November 2005 I Issue 7
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Revisiting Smallpox Page 4
Jaron Chong
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Research Articles
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Revisiting Smallpox: Is a Now'Dead' Virus Still a Threat? Niranjan Vijayakanthan, Mohammad Zubairi, Hamilton Candundo, Brent Mollon, Gregory Agate
Synthesizing Human Antimicrobial Peptides: Harmful or Helpful?
12
Jaron Chong
West Nile Virus
Synthesizing Human Antimicrobial Peptides Page 9
Avian Flu H5N1: The Edge of Pandemic J_2_}f 'B'VWWWWW %vL Page 12 ~=| H A V W W W S A rr~~
Elena Igwe
Avian Flu H5N1: The Edge of Pandemic
Mosquito vector 17
Culexpipiens complex
West Nile Virus Page 17
Joseph Catapano
Avian Influenza Pandemic: Fight or Flight? RomyCho
21
References References
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Avian Influenza Pandemic: Fight or Flight? Page 21
About The McMaster Meducator The McMaster Meducator adopts an educational approach to our publication. Despite our efforts to ensure correctness, w e recognize that the publication m a y be subject to errors and omissions. In light of these potential errors and n e w developments in the medical field, w e invite you to partake in feedback and constructive discussion of the content herein for the purpose of furthering your understanding of the topic - in the n a m e of education and discovery. A n online discussion forum for each article is available on our website: www.meducator.org. Students and professors alike are welcome. Please enjoy the Meducator online experience! Disclaimer: The views represented in the articles do not necessarily reflect those of the McMaster Meducator and should not be substituted for medical advice.
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N o v e m b e r 2005 I Issue 7
Dear Reader,
M e d u c a t o r Staff
It always begins the same way. First a sniffle. Then a runny nose. Maybe bleary eyes and aPresident headache. But within a week you feel better and get on with your life. Jaron Chong Vice-President Brent Mollon
The sniffles were not what we had in mind. This issue promises to be one of our most hard-hitting to date as w e tackle this year's first theme: infectious diseases. Join Joseph Catapano as he examines West Nile Virus, an endemic and emerging disease in North America. Elena Igwe delivers a potent response to antibiotic resistance with n e w developments in anti-microbial peptides. As of late, Avian Bird Flu has been all over the news. Explore influenza H5N1's potential for pandemic in m y article and learn about probable treatments with R o m y Cho's report on influenza vaccines and antiviral medications. Finally, in a collaborative article by Niranjan Vijayakanthan, M o h a m m a d Zubairi, Hamilton Candundo, Brent Mollon, Gregory Agate, they discuss the possibility of the vanquished smallpox virus re-emerg ing as a looming biological weapon.
Creative Director Anthony Collini VP Medical Research and Health Ethics Jeannette So Shama Sud Jonathan Liu VP Public Relations Amandeep Rai VP W e b Design Edwin Ho Fify Soeyonggo
The sniffles were definitely not what w e had in mind. Every issue owes its creation to the brilliant cooperation of post-graduate editors, writers, and staff members. First and foremost, I would like to extend our immense gratitude to our post-graduate editors, w h o have taken time out of their busy schedules to provide our writers with valuable insight and feedback. Over the years, the Meducator has had unwavering and tremendous support from Dr. Del Harnish and the Bachelor of Health Sciences Program, for which w e are truly grateful. Our publication would be a collection of blank pages without the thoughts and words of our writers, whose extraordinary efforts are presented to you today. Over the past two years, I have had the distinct honour of working with the finest Executive assembled: Brent Mollon, Vice-President, for your leadership and impeccable attention to detail. Jeannette So, Shama Sud, and Jonathan Liu, VP's of Medical Research & Health Ethics, for your close work with both post-grad editors and writers, as well your exemplary editing acumen. Anthony Collini, Creative Director, for your dazzling artistic talent and keen sense of aesthetics. A m a n d e e p Rai, VP of Public Relations, for your amazing ads, tenacious correspondence, and the smarts to put technical and interpersonal skills together. Edwin Ho, VP of W e b Design, for your incredible c o m m a n d of all things w e b related. Finally, I would like to extend a w a r m welcome to our newest executive members: Harjot Atwal, Crystal Chung,Tyler Law, Joshua Ng, Sarah Mullen, and Fify Soeyonggo.
