer
neutralised by anti-SARS-CoV1\unskip\break 
antibodies or protected by anti-SARS
vaccines~\cite{24}. It was concluded that there is a potential risk of
re-emergence of SARS-CoV1.

In 2014, it was shown that the MERS-CoV Spike protein has two furin
sites that sensitise it to cellular proteases~\cite{27}. In 2017, an
American team carried out GoF experiments on MERS-CoV. The wild-type
virus propagated in human DPP4 receptor-expressing transgenic mice
initially causes no disease, but after 30 passages, the virus acquires
the property of growing 100~times more in the lungs than the parental
virus, leading to lethal infection in mice. Genetic analysis of the
MERS~viruses after passages shows the acquisition of 13 to
22~mutations, including several in Spike~\cite{28}. In 2021, an
international team showed that deletion of gene~5 in the MERS-CoV virus
led to hypervirulence. This gene normally stimulates interferon
production during infection of DPP4~transgenic mice~\cite{29}. This
shows that a gain-of-function can be obtained by deleting\break
a gene.

Recently in 2023, an American team carried out a Gof experiment on the
Omicron variant of SARS-CoV2. This virus carrying numerous mutations in
the Spike RBD escapes the humoral immunity of vaccine and is more
contagious and less virulent than the original Wuhan-Hu-2 virus from
2020. The Omicron variant causes a moderate non-lethal infection in
ACE2-transgenic mice. Replacing the Omicron \textit{spike} gene by that
of the Wuhan-Hu-1 (D614G) created a chimeric virus of omicron virus
that causes a severe infection in mice with 80\% mortality. Further
investigation showed that mutating non-structural protein 6 (nsp6) in
addition to the S~protein was sufficient to recapitulate the attenuated
phenotype of Omicron~\cite{30}.

\section{Synthetic biology}\label{sec2}
In 1965, the first deciphered sequence of a natural polynucleotide was
a yeast tRNA-Ala (76~nt)~\cite{31}, which was then synthesised in 1970
in the form of double-stranded DNA~\cite{32}. Chemists then developed
solid-phase oligonucleotide synthesis, which enabled the first
nucleotidic syntheses by segments of 40 to 80~nt~\cite{33}. The first
functional gene synthesised by this way was a 207~bp DNA encoding
tyrosine suppressor tRNA  from \textit{E.~coli}~\cite{34}. Larger genes
were then synthesised by assembling large polynucleotides obtained by
ligating overlapping \mbox{oligonucleotides~\cite{35,36},}
a tedious and
costly \mbox{process.} A 2700~bp plasmid containing the
$\beta$-\textit{lactamase} gene~\cite{37} and the 4917~bp gene
encoding the merozoite surface protein (MSP-1) of \textit{P.
falciparum}~\cite{38} were synthesised. This was followed by
considerable advances in nucleotide sequencing and synthesis with the
setup of high-throughput platforms, which considerably reduces time and
costs~\cite{39}. It is now possible to synthesise fragments of
8--30~kb, enough to reconstitute most RNA viruses. Several bacterial
genomes have now been chemically synthesised and assembled. In 2008,
the 582,970~bp genome of \textit{M.~genitalium} was
synthesised~\cite{40}, followed in 2010 by the 1.08~Mb genome of
\textit{M. mycoides}~\cite{41} and in 2017 the creation of nine strains
of \textit{Saccharomyces cerevisiae} in which one or two of the
16~chromosomes have been replaced by synthetic DNA~\cite{42}. Finally
the complete recoding of the \textit{E.~coli} genome was achieved in
2019~\cite{43}. Any microorganism genome can now be synthesised and
assembled from the \textit{in silico} sequences. 

