Science of Biological warfare and Biopreparedness:

Germ warfare refers to activities that intend to induce mortality and morbidity of living beings by the application of bacteria, virus, fungi and their derivatives. Advances in the field of biotechnology have opened new avenues for the development of airborne, highly dispersible lethal biological weapons that may cause the death of millions of people. The SARS-CoV-2 virus which originated in Wuhan, China and caused global pandemic has led to speculation about the origin of the virus and the possibility that the virus has laboratory-based origins. While the use of biological weapons in war is not new, the impact of biological warfare on societies would be devastating. Besides state actors, such weapons falling into the hands of terrorist groups and militant organisations would also pose serious security challenges across the globe, with unimaginable consequences. This article aims to discuss the scientific and biotechnological prospective of biological agents and various microorganisms and the molecular mechanism of their potential candidature as bioweapons.

Biological weapons (BW) are weapons which contain replicating infectious and lethal forms of life including bacteria, viruses, fungi, protozoa, prions, or poisonous chemical toxins produced by living organisms. They have a strategic and technical advantage in wars because of their easy availability, low production costs, easy transportation and dispersal, and non-detection by basic security systems. These biological warfare agents (BWAs) multiply in the host and get transmitted to other individuals leading to a widespread disease with high morbidity and mortality. These disease-causing biological agents have been used to degrade combat capabilities of enemy forces at the war front. In the last few decades, several incidences of bioterrorism and biological warfare research and development have been recorded. As the world witnesses rapidly evolving geopolitical power shifts and competition, some countries, despite being signatory members of the 1972 Biological and Toxin Weapon Convention (BTWC, 1972) have started showing interest in biotechnological, genetic engineering and synthetic biology tools to develop highly potent and deadly chimeric biowarfare agents. Extensive covert research is getting established under vaccine and enzyme development programmes to modernise and weaponise the genetically engineered human pathogens to develop highly contagious strains that would defeat all the barriers of immune systems and current medical treatments. These researches include weaponising highly contagious, antibiotic-resistant recombinant novel strains and synthetic chimeric viruses to aerosolise and develop powder formulations for direct loading into munitions and cluster bombs.

Next Generation Biological Weapons

The technical application synthetic biology and genetic engineering tools can be strategically misused to transform harmless bacteria and viruses into lethal warfare agents with enhanced infectivity, pathogenicity, virulence, survivability and drug resistance. Political and military leaderships need to be made cognizant of the risks, threats and the impact of offensive biotechnological warfare attacks by potential state/non-state bio-terrorists, so that response activities for early prevention, detection, assessment, rapid response and recovery can be implemented.

The biowarfare agents are classified into six major groups^[i]:

1. Binary biological weapons: This includes a dual component system, consisting of a pathogenic host strain and a plasmid bearing virulence genes. These are first individually propagated at a large scale and then mixed for transformation within the munition, acting as a bioreactor and subsequently deployed as a bioweapon. This technique can be misused to enhance the virulence of human pathogens, causing anthrax, dysentery and plague etc.
2. Designer genes: Decoded and available whole-genome sequence data of pathogenic microorganisms, advanced genetic engineering tools and techniques can be misused to design, reconstruct desired virulence genes for creating novel lethal pathogens.
3. Designer diseases: Advanced molecular and cellular biology understanding can be misused to create designer pathogens to develop designer diseases with desired symptoms of a novel hypothetical disease. Somatic or germ cells can be targeted through inducing immune suppressive effects or inducing apoptosis, enhanced cell proliferation causing major tissue or organ system destruction.
4. Gene therapy based bioweapons: Retroviruses can be misused as vectors to introduce the desired gene in mammalian cells. These viruses integrate into the human genome while overcoming all the barriers of the natural defence system of the human body.
5. Host swapping diseases: Zoonotic diseases where a pathogenic virus has a natural animal reservoir can be swapped to humans through codon manipulations. Animal viruses can be humanised by genetically modifying to utilise preferential human codons.
6. Stealth viruses: Viral agents bearing human oncogenes can be illicitly transferred to human genomes. Exposing stimulus to initial dormant transduction can activate oncogenic determinants present on the stealth viruses which can destroy the human population.

Construction of synthetic infectious agents:

Living systems can be engineered with novel pathways by redesigning natural biological processes using synthetic biology tools. Whole-genome sequence data can be used to artificially synthesise, design, reconstruct virulent effector elements and genes with requisite pathogenicity to create infectious dwarfed genomes or genomes resembling natural human pathogens (for example synthesis of bacteriophage and mycoplasma genome). The first artificial bacteriophage, φX174 of 5386 bp genome was synthesised and stitched to produce biofuels^[ii]. T7 bacteriophage of 39,937 bp genome was redesigned by refactoring to generate chimeric bacteriophages by removing and replacing genetic segments maintaining replicative and functional activities^[iii]. Systematic mutagenesis  researches helped in understanding the minimal genome content essential for maintaining cell viability and supporting cell replication for Mycoplasma genitalium ^[iv]. The study led to the synthesis of the first dwarfed 582,970bp genome of Mycoplasma genitalium and construction of a slow growing M. genitalium to a synthetic, prolific designer strain M. mycoides^[v].