Junior Executives Harjot Atwal Crystal Chung Tyler Law Joshua Ng Meducator Founder Jonathan M. Ng
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Dr. Karen Mossman, Ph.D. On behalf of all of the writers and staff, we hope you enjoy this issue of the McMaster Dr. Jan Sargeant, Ph.D., Meducator. D.V.M.
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November 2005 I Issue 7
Revisiting Smallpox: Is a Now'Dead'Virus Still a Threat? Niranjan Vijayakanthan, M o h a m m a d Zubairi, Hamilton Candundo, Brent Mollon, Gregory Agate
n 1979, the World Health Organization ( W H O ) bioterrorism has halted this destruction (Friedman announced what is arguably one of the greatest et al., 2004) and rekindled debate within the World achievements in modern medicine: the world-wide Health Assembly ( W H A ) regarding the future uses of eradication of the virus which causes smallpox ( W H O , these stockpiles (Friedman etal., 2004; Enserink, 2005). 1980; Friedman & Isaacs, 2004). The importanceof such S o m e scientists argue that these stockpiles should be an accomplishment is realized w h e n one considers used to support research into n e w antiviral therapies that smallpox has resulted in more recorded h u m a n and safer vaccines. In addition, the destruction of deaths than all other infectious diseases combined these stockpiles would not necessarily guarantee the (McFadden, 2005). complete disappearance of variola (Enserink, 2005). Despite a mortality rate of 3 0 % in unvaccinated Indeed, current biotechnology has enabled scientists individuals, several factors m a d e this eradication to reproduce the 1918 Spanish Influenza virus from of smallpox possible (Friedman et al., 2004; Fenner, frozen corpses for the purpose of determining the Henderson, & Arita, 1988). These factors included source of its virulence (Tumpey etal., 2005). Forsimilar the availability of an effective vaccine, the lack of an reasons, the W H A has recently authorized a series of animal host, and the fact that all those infected with research studies on the smallpox virus, and both the the smallpox virus displayed symptoms of the disease United States and Russia have currently expanded (Friedman et al., 2004; Fenner et al., 1988). Thus, it wastheir research programs studying this virus (Enserink, possible to identify and isolate all infected individuals 2005). Although this virus is currently deemed as while fostering wide-spread immunity through the use of an available and easily produced vaccine eradicated, it is important to examine what threat it m a y pose should it re-emerge. In addition, if redeveloped from a less virulent poxvirus. It must be noted, simply because the W H O emergence does occur, h o w prepared are national declared this virus'eradicated'does not m e a n that live and global health agencies in containing the spread smallpox viruses (termed variola) have disappeared of this agent? altogether. O n the contrary, it is well publicized that two laboratory samples of the variola virus still exist THE BIOCHEMISTRY, PATHOGENESIS (Enserink, 2005). These samples, present in the Center AND PATHOLOGY OF SMALLPOX for Disease Control in theUnitedStatesand the Russian State Research Center of Virology and Biotechnology, The variola virus, which causes smallpox, is an were originally set to be destroyed June 30, 2002 Orthopoxvirus that belongs to the Poxviridae family (Friedman et al., 2004). However, post-9/11 fears of (Flint, Enquist, Racaniello, & Skalka, 2004). Historically,
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November 2005 I Issue 7
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Figure 1: While the mechanisms that determine host tropism in the variola virus are still not understood (McFadden, 2005), the vaccinia virus (the virus which is used to vaccinate individuals against variola) can be used as a template to explore Orthopoxvirus replication. The virus initially fuses with cellular membranes, ultimately releasing the viral core into the cytoplasm. The presence of viral DNA-dependent RNA polymerase and transcription factors results in the production of early mRNA, which produces the early proteins that facilitate genome replication. Once the DNA genome has been replicated, viral initiation proteins and the products of early genes result in the translation of intermediate mRNAs. Once translated, the intermediate proteins help stimulate the production of late mRNA, which codes for structural proteins and additional enzymes that must be packaged within the newly replicated virus. Once assembled, the virus matures into a brick-shaped intracellular mature virion (IMV), which can be released if the cell lyses. If lyses does not occur, the IMV will acquire a second double membrane from the Golgi to form the intracellular enveloped virion (IEV), which fuses with the cell membrane to bud the virus from the cell (Flint et al., 2004).