\subsection{Virus synthesis}\label{sec2.1}
It was possible to produce viruses very early on by enzymatic synthesis
from viral genomes, using a DNA polymerase for DNA viruses or a reverse
transcriptase for RNA viruses. In 1978, the genome of the RNA phage
Q$\rmbeta$  (4127~nt) was converted into double-stranded cDNA~\cite{44},
incorporated into a plasmid and transfected  into \textit{E.~coli} to
produce functional phages. This approach, which subsequently allowed
mutations to be introduced into the genomes of RNA viruses, has
revolutionised our understanding of their biology~\cite{45}. A few
years later, the cDNA of a poliovirus inserted into a plasmid was
introduced into HeLa cells, enabling the production of infectious
polioviruses in very small quantities~\cite{46}. Thus, cDNAs from RNA
viruses can be easily prepared and used to regenerate either
positive-strand (mRNA polarity) or negative-strand RNA viruses,
depending upon the virus. This approach was widely used to synthesise
numerous RNA viruses, including rabies virus~\cite{47}, respiratory
syncytial virus~\cite{48}, influenza A virus~\cite{49,50}, measles
virus~\cite{51}, Ebola virus~\cite{52}, bunyavirus
(arbovirus)~\cite{53} and\break rotavirus~\cite{54}. 

In 2002, the Spanish flu virus was sequenced using viral sequences
obtained by reverse transcriptase from multiple human samples dating
back to 1918. The virus was then reconstituted into \mbox{fragments}
\mbox{reassembled} into cDNA corresponding to the 8 fragments of the virus.
These fragments were transferred into monkey MDCK cells or chicken
embryo chorioallantoic cells, previously infected with low
virulence~H1N1 viruses~\cite{55,56}. The viable H1N1 Spanish flu virus
was resurrected in this way, as highly virulent viruses~\cite{57}.
Other RNA viruses have also been reconstituted from viral fragments.
One can even reconstitute the genomes of retroviruses that have been
part of our chromosomes since time immemorial. In 2006, the entire
genome (9472~nt) of a HERV retrovirus was successfully synthesised
from endogenous retroviral remnants that had been inserted into human
chromosomes for over one million years~\cite{58}, followed by another
infectious HERV-K provirus (designated \textit{Phoenix})~\cite{59}.
These reconstituted retroviruses could replicate in cultured human
cells. Similarly in 2007, using viral DNA fragments obtained by RT-PCR
from chimpanzee faeces, it was possible to reconstitute \textit{de
novo} an infectious simian immunodeficiency virus (SIVcpz), which is
the virus most closely related to HIV-1~\cite{60}. The genomes of large
RNA viruses, such as coronaviruses (29~kb), are difficult to clone and
manipulate in \textit{E.~coli} due to their size and genome
instability. Viral fragments transcribed in cDNA are generated from
viral isolates, cloned viral DNA, clinical samples or synthetic DNA.
These fragments are then reassembled in a single step and cloned into
the artificial chromosome of the yeast \textit{S.~cerevisiae}. T7 RNA
polymerase was then used to generate the viable virus in yeast. The
SARS-CoV2 was thus reconstructed from chemically synthetized
clones~\cite{61}. 

Any virus genome can also be synthesised by \textit{de novo} chemical
synthesis from the \textit{in silico} sequence. In 2000, the first
replicative structure obtained in this way was a hepatitis~C virus
replicon lacking the structural protein genes~\cite{62}. In 2002, the
first complete synthesis of a poliovirus RNA virus (7500~bp) was
achieved in the absence of a molecular template~\cite{63}. The cDNA
obtained was transcribed \textit{in vitro} into infectious viral RNA
after incubation in HeLa cell extract. At the same time, the complete
genome of a DNA virus, phage $\Phi$X~174 (5386~bp), was synthesised in
a fortnight by chemical synthesis. The DNA was then transfected into
\textit{E.~coli}, which produced viable bacteriophages~\cite{64}.

\subsection{Remodelling entire genomes}\label{sec2.2}
The design of new microorganisms involves engineering and modifying
synthetic microbial frames, which is one of the best ways of
discovering the fundamental principles of life, leading to improved
applications in many fields, including medicine and industry. The
principle is inspired by the practice of refactoring used in computing
in order to improve existing software. The general aim is to improve
the internal structure of an existing system for future use, while
maintaining the main functions. 

