Proposed new cover page for TUJM

Coming up Next-TUJM

Tribhuvan University Journal of Microbiology-Vol 4(1)

Bioterrorism; An emerging threat to World

Bioterrorism

A bioterrorism attack is the deliberate release of viruses, bacteria, or other germs (agents) used to cause illness or death in people, animals, or plants. These agents are typically found in nature, but it is possible that they could be changed to increase their ability to cause disease, make them resistant to current medicines, or to increase their ability to be spread into the environment. Biological agents can be spread through the air, through water, or in food.  Terrorists may use biological agents because they can be extremely difficult to detect and do not cause illness for several hours to several days.  Some bioterrorism agents, like the smallpox virus, can be spread from person to person and some, like anthrax, cannot.

History

—  In 1984, in The Dalles, Oregon, U.S., a group of extremist followers of Bhagwan Shree Rajneesh (also known as Osho) contaminated the salad in 10 different salad bars with the pathogen of salmonellosis, Salmonella thyphimurium, in order to disable the population.  A total of 751 people contracted the disease and several of them were hospitalized. Although there were no fatalities, this terrorist act is considered the largest bioterrorist attack in the history of the U.S. (Török et al., 1997).

—  In the 1990s, the Japanese cult of Aum Shinrikyo tested different bioweapons, including botulin toxin, anthrax, cholera, and Q fever.  

—  In 1993, during a humanitarian mission in Africa, it tried to obtain samples of the Ebola virus.

—  Between 1990 and 1995, the cult attempted to carry out several bioterrorist acts in Tokyo using vaporized biological agents, including botulinum toxin and anthrax spores. Fortunately, the attacks were unsuccessful (Olson, 1999).

—  A significant bioterrorist event occurred in the U.S. contextually to the dramatic attacks to the World Trade Center in New York in September 2001. The release of Bacillus anthracis spores through the U.S. postal system was carried out with letters addressed to the press and to government officials. There were 22 confirmed cases of anthrax contamination, consisting of 12 cutaneous and 10 inhalational cases. The 12 cutaneous patients responded positively to antibiotic treatment, while of the 10 inhalational cases, 4 were fatal (McCarthy, 2001).

Figure showing Confirmed anthrax cases associated with bioterrorism: U.S., 2001.
A. Geographic location and clinical manifestation of the 11 cases of confirmed inhalational and 7 cases of confirmed cutaneous anthrax.
B. Epidemic curve for the 18 confirmed cases of inhalational and cutaneous anthrax and 4 cases of suspected cutaneous anthrax.

—  In 2002, in Manchester, U.K., six terrorists were arrested for being found in possession of ricin, and in 2004, traces of the same toxin were found at the Dirksen Senate Office Building in Washington D.C. (Bhalla & Warheit, 2004).

—  It appears evident then that the use of biological agents has moved, in recent times, to terrorist groups.

—  This creates very strong concerns that the use of bioweapons by terrorists can create unexpected scenarios characterized by massive destructive potential

Bioterrorism agents’ important features of a perfect BW are:

1. Highly infectious and highly effective.
2. Easily produced with a long shelf life.
3. Efficiently dispersible.
4. Readily grown and produced in large quantities.
5. Stable on storage.
6. Resistant enough to environmental conditions.
7. Resistant to treatment
8. High morbidity and mortality
9. Potential for person-to-person spread
10. Low infective dose and highly infectious by aerosol
11. Lack of rapid diagnostic capability
12. Lack of universally available effective vaccine
13. Potential to cause anxiety
14. Availability of pathogen and feasibility of production
15. Database of prior research and development
16. Potential to be “weaponized”

Category of Bioterrorism by Centers for Disease Control and Prevention (CDC):

The U.S. Centers for Disease Control and Prevention (CDC) defines a bioterrorism attack as “the deliberate release of viruses, bacteria or other germs (agents) used to cause illness or death in people, animals, or plants” (CDC, 2013).  It classifies biological agents into three categories

Category A:

·         The U.S. public health system and primary healthcare providers must be prepared to address various biological agents, including pathogens that are rarely seen in the United States.