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November 2005 I Issue 7 there are two forms of the virus; variola major and variola minor. These two types are distinguished based on fatality rates, with the major form resulting in mortality in 3 0 % of cases while the minor form is associated with a 1 % mortality rate (Centers for Disease Control and Prevention, 2004a). The Poxviridae family consists of double stranded D N A viruses with a virion diameter of 170-200 x 300-450 n m . They also have 130-280 kb genomes, which are large enough to code for all proteins necessary for replication. Consequently, these viruses are minimally dependent on the host cell and are capable of utilizing their o w n proteins to synthesize D N A and produce viral m R N A s in the cytoplasm (Flint et al., 2004; see Figure 1 for an overview of the Orthopoxvirus replication cycle). Variola transmission occurs via inhalation of airborne variola virus, which then replicates within macrophages residing in the respiratory tract. In addition, the virus m a y be transmitted through direct contact with infected lesions, bodily fluids or contaminated objects and surfaces (Constantin et al., 2003). Once infected with the virus, macrophages migrate to lymph nodes where additional viral replication occurs. The presence of virus in lymph nodes and the circulatory system, k n o w n as viremia, allows the virus to spread to the rest of the body. Once the infection reaches small dermal blood vessels, it can result in infection of epidermal cells and endothelial swelling. W h e n the infection spreads to the deep vascular inner layer of the skin and sebaceous glands, the patient develops pockmarks which later heal to produce the scars characteristic of smallpox (Friedman et al., 2004). To b e c o m e infected with smallpox an individual would only need to be exposed to an initial 10-100 virus particles (FIRSTConsult, 2005). Normally, the time it takes for the virus to establish itself in its host prior to the onset of symptoms is anywhere from 10 to 14 days, during which the infected person is non-contagious (Constantin et al., 2003). In the following prodromal phase, which lasts between 2 to 4 days, initial symptoms appear and the individual m a y be contagious. Initial symptoms are described as influenza-like and most c o m m o n l y include a high fever, as well as body and headaches. Once the prodromal phase has passed, red spots appear on the tongue and mouth, which develop into sores. Subsequently, rashes appear on the skin, which then
6 WEWJWE
b e c o m e raised b u m p s and fill with fluid to become pustules. These pustules will eventually scab and fall off (CDC, 2004a). Despite the well documented progression of smallpox in the literature, less is k n o w n about the biochemical factors which result in the high virulence of the variola virus relative to other m e m b e r s of the Orthopoxvirus genus. For example, an analysis of the D N A g e n o m e of a Bangladesh variola strain has noted 187 putative proteins which might contribute to pathogenicity (Massung et al., 1994; Massung et al., 1993). Of these proteins, 37 were found to be notably different compared to the less virulent vaccinia virus. Thesedifferences m a y result in thecontrasting degrees of virulence between the variola and vaccinia virus, although the specific proteins involved are still largely u n k n o w n (Massung et al., 1994; Massung et al., 1993). O n e protein that has been identified, the smallpox inhibitor of complement enzymes (SPICE), has been shown to be 100 times more potent at inactivating the complement cascade protein C3b relative to the vaccinia counterpart (Rosengard, Liu, Nie, & Jimenez, 2002). In addition, it appears that the SPICE protein is more specific to h u m a n complement proteins, inhibiting the formation of the C3/C5 convertases which lead to pathogen destruction and opsonization (Rosengard et al., 2002; Janeway, Travers, Walport, & Shlomchik, 2005). Though routine vaccination is no longer c o m m o n practice, the recent threat of smallpox as a bioterrorist agent has resurrected the smallpox vaccine as a potential defensive measure. The United States has been at the forefront of developing a smallpox response plan, outlining immunization procedures and potential health risks (see CDC, 2005).
