Ebook: Emerging Biological Threat

Ladies and Gentlemen,

It is my privilege to welcome you on behalf of the Head of the Health Service of the Hungarian Defense Forces, on behalf of Major General László Svéd, M.D.

The date 6th of October is a historical Anniversary in the Hungarian Republic. In 1849 the Army of the Russian Czar defeated the defense forces of the Hungarian Democratic Revolution raised against the Austrian Habsburg Empire, and on the 6th of October 13 Generals were sentenced to death and executed by the Austrian government in the city of ARAD, located since 1919 in Romania.

The title of this 2003 conference is “Emerging Biological Threat”. We believe that this advanced research workshop will be one of the best initiated by the NATO Scientific department, and organised by Co-Directors Akbar Khan and George Berencsi. The topic itself has Hungarian roots.

The first microtechnique in microbiology was developed by Dr. Takátsy in the early fifties. The microplates and spiral loop system used for many decades for serological diagnostic has Hungarian origins.

This Advanced Research Workshop has limited funding. The title is very progressive, since emerging infections are concerned, but top experts in this topic are available in the United States. The Sponsor decided not to prefer training courses held by US professionals for Hungarian and eligible countries. In spite of this, we believe that this meeting will be very advantageous for participants.

There are many reasons for optimism. American, European and Hungarian participants are represented in equal proportions. I have to mention, that in 2003 the first Hungarian writer Imre Kertesz, was awarded with the Nobel Prize. Natural sciences have to aim to be at the same level.

I am sure that the material of the Workshop will be of outstanding scientific quality. I should like to wish you success in your work, and please prepare your manuscripts in time to be included in the edited proceedings of this meeting.

Budapest, 6 October 2003, Colonel István Kopcsó, MD, Hungarian Defence Forces, Institute of Health Protection

Respiratory infections have been reported in Hungary since 1932 on monthly basis to the National Institute of Public Health. These infections were complicated influenza like illnesses, measles, varicella-zoster, scarlet fever, epidemic meningitis, pertussis and diphtheria. The reexamination of these epidemics indicated, that the influenza epidemics were associated with the significant reduction of the number of clinical illnesses caused by other respiratory virus infections. The spread of enteroviruses during summers contributed probably to the decrease of infections caused by the above viruses, too. Epidemics of respiratory illnesses of bacterial etiology were not influenced by influenza epidemics, with the exception of N. meningitidis epidemics, which had been positively influenced by influenza and probably by other viruses (i.e. Respiratory Syncytial Virus). Combinations of microorganisms may significantly increase or reduce virulence or increase pathogenicity of each other. Combination of microorganisms may result in or interfere with emerging infections at the level of microbial physiology.

The global HIV/AIDS epidemic killed more than 3 million people in 2003, and an estimated 5 million acquired the human immunodeficiency virus (HIV) – bringing to 40 million the number of people living with the virus around the world. Every day about 14.000 persons in the world contract HIV [1]. This report concentrates to the preventive efforts introduced and run in Lithuania.

High prevalence of drug use during last years resulted in the significant increase in morbidity with hepatitis B and C and concentrated HIV epidemic in Estonia. Even the onset of the hepatitis A virus epidemics were shown to be associated with intravenous drug use. Characteristics of these emerging viral infections are summarised below.

Influenza, which are yearly circulating in the cities registered from the last decade of 19^th century. The same time it is an emerging threat, because influenza A subtypes periodically are changing their genetic and antigenic structures by reassortment of the RNA segments called “shift” variations.

Especially peculiar epidemic behavior have been observed in the case of influenza A/H1N1 infections during the last 40 years. Variants circulating in the world during 1947–57 disappeared due to the emergence of subtype influenza A/H2N2. The latter has been also eliminated by the emerging next pandemic strain influenza A/H3N2 in 1968–69.

Influenza A/H1N1 reemerged, however, in 1977 causing pandemic among people mostly younger then 30 years of age. Elder cohorts had been protected probably by residual immunity since it happened that the antigenic structure of A/USSR/90/77-like A/H1N1 viruses possessed nearly identical antigenic properties than those circulating in 1950^th.

There were four epidemics of influenza A/H1N1 in Russia after 1977–78 (in seasons 1981–82, 1984, 1986–87 and 1989). Influenza A/H1N1 viruses continued to undergo antigenic “drift” in comparison to the reference strains from A/USSR/90/77-like strains to A/Singapure/6/86-like variants. The spread of influenza A/H1N1 viruses did not prevent the circulation of A/H3N2-caused influenza A epidemics. The epidemics caused by influenza A/H1N1 variants alternated with epidemics caused by influenza A/H3N2.