·         High-priority agents include organisms that pose a risk to national security because they can be easily disseminated or transmitted from person to person;

·         result in high mortality rates and have the potential for major public health impact;

·         might cause public panic and social disruption; and

·         require special action for public health preparedness.

Groups	Diseases	Agents
A	Anthrax	Bacillus anthracis
	Botulism	Clostridium botulinum toxin
	Plague	Yersinia pestis
	Smallpox	Variola major
	Tularemia	Francisella tularensis
	Viral hemorrhagic fevers	Filoviruses (e.g. Ebola, Marburg) and Arenaviruses (e.g. Lassa, Machupo)

Category B:

—  Second highest priority agents include those that are moderately easy to disseminate;

—  result in moderate morbidity rates and low mortality rates; and

—  require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance.

Groups	Diseases	Agents
B	Brucellosis Epsilon toxin	Brucella spp. Clostridium perfringens
	Food safety threats	Salmonella spp., E.coli O157:H7, Shigella
	Glanders	Burkholderia mallei
	Melioidosis	Burkholderia pseudomallei
	Psittacosis	Chlamydia psittaci
	Q fever	Coxiella burnetii
	Ricin toxin	Ricinus communis
	Staphylococcal enterotoxin B	Staphylococcus spp.
	Typhus fever	Rickettsia prowazekii
	Viral encephalitis	Alphaviruses (e.g. Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis
	Water safety threats	Vibrio cholerae, Cryptosporidium parvum

Category C:

—  Third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of availability;

—  ease of production and dissemination; and

—  potential for high morbidity and mortality rates and major health impact.

Groups	Diseases	Agents
C	Emerging infectious diseases	Nipahvirus and Hantavirus

Other classifications:

Generally, biological agents (included those used as bioweapons) can be further classified according to certain characteristics that define the hazard to health (NATO, 1996):

a.       Infectivity: The aptitude of an agent to penetrate and multiply in the host.

b.      Pathogenicity: The ability of the agent to cause a disease after penetrating into the body.

c.       Transmissibility: The ability of the agent to be transmitted from an infected individual to a healthy one

d.      Ability to neutralise: Its means to have preventive tools and / or therapeutic purposes.

Transmissions:

Biological agents can be transmitted through one or more ways.  The transmission modes are the following:

1. Parenteral: Agents that are transmitted through body fluids or blood.
2. Airway (by droplets): Agents that are emitted by infected people, which can then be inhaled by surrounding people.
3. Contact: Through which the agents present on the surface of the infected organism can infect another organism.
4. Oral-faecal route: Through objects, foods or other items contaminated with the faeces of infected patients, or through sexual contact.

Impacts of Bioterrorism:

—  Economic impact of a bioterrorism attack could be devastating.

—  Cost $23 million to decontaminate a government building after 2001 anthrax attacks in the US.

—  Early intervention can significantly decrease the costs resulting from a bioterrorist attack.

—  Still expensive to provide prophylactic antibodies to a large number of individuals

—  Reduction in hospital admissions greatly outweighs initial costs

Warning signs:

—  In any location hit by a bioterrorism act the public health system will probably be first to detect and respond.

—  May not be realistic to wait for confirmation of diagnosis.

—  Delay increases the potential for spread.

—  Emergency response may need to be activated on basis of patterns and timing of patient presentation.

—  Important clues that can help alert hospitals to bioterrorist attack

—  Every health care professional should be suspicious of any unusual activity.

—  It will take many people in a variety of fields to control the impact of a biological attack.

—  Veterinarians –many infectious diseases are zoonotic

—  Scientists, epidemiologists, doctors, and nurses will need to work together.

—  Law enforcement –reporting disease and controlling public reaction

—  Bioterrorism is a matter of national and international security.