SMALLPOX VACCINATION
The current licensed vaccine in the United States i DryvaxÂŽ, which contains a live vaccinia virus that that produces neutralizing antibodies that are protective against viruses within the Orthopoxvirus genus. The DryvaxÂŽ vaccine is acquired by draining the lymph nodes of calves infected with the vaccinia virus (Rosenthal et al., 2001). Calf-lymph derived vaccines were used in the global eradication of smallpox (Arita, 2005). w w w . m e d ucator.org
November 2005 I Issue 7
The vaccine is administered using a bifurcated needle, dipped in the vaccine, and pricked on the skin surface in the upper arm (CDC, 2004b). The length of protection has traditionally been thought to vary between three to five years. However, Hammarlund et al. recently found immunity to the vaccinia virus in individuals immunized more than 25 years ago against smallpox, indicating that the vaccine m a y show effectiveness over longer durations than once thought (2003). Normal reactions to immunization with the vaccine include fever, body aches, and a scar from a scabbed pustule. Unfortunately, there are concerns over the adverse side effects of Dryvax速. As mentioned above, the smallpox vaccine contains a live form of the vaccinia virus, which poses the risk that the virus m a y spread to other parts of the body. Specifically, adverse cases are k n o w n to occur in persons with eczema, which is a chronic skin condition characterized by hardening of the skin, redness, inflammation, and flaking. In addition, individuals with suppressed or weakened i m m u n e systems face the risk of a progressive infection characterized by developing necrosis at the site of vaccination (Hong, 2005; see Figure 2). Pregnant individuals are also advised against immunization since there is a great risk to the fetus (Recommendations of the Advisory Committee on Immunization Practices, 2001). In addition to the adverse side-effects of Dryvax速, there is a need to find a replacement for the limited stocks of this vaccine, since its manufacture is no longer acceptable. The reasons for this include: the development process of Dryvax速 lacked controls, the risk of bacterial contamination in isolating the virus from calves, and the risk of diseases like bovine spongiform encephalitis from a bovine intermediary (Greenberg etal., 2005).
contain an outbreak of smallpox. The effectiveness of this plan in containing smallpox rests on the ability of the C D C to mobilize and deliver vaccines and personnel to areas of confirmed outbreak. Being able to isolate and vaccinate individuals with confirmed and suspected smallpox infection (a process called ring vaccination), along with those at high-risk of contracting the disease, is part of the plan's general strategy.The C D C realizes that certain barriers exist in the implementation of the above response plan. L ow residual immunity, lack of routine vaccination, health personnel's unfamiliarity with the disease and a growing population are factors that m a y contribute to the rapid spread of smallpox, making its containment difficult (CDC, 2004c; CDC, 2005).
Figure 2: Progressive vaccinia is a potential adverse effect due to smallpox vaccination. In this immunodeficient child, the vaccination site expanded with rapid necrosis instead of healing normally (Hong, 2005).