The A/H1N1) viruses disappeared from open epidemic circulation for 5 years from summer 1989 till summer 1995. The A/H1N1 viruses caused mostly local outbreaks among schoolchildren and youth during the epidemic season 1995–96. The isolated influenza A/H1N1/1995 strains were A/Texas/36/91-like. Their hemagglutinins (HA1) were different in 8–10 amino acid positions from those of strains isolated between 1986–1989. The antigenic “drift” during 1990–1995 moved into a different direction than those isolated from 1950 to 1956.

After 1996 no open epidemics were caused by influenza A/H1N1 again for 5 years. The influenza A/H1N1 reemerged in the epidemic season in 2000–2001. Antigenic modifications of these variations are discussed.

Studies of the interactions of vertebrates, viruses and arthropod vectors of these viruses were monitored in terms of different ecological groups of viruses transmitted by mosquitoes and ticks in Northern Eurasia in an area encompassing more than 15 million km². About 90 viruses were isolated, including 24 new to science. Newly recognized infections of vertebrates, including humans, were described. Many unusual epidemic situations were analysed. Permanent efforts were established to prevent bioterrorist activities and their consequences. Extensive epidemic outbreaks of West Nile fever (WNF; i.e., fever caused by West Nile virus) and Crimean-Congo hemorrhagic fever (CCHF) with unusual high mortality appeared in the last four years in southern Russia. Infection rates in humans, domestic and wild animals, mosquitoes and ticks from natural and anthropogenic biocenosis had been determined. CCHF virus strains were phylogenetically similar to strains isolated in this area 35 years ago but different from Central-South-Asian and African strains. Before the outset of the current emergence of epidemic WNF, three genetic variants of this virus had been isolated in USSR, two African and one Indian. Phylogenetic analysis of complete genome sequences of epidemic strains demonstrated considerable similarity to strains from USA and Israel and differences from strains isolated in the same USSR areas 20–30 years before. In addition to strains of genotype 1, we isolated strains of second and third lineages and a strain of a fourth genetic variant. Nucleotide differences of these strains from all three genotypes was about 30%. The emerging WNF situation in Russia for the last 4 years probably has been the result of not only by natural and social factors but also by evolution of the virus.

The most important duty of a clinical virologist working in a public health laboratory is to find out the aethiological diagnosis of infectious diseases, especially of outbreaks of emerging/re-emerging diseases. In the case of zoonotic diseases search for natural foci and surveillance constitute the basis of the work that complete this requirement. The same preparedness is indispensable for the recognition of an accident involving biological and toxin warfare (BTW) agents [1].

Veterinary medicine had many practical problems with coronaviruses discovered in the thirties. Molecular biologists had to confront new problems when the unique size and replication strategy of the virus had been recognised first. The majority of the medical society became aware on the importance of the Coronavirus Family only upon the emergence of the SARS-CoV.

Herpes B virus (HB, Cercopithecine Herpes Virus 1) is a primate herpesvirus that has been reported to cause lethal infections in humans. To date, about two dozen well-documented cases of human infections with HB have been reported, with mortality of 75%. HB is indigenous to macaque monkeys, and macaques used in biomedical research including both rhesus (M. mulatta) and cynomologus or long-tailed macaques (M. fascicularis) are commonly infected with this agent without showing any visible signs of disease. Following primary infection, macaques may develop oral or genital herpetic lesions much like those caused by herpes simplex virus (HSV) in humans, and the virus subsequently establishes a latent infection in sensory ganglia. Latent HB virus may reactivate in response to stress and be shed in various body fluids. HB transmission to humans usually occurs from direct contact with body fluids such as saliva from infected monkeys. Primary human disease is characterized by vesicular eruptions on the skin or mucous membranes that are indistinguishable from herpetic lesions caused by HSV1 or HSV2. Unlike typical HSV infections that rarely lead to central nervous system involvement, HB causes a rapidly ascending myelitis and encephalitis that almost always leads to death.

Ticks are obligate hematophagous parasites attacking every class of vertebrate throughout the world. Currently, there is an increasing awareness of tick-borne diseases. Since the identification of Borrelia burgdorferi, eleven tick-borne bacterial pathogens have been described in humans. This review show that Central Europe is a heavily infested area where a new tick-borne rickettsiosis was described.