—  Require the coordination of local, state, federal, and international agencies

Individual role

—  It is imperative that you understand your role.

—  Prepare ahead of time.

—  Become familiar with the location of important telephone numbers and resources.

—  Then you will be ready to assist at a moment’s notice.

—  Your day-to-day responsibilities may be much different during the response to a bioterrorist attack.

—  First step is notifying the proper officials.

—  Know how to contact these agencies in advance.  

—  This may save crucial minutes during a time of chaos.

DNA-THE DEOXYRIBONUCLEIC ACID

THE STRUCTURE AND FUNCTION OF DNA

Biologists in the 1940s had difficulty in conceiving how DNA could be the genetic material because of the apparent simplicity of its chemistry. DNA was known to be a long polymer composed of only four types of subunits, which resemble one another chemically. Early in the 1950s, DNA was examined by x-ray diffraction analysis, a technique for determining the three-dimensional atomic structure of a molecule. The early x-ray diffraction results indicated that DNA was composed of two strands of the polymer wound into a helix. The observation that DNA was double-stranded was of crucial significance and provided one of the major clues that led to the Watson - Crick Model for DNA structure. But only when this model was proposed in 1953 did DNAs potential for replication and information encoding become apparent. In this section we examine the structure of the DNA molecule and explain in general terms how it is able to store hereditary information.

HISTORY OF DNA EVOLUTION

·         In 1868: F. Miescher isolated nucleic acids from white blood cells that were acidic in nature to which he called nuclein.

·         In 1880: Fischer isolated purines and pyrimidines.

·         In 1881: Zacharis identified nuclein with chromatin.

·         In 1899: Altaman replaced the term nuclein with nucleic acid.

·         In 1900s: Kossel identified the presence of histones and protamines with nucleic acids (Nobel laurate).

·         In 1910s: P. A. Levene discovered phosphate and pentose sugars callled deoxyribose molecule.

·         In1928: Frederick Griffith demonstrate the existence of a chemical in bacteria that caries genetic information

·         In 1943: Three American Microbiologist; Ostawald Avery, Colin MacLeod and Maclyn McCarty for the first time presented the evidence that DNA is the genetic material and is made up of genes.

·         In 1944: Oswald Avery showed that degradation of DNA and not protein resulted in loss of genetic information.

·         In 1950s: Rosalind Franklin and her supervisor Maurice Wilkins were working on the X-ray diffraction model for DNA.

Rosalind Franklin (1951):

·         Generated  X-ray crystallography data suggesting a double helix with phosphates on the outside

• Rosalind Franklin who actually proposed the concept of double helix was deprived of Nobel prize due to cruel death in 1958.
• Great revolution in DNA Biology
• In 1953 February: Pauling and R. B. Corey gave a triple helix model of the DNA molecule. However he couldn’t explain the process of DNA replication.
• They were near to present about the double helix model.

• In 1953 April: J.D. Watson (an American Biologist) and F. H. C. Crick (a British Physicist) presented the double helix model of DNA (published in Nature entitled “A structure for deoxyribose nucleic acid’).
• Nobel prize awarded to

o   Watson, Crick and Wilkins

o   in 1962.

DNA STRUCTURE

DNA is composed of nitrogen bases, deoxyribose sugars and phosphate. Adenine and guanine are purine bases while cytosine and thymine are pyrimidine bases. The phosphodiester bond between sugar and phosphate molecules form the backbone of DNA. The glycosidic bond is formed between nitrogen bases and sugar molecules.