ESTABLISHING A RESPONSE PLAN This defensive initiative moves beyond the stockpiling With the many risks involved with vaccination, one of vaccines to include support for research initiatives. might ask h o w prepared is a nation like the U S A in M a n y scientists n o w claim that genetic modifications dealing with a potential re-emergence of smallpox? At of the variola virus could speed up the development of presentthe United States has enough stockof Dryvax速 more effective vaccines and antiviral drugs (BBC News, to immunize its current population (CDC, 2004c). The 2005). Facilitating such therapeutic and preventative Centers for Disease Control and Prevention (CDC) developments is a growing understanding of the Smallpox Response Plan is a set of guidelines to biochemical pathways involved in a smallpox infection. www.meducator.org
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November 2005 I Issue 7
Recently, Yang et al. published an article which explores host cellular pathways facilitating smallpox viral replication (2005). Here, they note that Smallpox Growth Factor (SPGF) binds to host receptors of the tyrosine-kinase superfamily. In particular, binding of SPGF to Erb-1 receptors stimulates intracellular signal pathways that ultimately aids in viral pathogenesis. This particular study examined the effects of a tyrosine-kinase inhibitor k n o w n as CI-1033 in variola and vaccinia infected m o n k e y liver cells.The inhibitor had no effect on the overall quantity of newly m a d e virus in experiments in which all cells were infected simultaneously. However, it did have an effect in the transmission of virus in the experiments that infected a single cell in culture. Plaques were minimized, indicating that the single infected cell was hampered in its ability to transmit the virus to unaffected cells (Yang et al., 2005). Although this example highlights s o m e of the antiviral research being conducted, more work needs to be done before the results of biochemical studies m a y be applied in a clinical setting.
of vaccination or potential for antiviral interventions (Weiss et al., 2004), any such predications would be clouded in uncertainty or prone to erroneous assumptions. Also, given the fact that the great majority of the population has never been exposed to smallpox, and that there is still debate as to whether past vaccination still provides immunological protection, past estimates of viral spread m a y also be misleading (Weiss et al., 2004). Nonetheless, the old maxim that'it is better to be safe then sorry'holds true w h e n evaluating the smallpox threat. It is by exploring the m a n y different biological agents which m a y be purposely used to take lives, and designing protocols to address these threats, that a country m a y best protect the health and wellbeing of their citizens in the gravest of circumstances. M
A special thank-you goes to Dr. Karen Mossman and Dr. Brian L chty for their aid in manuscript reviewing and guidance in current smallpox research.
CONCLUSION
Craig Venter, co-founder of Celera Genomics Corporation and adviser to former U S president Bill Clinton, addressed the American Association for the Advancement of Science on the on the threat of genetically engineered weaponry. During this speech, he suggested that cracking the genetic code of every bacteria and virus w a s no longer just a biomedical inquiry, but a threat to national security (Ellis, 1999). Although the variola virus no longer occurs naturally, its genetic sequence is well k n o w n and studied. This information m a y potentially be utilized to recreate the virus, and in the wrong hands it provides access to a lethal biological weapon. As w e have outlined above, s o m e countries, including the US, have b e c o m e apprehensive and developed a series of policies and procedures to be followed and implemented in the event of a biological attack. Although the potential to use smallpox as a w e a p o n exists, it would be impossible to accurately predict the d a m a g e the variola virus would inflict if reintroduced. With variables like method of dispersal, epidemiology of viral spread, effectiveness
8
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November 2005 I Issue 7
Synthesizing H u m a n Antimicrobial Peptides: Harmful or Helpful?
Elena Igwe
W i t h the emergence of antibiotic resistance to bacteria, researchers are investigating the potential impact of Anti-Microbial Peptides (AMPs) as a counter-measure. The isolation of these peptides, mechanisms of bacterial resistance to these agents, and h o w A M P s fight against bacteria must be understood before the use of h u m a n A M P s to treat diseases can occur. Although there is potential risk for bacteria to develop resistance against AMPs, research on these peptides looks to be a promising endeavour in increasing the number of available strategies to treat infectious disease.