Synthesis of native or chimeric viruses: Synthetic virology tools assist in the construction of chimeric viral genomes with designer elements, in-vitro phage assembly, and development of efficient delivery systems.

1. Synthesis of the 1918 Spanish flu virus: Gene sequencing and RT-PCR technique was applied to reconstruct the first genome of the 1918 Spanish Flu from eight viral RNA segments recovered from lung tissue autopsy samples of pandemic victims^[vi]. Later using reverse genetics, the first synthetic virus was constructed. Different variants were reconstructed and studied for factors contributing to the severity of the disease, antigens and glycoproteins for attachments, mutations linked to epidemics in humans and birds, components of viral capsids required for assembly etc.^[vii]
2. Synthesis of poliovirus: First artificial poliovirus was constructed using cDNA synthesis. Twenty-five different mutations were investigated in cell lines and animal models for infectivity, pathogenicity, virulence and oncological features associated with the viral genome^[viii].
3. Synthesis of human endogenous retrovirus (HER): HERs includes a class of degenerate human retroviruses that infested human genome million years ago. Using synthetic consensus sequence and site directed mutagenesis, infectious proviral particles of HERV were generated^[ix]. Further, using whole-genome synthesis, another proviral clones of HERV were generated and studied for infection on human cell lines^[x].
4. Synthesis of the human immunodeficiency virus (HIVcpz): Viral nucleic acid strings were isolated from faecal samples of wild chimpanzees, and by deriving consensus viral sequences an artificial simian immunodeficiency virus (SIVcpz) was synthesised. This was further used to produce infectious molecular clones of immunodeficiency virus and investigated for cross-species transmission and host adaptive responses to viral infections^[xi]
5. Synthesis of SARS-like coronavirus: Severe acute respiratory syndrome virus coronavirus (SARS-CoV) and artificial clones of SARS-CoV were created by exchanging the receptor-binding domain (RBD) with that of human SARS-CoV capable of infecting human cell lines and animal model mice.^[xii] The repertoire of acquired research on human adaptation, virulent genetic loci and assembly of the designer pathogen can be misused to design deadlier viruses and pathogens.

In vitro packaging of viral genomes:

Arming DNA synthesis, and sequencing technologies in the genetic engineering arsenal have advanced the construction of the whole genome of viruses with desired pathogenic properties. Biological understandings of host-pathogen interactions, mechanism of infection, detailed mechanism of the packaging of viral genomes can be used to synthesise host-specific chimeric constructs with enhanced infectivity. Researches are accessible which are used for in vitro packaging of chimeric viral genomes for the assembly of infectious viruses.

All the researches shared above are published on various web research portals with experimental details and protocols which are accessible internationally. The initial intent of these studies is to use biotechnology for saving lives by understanding the mechanism of host-pathogen interaction for the development of vaccine, antimicrobials, therapeutics, biofuels etc., but the threat & risks associated with dual-use remains. The biodefense, biological security strategy and associated preparedness measures starts when the associated dual risks are understood, and the understanding is advanced and a step ahead to proactively prepare and engage in countering, preventing, mitigating the threats associated.

Biodefense and Bio-preparedness: technologies and strategies for Biowarfare agent detection

Biowarfare is an evolving and emerging national and global security threat with a potentially catastrophic economic, psychological, and social impact. To counter this, several countries have proactively established their comprehensive biodefense institutions and security strategies to strengthen early and efficient detection, protection, and decontamination of biowarfare agents^[xiii]. Advanced molecular and microbiological sensing techniques such as antibody-based immunoassays, cellular fatty acid profiling, flow cytometry, nucleic acid-based detection, mass spectrometry, microbiological culturing, and genomic analysis can be used for primary identification of biological agents. Efforts are being made across the globe for the development of highly efficient, reliable, sensitive, and selective technologies and system for detection and identification of BWAs.