Figure 1: Nitrogen bases

Figure 2: Structure of nucleotide showing phosphodiester bond and glycosidic bond

Figure 3: Hydrogen bonding between nitrogen bases

CHARGAFF EQUIVALENT RULE

In 1948, a chemist Erwin Chargaff, on the basis paper chromatography experiment, analyze the base composition of DNA. In 1950, he discovered that: In a DNA molecule of different types of organisms, the total no. of purines is equal to the total no. of pyrimidines. A/T=G/C

Number of Purines (A+G) = Number of Pyrimidines (C+T)

WATSON AND CRICK MODEL FOR DNA

The was proposed by Watson and Crick which was published in Nature in 1953. It is also known as ‘double helix model for DNA’ molecules. However, the photograph for model was taken from X-ray diffraction photograph from Rosalind Franklin.

Figure 4: Double helix structure of DNA

According to the Model:

·         DNA molecule consists of two strands which are connected by H-bonding and they are helically twisted.

·         Each step in one strand consists of nucleotide of purine base which alternately pair with pyrimidine base.

·         DNA is a polymer of four nucleotides (A T G C).

·         Adenine pairs to thymine with 2-H bonding (A=T).

·         Gaunine pairs to cytosine with 3 H-bodings (GºC).

·         Two strands apart 20 A from each other.

·         Helix coils in right hand i.e. clockwise direction and completes at every 34 A distance.

·         Two strands are complementary to each other.

·         One strand runs 5’®3’ while the complementary strand runs 3’®5’.

·         The polarity of DNA is due to direction of phosphodiester linkage.

·         Turning results in deep and wide major groove which is the site of bonding of specific protein.

·         The distance between two strands form a minor groove.

·         One turn of double helix at every 34 A distance includes 10 nucleotides.

·         Each nucleotide is situated at a distance of 3.4 A.

·         Sugar phosphate makes the back bone of double helix of DNA molecules.

·         The DNA model also suggested a copying mechanism of the genetic material which is semi conservative in nature.

·         Experimentally proved by Mathew, Meselson and Frank W. Stahl in 1958.

·         Universally accepted.

DIFFERENT FORMS OF DNA

Three different forms of DNA are found i.e. A form, B form and Z form. The B form (10 bp/turn), which is observed at high humidity, most closely corresponds to the average structure of DNA under physiological conditions. A form (11 bp/turn), which observed under the condition of low humidity, presents in certain DNA/protein complexes. RNA double helix adopts a similar conformation.  Z form (12 bp/turn) more loosely arranged DNA is found during DNA replication.

Figure 5: Different forms of DNA

A DNA Molecule Consists of Two Complementary Chains of Nucleotides:

A deoxyribonucleic acid (DNA) molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits. Each of these chains is known as a DNA chain, or a DNA strand. Hydrogen bonds between the base portions of the nucleotides hold the two chains together. The nucleotides are composed of a five-carbon sugar to which is attached one or more phosphate groups and a nitrogen-containing base. In the case of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group (hence the name deoxyribonucleic acid), and the base maybe either adenine (A), cytosine (C), guanine (G), or thymine (T). The nucleotides are covalently linked together in a chain through the sugars and phosphates, which thus form a "backbone" of alternating sugar-phosphate. Because only the base differs in each of the four types of subunits, each polynucleotide chain in DNA is analogous to a necklace (the backbone) strung with four types of beads (the four bases A, C, G, and T). These same symbols (A, C, G, and T) are also commonly used to denote the four different nucleotides-that is, the bases with their attached sugar and phosphate groups. The way in which the nucleotide subunits are linked together gives a DNA strand a chemical polarity. If we think of each sugar as a block with a protruding knob (the 5'phosphate) on one side and a hole (the 3'hydroxyl) on the other, each completed chain, formed by interlocking knobs with holes, will have all of its subunits lined up in the same orientation. Moreover, the two ends of the chain will be easily distinguishable, as one has a hole (the 3'hydroxyl) and the other a knob (the 5'phosphate) at its terminus. This polarity in a DNA chain is indicated by referring to one end as 3' end and the other as the 5' end. The three-dimensional structure of DNA-the double helix-arises from the chemical and structural features of its two polynucleotide chains. Because these two chains are held together by hydrogen bonding between the bases on the different strands, all the bases are on the inside of the double helix, and the sugar-phosphate backbones are on the outside. In each case, a bulkier two-ring base (a purine) is paired with a single-ring base (a pyrimidine); A always pairs with T and G with C.