Dangerous viruses are highly contagious and pathogenic, i.e. they reach the target organ quickly causing severe morphologic changes. The tools of virus pathogenicity involve the capsid or envelope proteins that mediate adsorption and penetration to susceptible cells. In addition, virus-coded non-structural proteins trigger the shut-off of the host cell proteosynthesis and/or inhibit the host cell mRNA formation. In many cases, the virus-coded enzymes determine the rate of virus DNA or RNA synthesis, the destruction of host cell organelles and the assembly of new virus particles. The mechanisms selecting strains of higher or lower virulence among the virion progeny, follow the basic principles of genetics and apply equally to agents and their hosts. The outcome depends on the portal of entry into the human body, the route of transmission, the virulence of the agent and of its resistance to environmental conditions. Immobilization of the host by an average air-borne respiratory virus would create disadvantage for the spread of the given agent. Unless extremely contagious, it needs time to replicate and disseminates before killing the host. On the other hand, arboviruses transmitted by vectors may immobilize the host without influencing the frequency of transmission. The emerging viruses have not occurred before (HIV/AIDS, coronavirus/SARS) or they escaped attention, when affecting small and remote communities (Ebola and Lassa viruses). Some viruses, though certainly occurring before, had not been recognized to cause disease (hantavirus pulmonary syndrome, hepatitis C virus). These agents do not emerge as a result of mutations or recombinations, but rather represent a new germ, which survived in an animal reservoir. Re-emerging infections are diseases that once were a major public health problem, later on declined, but recently have become important again (reappearance of new influenza strains, dengue fever, rabies, West Nile virus and others). The control of emerging infectious agents by inhibiting the spread of particularly virulent variants of pathogens needs special control programs and surveillance systems (i.e. ProMED, Eurosurveillance). In addition, effective measures for fighting poverty and malnutrition, special vaccination and education programs, available medical care and treatment, and last but not least, alternative molecular methods for DNA or RNA identification (16S rRNA sequencing, broad range PCR and RT-PCR, representational difference analysis, novel toxin bioassays, comprehensive host gene expression profile etc.) are desirable. Increased urbanization and population density, social and political factors, local armed conflicts, increased migration and travel, natural and environmental changes and unexpected climate disasters make outbreaks of new infections possible and unpredictable.

Although commercially available nucleic acid amplification assays exist for three most important parenterally transmissible viruses, e.g. human immunodeficiency virus type 1 and hepatitis B and C viruses, the relatively high price of these assays obviates their use in many parts of the world. Among recent technical improvements in the field of molecular virology, real-time polymerase chain reaction (PCR) is one of the most exciting and is expected to be an alternative and a cheaper diagnostic tool for the detection of parenterally transmissible viruses. Real-time PCR allows a simultaneous amplification and quantification of specific nucleic acid sequences. This is achieved by a combination of rapid thermal cycling and cycle-by-cycle basis detection of the reaction kinetics by means of fluorimetry.

The U.S. Army Medical Research Institute of Infectious Disease's program for development of diagnostic technologies for defense against biological weapons is focused on supporting unit readiness by improving the health of soldiers in the field. The program explores the use of orthogonal technologies to detect multiple agent biomarkers to provide a high level of confidence to the diagnostic questions being asked. Currently, the program research goals are focused in four major areas: assay development, biological target identification, technology assessment, and testing and evaluation. Research conducted over the last seven years has led to the fielding of a large number of real-time nucleic acid detection assays and improved immuno assays capable of detecting all of the major biothreat agents. Future research efforts are focused on integration of technologies into a single device capable of providing high confidence diagnostics to forward deployed troops.

Infrequent infections of domestic animals and humans by Yersinia pestis, an endemic bacterial pathogen in many regions of the world, is a result of transmission by the bite of an infected flea and usually terminates with regional lymphadenitis (bubonic plague). However, progression to bacterial septicemia may result in lung colonization, organ failure and death in a high percentage of patients unless infection is controlled by early intervention with antibiotics. Due to the highly virulent nature of infections caused by inhalation of bacterial aerosols Y. pestis is listed by federal agencies as a Biodefense Category A pathogen. The protected intracellular bacterial growth of the earliest stage of infection suggest that cytolytic T cells (CTL) and innate immunity are critical to bacterial clearance. Several protein antigens recognized by CTL or antibodies are expressed by Y. pestis as components of the type III secretion system. Vaccine or therapeutic strategies targeting this virulence assembly may have the added benefit of providing cross-species protection.