AMP
CLASSIFICATIONS
Although there are different types of AMPs, they are generally small (less than 10 kDa), cationic, and hydrophobic, with activity at cell membranes (Hultmark, 2003). There are two main types of A M P s produced in the h u m a n body: cathalicidins and defensins. The latter are composed of p-sheets and three disulphide bridges, and can be separated further into two groups based on patterns of disulfide bridging and amino acid sequence motifs (Lehree & Ganz, 2002). a-Defensins are produced by neutrophils and (3-defensins are found in epithelia and skin. H u m a n s produce only one type of cathalicidins, HUMAN ANTIMICROBIAL PEPTIDES termed a-helical LL-37. This A M P is found in various With the introduction of antibiotics in the 1940's,neutrophils and epithelia (Pescel, 2002). A M P s bacterial resistance to these n e w drugs was originally also exhibit fungicidal, tumouricidal, and virucidal dismissed because it was thought to require an properties along with activity against Gram positive unusually high rate of advantageous adaptive and Gram negative bacteria, including antibiotic mutations (Bel & Gouyon, 2003). This premise resistant strains, which m a k e them good candidates turned out to be inaccurate, as antibiotic resistance for therapeutic drugs (Bals, 2000). is currently one of the largest problems confronting the health care sector. Scientists looking for n e w ways to fight bacteria have identified AMPs, a n e w class of self-protection proteins. A M P s act in conjunction with other factors as part of the innate i m m u n e system, which is the first line of defence against pathogenic attack (Bals& Wilson, 2003).
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MECHANISMS OF ACTION OF HUMAN ANTIMICROBIAL PEPTIDES Research into the functional mechanisms of AMPs has been an important area of study because it provides the basis for examining their pharmacological potential. There are three general models to explain the mechanisms of action of h u m a n cathalicidins and defensins (Figure 1). The first model is termed
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November 2005 I Issue 7 barrel stave. In this model the bacterial m e m b r a n e is perpendicular to the amphipathic peptides that align themselves so their hydrophobic side chains face outward into the lipid environment, while transmembrane pores are formed by the polar side chains that are aligned inward. These pores allow leakage of cytoplasmic components that disrupt the m e m b r a n e and kill the bacteria (Ehrenstein & Lecar, 1977). In the second model, termed the carpet, peptides are not inserted into the membrane, but are aligned in parallel to the bilayer while remaining in contact with the lipid bilayer and lipid head groups, thus coating the surrounding area. This model causes m e m b r a n e cracks, leakage of cytoplasmic contents, m e m b r a n e potential disruption, and eventually the disintegration of the m e m b r a n e (Pouny et al., 1992). The third model is the micellar aggregate model. This model suggests that an informal membrane-spanning micellar arrangement is formed by the peptide's orientation and association with the bilayer and can be used to explain translocation into the cytoplasm by the collapse of the micellar aggregates (Powers & Hancock, 2003). Any three of these models m a y be used by varying cathelicidins and defensins. However, the net result is the same in all cases - the disruption of bacterial m e m b r a n e integrity causing rapid depolarization of the cell membrane, leading to cell death (Powers & Hancock, 2003).
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Figure 1: Mechanisms of action of AMPs. After electrostatic interactions between the negatively charged bacterial wall and the positively charged peptides (a), the peptides associate with the membranes, leading to a destabilization of the m e m b r a n e and subsequent cell death (b). b1, barrel stave model; b2, aggregate channel model and b3, carpet model (Bals & Wilson, 2003).