DNA TOPOLOGY

In order to fully understand DNA topology, students need to familiarize themselves with three key mathematical concepts: twist (Tw), writhe (Wr), and linking number (Lk). Twist represents the total number of double helical turns in a given segment of DNA. By convention, the right-handed twist of the Watson-Crick structure is assigned a positive value. Writhe is a property of the spatial course of the DNA and is defined as the number of times the double helix crosses itself if the molecule is projected in two dimensions. The helix-helix crossovers (i.e., nodes) are assigned a positive or negative value based on the orientation (i.e., handedness) of the DNA axis. The numerical term that describes the sum of the twist and the writhe is called the linking number, which represents the total linking within a DNA molecule. Mathematically, these properties of DNA can be expressed as:

Lk =  Tw +  Wr

Why is DNA supercoiling important? Duplex DNA is merely the storage form for the genetic information. In order to replicate or express this information, the two strands of DNA must be separated. Since the global underwinding of the genome imparts increased single-stranded character to the double helix, negative supercoiling greatly facilitates this process. As a result, replication origins and gene promoters are more easily opened, and rates of DNA replication and transcription are greatly enhanced.

While negative supercoiling promotes many DNA processes, positive supercoiling inhibits them. When tracking systems, such as replication or transcription complexes travel along the double helix, they do not spiral circumferentially around the DNA. Rather, they move linearly through the DNA and the double helix spins to accommodate this motion. Recall from the earlier discussion that the ends of chromosomal DNA are not free to rotate. As a result, the number of turns of the helix remains invariant unless the nucleic acid chain is broken. Thus, the linear movement of tracking enzymes through DNA does not change the number of turns, but merely compresses them into a shorter segment of the genetic material. Consequently, the double helix becomes increasingly overwound ahead of tracking systems. DNA overwinding, or positive supercoiling, makes it more difficult to open the two strands of the double helix and ultimately blocks essential nucleic acid processes if not alleviated.

Figure 6: DNA supercoiling

Topoisomerases I

Type I topoisomerases are denoted by “odd” numbers (topoisomerase I, III, etc.). These enzymes are monomeric in nature and require no high-energy cofactor. There are two subclasses of type I enzymes, type IA and type IB. Type I topoisomerases act by creating transient single-stranded breaks in the double helix, followed by passage of the opposite intact strand through the break (type IA) or by controlled rotation of the helix around the break (type IB). Type IA enzymes require divalent metal ions for catalytic activity and covalently attach to the 5’-terminal phosphate of the DNA. In contrast, type IB enzymes do not require divalent metal ions and covalently attach to the 3’-terminal phosphate

Topoisomerases II

Type II topoisomerases are denoted by “even” numbers (topoisomerase II, IV, etc.). These enzymes contain multiple polypeptide chains and require ATP for overall catalytic activity. Prokaryotic enzymes have an A₂B₂ structure and eukaryotic enzymes are homodimers in which the bacterial A and B subunits have merged. Based on the structure of the archetypical bacterial type II enzyme, gyrase (see below), the A subunit (or domain) contains the active site tyrosyl residue that links to DNA during the cleavage event and the B subunit (or domain) contains the site of ATP hydrolysis.

Type II topoisomerases modulate DNA topology by generating a transient double-stranded break in the DNA backbone, passing a separate double helix through the opening, and resealing the break. All bacterial and eukaryotic type II enzymes require divalent metal ions for activity and those examined so far appear to utilize a two-metal-ion mechanism similar to that of DNA polymerases and primases. The cleavage reaction of type II topoisomerases generates DNA intermediates with 4-base, 5’-cohesive ends that are covalently attached to the enzyme through their 5’-terminal phosphates