\subsubsection{Genome recoding}\label{sec2.2.1} 
On the scale of the entire genome, the genetic code of a virus or a
bacteria can be modified. As a result of the degeneracy of this code,
there are several synonymous codons for the same amino acid, some of
which are used preferentially in the genomes of different
microorganisms. Introducing a codon bias can slow down protein
production, due to the use of rare codons. This process has been used
to attenuate the proliferation and virulence of polioviruses~\cite{65}.
This can also be done in a targeted way to restrict the expression of
certain genes (de-optimisation), without modifying a single amino acid
of proteins encoded by genes~\cite{66}. One can also use the bias of
codon pairs which are unequally distributed according to the various
genomes, with certain highly under-represented, impacting the rate of
viral replication~\cite{67}. In 2019, it was possible to synthesise and
recode the entire \textit{E.~coli} genome using 59~codons instead of 64
for the wild-type strain~\cite{43}.

To improve the performance of a microorganism for industrial purposes,
efforts have been made to identify non-essential genes by modifying the
microbial chassis. This allows to understand which genes are essential
for cellular properties and required to maintain cellular life. In
general, a number of computer analyses are first used to define the
genes essential for maintaining life. These genes are usually involved
in basic metabolism, cell wall metabolism, cell division and DNA
metabolism. Through synthetic biology, it is thus possible from natural
bacterial or viral genomes to redesign and create new biological
entities that do not exist in nature, mainly for medical or industrial
applications. It can help to design new viruses by manipulating
sequences \textit{in silico}, possibly with the help of Artificial
Intelligence.

Because of the widespread use of bench-top synthesizers, this
biological synthesis approach needs to be controlled, as it can be a
true threat. Engineering and modifying synthetic microbial chassis is
one of the best ways not only of discovering the fundamental principles
of life, but also of improving applications in the fields of health,
medicine, agriculture, veterinary medicine and public health. But it
also offers the prospect of malicious use on pathogens. 

\subsubsection{Construction of microbial chassis}\label{sec2.2.2} 
There are two strategies for building microbial chassis, top-down or
bottom-up~\cite{68}. The top-down strategy consists of progressively
reducing the microorganism's genome. This process makes it possible to
identify the genes that are essential for the survival of the organism 
by combining computer analysis of the systems, experimental data and
models such as those of metabolic, regulatory and signaling networks. A
framework is then constructed and modified by inactivating or deleting
non-essential genes using various strategies, including the use of
DNA-mediated procedures and site-specific recombinases, transposon
mutagenesis or CRISPR/Cas system. Examples of this approach include
genome reduction in \textit{E.~coli} and \textit{B.~subtilis}. This has
resulted in a faster-growing strain of \textit{E.~coli} than the
parental strain~\cite{69} and elucidation of the genetic basis of
secreted proteins in \textit{B.~subtilis}~\cite{70}. 

The bottom-up strategy uses low-cost chemical synthesis of DNA segments
from various genomes that will be fused, making it possible to
construct versatile chassis from different biological entities. The new
synthetised DNA fragments were assembled and transplanted into
organisms (cells, bacteria, etc.) to create and produce entire genomes.
Genetic entities can easily be designed by synthetic biology with the
help of computers. For example in 2016, a viable bacterium derived from
\textit{M.~mycoides} was successfully constructed, harboring a minimal
bacterial genome synthetised from an \textit{in silico} sequence of
531~kb instead of the 1079~kb of the wild-type strain~\cite{71}. This
strategy was also used in 2019 to synthesise a minimal genome of the
bacterium \textit{Caulobacter crescentus}~\cite{72}. Additionally, in
this way, personalised microorganisms can be constructed to produce
molecules for industrial use. It is also possible to reformat viruses
to improve their performance starting from known genomes. This was done
in 2005 with bacteriophage~T7, which was physically separated into
genomic segments that were then reassembled into an ergonomic virus
with improved performance. The chimeric virus is viable with all the
properties of the wild-type virus, but is much easier to
manipulate~\cite{73}. Viral genomes can therefore be easily
reconstructed to improve performance. Using the known viral sequence of
the horsepox virus~\cite{74}, researchers succeeded in 2018 in
synthesising the horsepox virus (212~kb) by fusing ten large
synthesised DNA fragments of 10--30~kb~\cite{75}. The resulting DNA was
introduced into cells infected with a related poxvirus, the Shope's
fibroma virus. Thus, a live horsepoxvirus is produced in cell culture.
This virus is less virulent in mice than the vaccinia virus and induces
a protective response against it. This is the first complete synthesis
of a poxvirus using synthetic biology. This publication is a debatable
technological achievement which has been heavily criticised, as it
reveals the strategy for constructing in the laboratory a very
dangerous virus, the smallpox\unskip\break 
virus~\cite{76,77}.