Major technologies available for detection include:

1. Microbiological culturing: Microbiological culturing is the conventional, highly reliable and specific method for the isolation and identification of biological agents such as bacteria, fungi, and viruses. Microbes are cultured on selective media, and viable microbes can be studied for morphological and biochemical characterisation.
2. Flow cytometry: This technique involves the scattering of laser light and emission of fluorescence by excitation of dyes linked with bacterial cells. Fluorescently labelled monoclonal antibodies are used for detection and identification of various Biowarfare agents such as anthracis, B. melitensis, botulinum toxin, F. tularensis, and Y. pestis.^{[xiv] [xv]}
3. Cellular fatty acid-based profiling: Bacterial strains can be identified based on the variability of their fatty acids structures and profiles. Cellular fatty acids are converted to fatty acid methyl esters which are analysed by gas-liquid chromatography. GC chromatograms generate fatty acid fingerprints that are specific and employed for the identification and characterisation of various biological agents such as anthracis, B. mallei, Brucella, B. pseudomallei, F. tularensis, and Y. pestis.^[xvi]
4. PCR based detection: This molecular biology technique is sensitive and rapid for identification of biowarfare as compared to conventional microbiological techniques. Polymerase chain reaction (PCR) is used to identify an organism based on the presence of specific DNA sequence(s) in the organism. PCR-based identification has been reported in the case of various biowarfare agents such as anthracis, C. burnetii, filoviruses, F. tularensis, Y. pestis, and chimeric viruses such as Zika virus, yellow fever virus, Ebola virus, and Mengla virus.^[xvii]
5. Immunological methods: This technique is based on antigen-antibody interactions for identification of BWAs. The cell surfaces posses specific antigens to which antibodies bind and form a detectable coloured complex. Enzyme linked immunosorbant assay (ELISA) for example, is used for the presence or quantitative detection of antigens present on the agent. It is efficient, economical and readily employed for the detection of biowarfare agents such as anthracis, B. pseudomallei, B. mallei, Brucella abortus, Ebola virus, F. tularensis, Marburg virus, toxins, and Y. pestis.^[xviii] Fluorescent microscopy can be used, where a fluorescent labeled antibody is attached to bioantigen present on the surface of the agent. Immuno-histochemical based methods have been used to detect CHKVs^[xix]. Other, hand-held immuno-chromatographic assays (HHIAs) performed on nitrocellulose or nylon membranes, based on lateral flow immunoassays can be used to detect B. anthracis, B. abortus, B. pseudomallei, botulinum, F. tularensis, smallpox virus, Ricin toxin, variola virus, and Y. pestis^[xx].
6. Next-generation sequencing (NGS): NGS techniques are highly specific and rapid can be used to sequence multiple DNA fragments of bacterial and viral BWAs from clinical or environmental samples simultaneously. This technology has been tremendously used in diagnostics development, for identification and differentiation of novel infectious agents. NGS has been used for anthracis and Y. pestis. F. tularensis detection in human clinical samples of unknown etiology.^[xxi]
7. Bio-sensors: These are analytical devices that generate an electrical signal when interacting with analyte present in BWAs. The biological response produced is converted to a detectable form by the transducer, which marks the presence of any biowarfare agent in the sample. Biosensors are highly specific, selective, efficient in electrochemical detection of biowarfare agents. Immuno-biosensor consisting of bismuth nanoparticles (BiNPs) has been developed for anthrax PA toxin detection in a particular sample.^[xxii] Other electrochemical immunosensor includes gold and palladium bimetallic nanoparticles, genosensor loaded with gold nanoparticles, and gold nanoparticles and graphene transducer etc.^[xxiii]

Surface plasmon resonance (SPR) is another rapid and specific technique that has been reported for detection of BWAs like B. anthracis, botulinum neurotoxin, Brucella, Staphylococcus enterotoxin, and Y. pestis.

Piezoelectric biosensors have been developed for detection of F. tularensis, and staphylococcal enterotoxin A in milk samples.^[xxiv]

Bio-preparedness against next-generation biological agents

Bio-preparedness against BWAs includes the development of effective and safe preventive and treatment measures against infectious diseases. Biotherapeutics includes vaccines, chimeric proteinacous toxins, specific proteins, oligonucleotides, ribozymes, peptide based drugs and RNAi based antivirals which by blocking viral entry, inhibiting viral replication, cleaving target RNAs and inhibiting mRNA translation selectively killing the infected cell.

Chimeric or designer viruses as candidates to develop a vaccine

Chimeric viruses are efficient, affordable candidates for the development of vaccines against contagious viruses. The dual potential of the chimeric virus as a biotherapeutic or biological warfare agent is a covert and overt challenge. Few examples of chimeric viruses to develop vaccine:

• Chimeric Zika virus (ZIKV): Zika virus is a single-stranded RNA virus transmitted by Aedes mosquitoes which causes congenital neurological complications. Recently, a chimeric virus was constructed by swapping antigenic surface glycoproteins, and capsid anchor of yellow fever virus with the corresponding sequence of pre epidemic ZIKV isolate^[xxv]. Various tissue culture adaptive mutants were made and tested in mice model. In the same year, another group constructed chimeric Zika virus strain which was integrated into yellow fever virus attenuated backbone. The chimeric strains were investigated for Neuro-invasiveness in cell line and animal model.^[xxvi]
• Chimeric West Nile virus (WNV): West Nile virus causes infection in blood samples of vertebrates. A chimeric virus was prepared by coexpessing Dengue serotype and West Nile^[xxvii]. This vaccine construct was investigated for mutations to improve immunogenicity and viability.
• Chimeric Chikungunya Virus (CHIKV): A chimeric CHIKV vaccine was constructed by using three recombinant viruses as the backbone, i.e., sindbis virus, vaccine strain of Venezuelan equine encephalitis virus, and eastern equine encephalitis strain expressing CHIKV structural protein genes.^[xxviii] This chimaera developed immunogenicity and robust neutralising antibody response in both immunocompetent and immunocompromised mice model. More chimeric vaccine candidates were prepared using structural genes of CHIKV and nonstructural protein genes of Venezuelan equine encephalitis virus. The chimeric constructs were less infectious in CHIKV vector Aedes aegypti with lower dissemination as compared to the wild strains.^[xxix]

Decontamination technologies

Traditional decontamination systems to minimise adverse effects caused by hazardous biological agents include bleach and decontamination solutions. Localised small-scale remediation can be done using decontaminant solutions such as hydrogen peroxide, chlorine dioxide gas dissolved in water, phenolics, sodium hypochlorite, and quaternary ammonium compounds, or decontamination foams. Large-scale remediation can be done by fumigating with chlorine dioxide gas. Other tested and reported decontamination agents include ethylene oxide, glutaraldehyde, hydrogen peroxide vapour, peracetic acid, ortho-phthalaldehyde, ozone, and para formaldehyde. The alcohol solution is useful for hard nonporous and 70% alcohol solution decontaminates almost biological contaminates.^[xxx]

Autoclaving, dry heat, thermal washer disinfection, ultrasonication and sterilisation are other commonly used decontamination procedures. Ionising and non-ionising radiations, thermal energy, and reactive gases produced by plasmas can also be used for the decontamination of biological agents.^[xxxi] A portable arc-seeded microwave plasma torch for decontamination of BWAs is available.^[xxxii] Highly reactive plasma in a highly energised state effectively oxidised and destroyed all the biological agents. Vacuum cleaning with HEPA filtration is also an effective decontamination method which reduces the particulate load to allow effective remediation.^[xxxiii]

Development of novel decontamination systems against biowarfare agents with a key focus on practical, economical, fast, nontoxic, and specific decontamination should be prioritised. Ideal and eco-friendly decontamination technologies that focus on selective and effective disinfection of biowarfare agents are still in the infancy stage.

Conclusion

The strategic use of technology like bioweapons can be camouflaged as a natural outbreak of diseases with the capability to destroy human population, livestock and crops and cause other economic damages. The dual potential of advancing genetic engineering and synthetic biology can be exploited for the synthesis of next-generation bioweapons, eventually increasing the risk of biological warfare. All critical biological data such as decoded genome sequences of pathogenic bacteria and viruses are accessible through various national and international depositories. Researches on essential genes, virulence factors, or synthetic constructs with humanised infectious elements are accessible, which can be misused to develop designer genes, designer disease and next-generation bioweapons for bio-terror attacks. At present, global biodefense technologies for detection, protection, and decontamination are limited. There is a massive gap in knowledge, technology and strategy for preparedness which needs attention.

The scientific community must proactively engage for competent and dedicated scientific collaboration required for the rapid development of biodefense solutions to counter any probable biological attack. Sharing of scientific knowledge within the scientific communities is the critical pillar of safe scientific development. Reported incidences and evidence indicate an asymmetric correlation between offensive and defensive biowarfare strategies. Domestic laws against the use of bioweapons need to be enacted. The Biological and Toxin Weapon Convention (BTWC) needs to be strengthened through a legally binding instrument. Strict vigilance, enforcement and compliance of the provisions of the BTWC, the dedicated national portal for bio-surveillance and extended bio-intelligence network for information exchange between the countries is needed. It is essential to develop a national decision theatre and a dedicated wing in civil and military administration for biodefense and health security network. Developing specialised biodefense laboratories, promoting community immunisation program and awareness campaigns are the key initiatives for effective management against and biological incidents and catastrophes. A comprehensive national biodefense strategy needs to be developed and operationalised to support the nation’s ability to proactively prepare and develop essential defensive tools such as diagnostics, vaccines, antibiotics and other therapeutics.

Dr Aakansha Bhawsar is Research Scholar at Jawaharlal Nehru University, New Delhi and Dr Sudeep Shukla is Principal Scientist, Environment Pollution Analysis Lab, Bhiwadi, Rajasthan.

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