Previous acts of biological terrorism involving release of B. anthracis spores within the United States logically prompted blood testing for the clinical detection of anthrax [1–3]. The mediastinitis that characterizes an initial course of inhalational anthrax is associated with a rapidly progressive bacteremia once efferent lymphatics become highly laden with organisms [2,4]. Therefore, PCR testing of blood should provide a rapid method for detection of anthrax in bacteremic patients [3].

The current revolution in biology especially genomics and proteomics, has identified genes encoding for new biological toxins and super-antigens. There is growing concern within both scientific defense and intelligence communities that this constitutes a serious potential for misuse as offensive biological weapons. Currently, sequences of close to 50 microbial genomes have been completed and the sequences of more than 100 genomes should be completed within the next 2 to 5 years. These sequences will encode a collection of >200,000 predicted coding sequences which will code for important functional proteins, as well as potential new biological toxins and super-antigens. Completed sequences of microbial genomes provide an excellent source to study the physiology and evolution of microbial species and expands our ability to better assign functions to the newly predicted coding sequences. Comparative analysis of sequences for multiple genomes will provide substantially more information on the emerging and re-emerging new biological toxins and super-antigens, and this information will be very valuable in the discovery of new signature sequences to enhance bio-detection, protection and treatment. A model comparative analysis using the complete genome sequence of an M1 strain of Streptococcus pyogenes, also known as group A streptococci (GAS) which is a strict human pathogen with no other known reservoir pr affected species will be discussed.

Distinguishing a particular microorganism from all others demands finding a target sequence that is unique for that organism, is conserved in all members of that species or strain, and has limited or no plasticity. Such targets can be a nucleic acid sequence, constitutive protein, or an antigenic epitope. The target can be probed with PCR-based, immunochemical, or mass spectroscopy assays. Genomics has played an important role in developing new generations of probes. The genomes of an organisms of which is the nucleic acid content has been sequenced and annotated is of invaluable aid in the identifications of targets. Post genomic disciplines such as proteomics and glycomics are and will usher in a new generation probes. The proteome can be defined as a set of proteins produced by an organism under a defined set of conditions. The presence of a protein is the ultimate proof that a gene is being expressed. The glycome represents the glycan groups or saccharide chains attached to proteins or lipids. Surface proteins (S-layer) are ideal targets. Since they are often found at the surface of a cell they are probed with antibodies without breaking the cell.

The “mysterious world of toxins” is connected not only to the force protection of military units, but also to our normal civil life. Several historical examples are important steps of our learning on microbial toxins. The illnesses caused by food poisoning during the last 5 years in Hungary are briefly mentioned [1,2]. Possible threat of emerging microorganisms has to be also taken into account. The demand for rapid detection has initiated the local development of reagents for the rapid detection of RICIN and S. aureus enterotoxin B. Emerging bioterrorism has to be coped by networks of military and civil laboratory facilities to be prone in preventing biological disasters.

One has to be first become aware that the possibility does indeed exist that “unconventional” biological agents be encountered in the field. There are a variety of strategies that could be implemented in either trigger or detection platforms that could be used to detect signatures from biological agents and possibly determine that they could present a danger to health and life.

However, we need to change our paradigms on how we think about the problem. The most important thing is to provide a detection and warning system that exploits credible signatures so that those in peril can take the appropriate protective measures.

The consideration of biological warfare has moved from the conventional military theatre to a terrorist operated bioterrorism event that's puts millions of people in cities across the globe under the threat of artificially acquired, life threatening infectious disease scenario. Biological warfare diseases are likely to present as one of a limited number of clinical syndromes. Plague, Staphylococcal Enterotoxin B and tularemia may present as pneumonia. Unfortunately many biological warfare diseases (Venezuelan Equine encephalitis, Q-fever, Brucellosis) may present as fever of unknown origin (FUO). Moreover, other diseases (anthrax, plague, tularaemia, smallpox) have undifferentiated febrile prodromes. Physicians must be able to identify early victims and recognize patterns of disease. The speaker will cover the clinical aspects of three most important infectious diseases caused by biological warfare agents: anthrax, plague and smallpox.

Recognition of need for local, regional, and national preparedness for threat against bioterrorism provides an opportunity to enhance the public health system and its linkages with current and new partners.