\section{Artificial intelligence}\label{sec3}
AI's ability to process large quantities of raw, unstructured data (DNA
sequences, proteins, etc.) has made it possible to reduce the time and
cost of certain experiments, to carry out others that were previously
unfeasible and to contribute to the wider field of genetic
engineering~\cite{78}. One of the applications of AI is the machine
learning, which allow to extract knowledge from data and learn from it
autonomously. By using algorithms to analyse large quantities of data,
this approach enables a machine to learn and improve automatically. We
can increase the human capacity to modify genetic material to obtain
specific functions. This raises concerns about potential biosafety
uses~\cite{79}. 

Applications to biological systems, in particular Machine Learning,
enable genetic systems to be modified and programmed with new
functions. Engineering principles and the use of systematic design
tools are used to reprogram cellular systems. The use of machine
learning has led to astonishing progress in the computational design of
proteins, enabling industrial and biomedical applications~\cite{80}.
Synthetic proteins have been designed to carry out cellular functions.
The corresponding genes can then be synthesised and inserted into the
microbial genome. This can be applied to virulence factors such as
toxins or adhesins in pathogenic microorganisms. For example, new
toxins can be designed from molecular models, and even dreadful weapons
can be created. A recent example is given by Collaborations
Pharmaceuticals, which uses an AI system called \textit{MegaSyn},
trained with pesticides, environmental toxins and drugs, in order to
find new medicines. Instead of looking for molecules with the lowest
possible toxicity, the researchers asked it to look for the highest
toxicity and bioactivity. In less than 6 hours, the AI generated more
than 40,000 neurotoxic molecules, each more toxic than the last. In
particular, it discovered VX, one of the most dangerous nerve agents
invented in 1952 and ten times more deadly than sarin. It has also
discovered other known chemical weapons and many as yet unknown 
molecules that are potentially more toxic than VX~\cite{81}. The
researchers said they had never thought before about how their tool
could be hijacked, and were very surprised by the results. With the
development of AI, the design of weapons that are still unimaginable
today might soon be within everyone's reach.

\section{Conclusion}\label{sec4}
Advances in biotechnology are ushering in a new era, one of
unprecedented scientific progress in our knowledge of living organisms,
but also one of dangerous biology, for which we must be extremely
vigilant. One can now easily modify the genome of microorganisms (loss
or gain-of-function) and synthesise gene sequences and even entire
microorganisms (bacteria and viruses), enabling major advances in many
areas of science. All these manipulations can be aided by the use of AI
to design \textit{de novo} manipulations and genome syntheses. These
include a better understanding of the evolution and properties of
dangerous pathogens. However, this has also implications in terms of
dual use and availability of highly dangerous germs that can be
synthesised from \textit{in silico} sequences available from databases.
In addition, genome synthesis of viruses offers unprecedented
possibilities for modifying natural genomes, allowing to create new and
potentially dangerous infectious microorganisms. As a result, the
Humanity could face the threat of pathogens far worse than anything
nature could create. The next big challenge will be to reconcile
scientific progress with biosafety and biosecurity.

\section*{Declaration of interests}
The author does not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and has
declared no affiliations other than his research institution.

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