10 LIEDMUOE
M E C H A N I S M S OF BACTERIAL RESISTANCE T O A M P S When AMPs are used, bacteria retaliate using their o w n defence mechanisms. One mechanism involves the disruption of AMP aggregation on the bacterial cell membrane, which causes the incorporation of components with reduced anionic charge and leads to obstruction of antimicrobial activity (Bel & Gouyon, 2003). Gram positive bacteria use this mechanism to substitute positively charged residues of the A M P s with the cell wall teichoic acids (Bel & Gouyon, 2003). The cell m e m b r a n e can also be altered to counter the effects of AMPs. This is accomplished by lowering the concentration of anioic phospholipids, which in turn, generates resistance. The negative surface charge of the bacterium Staphylococcus aureus is reduced by substitution of lysine into m e m b r a n e lipids, which minimizes loading of A M P s (Bel & Gouyon, 2003; Figure 2). Other mechanisms include the use of an efflux p u m p system to remove A M P s from the bacterial cytoplasm and the cleavage of a-helical A M P s by proteases (Guina, 2000). PHARMACEUTICAL CONSIDERATIONS Human immune cells and microbial organisms are in a constant battle for supremacy, however, the lytic properties of A M P s have tended to out-compete bacteria (Reddy et al., 2004). A M P s possess the ability to rapidly kill, within 1 to 2 minutes, a broad spectrum of microorganisms and pathogens, including bacteria that have been d e e m e d multi-drug resistant (Reddy et al., 2004). S o m e cathelicidins have already been found to prevent oral bacteria and yeastsfrom replicating and causing sickness (Guthmiller et al., 2001). Defensins have also shown a 9 9 % reduction in the formation of colonies of Mycobacterium tuberculosis (Miyakawa et al., 1996). These, and other h u m a n AMPs, are undergoing laboratory tests and clinical trials (Reddy et al., 2004). There are m a n y different strategies for A M P s therapeutic application: as single anti-infective agents, in combination with antivirals or antibiotics to induce any additive effects, as agents that enhance innate immunity, and as endotoxin-neutralizing
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November 2005 I Issue 7 Resistance Mechanism
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agents (Gordon et al., 2005). A salivary A M P used to THE FUTURE OF A M P S treat oral candidaisis affecting immunocompromised patientsandanAMPfoundinneutrophilsfortreatment Bacterial resistance has been linked to the overof severe pediatric meningococcaemia and Crohn's prescription of antibiotics. With careful control of disease are also a m o n g the A M P treatments in clinical A M P use, this novel discovery will be a promising trials (Paquette et al., 2002, Reddy et al., 2004). Inimex method for treating infections and diseases. S o m e Pharmaceuticals, a Vancouver based pharmaceutical disadvantages of synthesizing A M P s include high company, is developing immunoenhancement A M P s costs, patent exclusivity, sensitization and allergy that selectively upregulate innate immunity without after repeated application, confounding biological overstimulation of proinflammatory mediators functions, and most importantly, natural resistance (Gordon etal., 2005). by bacteria (Gordon et al., 2005). O n the other hand, To date there have been no published A M P s possess broad-spectrum activity, rapid onset of reports of commercial success in developing A M P s killing, potential low levels of induced resistance, and as therapeutic agents, but lab tests and clinical connection with broad anti-inflammatory activities trials are being conducted. The major concern is (Gordon et al., 2005). The next step for researchers whether bacteria will be able to develop resistance is to overcome the difficulties in synthesizing AMPs. to the synthesized drugs and in vivo h u m a n AMPs. A thorough understanding of A M P selectivity and If this were to occur, m a n y of the A M P responses to potential development of bacterial resistance to the bacteria m a y exhibit anergy. The neutrophil system peptides is necessary. Hopefully, continuing research could experience a higher rate and greater severity of in this area will provide us with a n e w therapeutic tool diseases due to chronic infection (Bel & Gouyon, 2003). to fight antibiotic resistant bacteria. M Although s o m e resistance to A M P s has been found, the impact on health depends on the m a n a g e m e n t of the problem.
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November 2005 I Issue 7
Avian Flu H5N1: The Edge of Pandemic
Jaron Chong
Figure 1 is a graphical representation of a typical influenza virion (a virus particle). The two most striking features present are the glycoprotein spikes located on the surface of the virion envelope as well as the eight g e n o m e segments located within. The spikes generally consist of two proteins: rodshaped hemagglutinin (HA) and mushroom-shaped neuraminidase (NA). Variants of H A and N A exist and are characteristic of an influenza subtype. Researchers label subtypes of influenza based upon these two proteins, combining the assigned H A number and NA number to form a n a m e like H5N1. The primary function of these surface proteins is to facilitate the transport of the virus to and from the host cell during influenza's life cycle (Figure 2). Hemagglutinin and neuraminidase are encoded by and translated from the 4th and 5th segments of influenza's RNA genome, appropriately named H A and NA. A high degree of genetic shift and reassortment will result in the various subtypes of influenza, a process greatly accelerated due to the modular nature of influenza's genome. Each surface INFLUENZA STRUCTURE AND FUNCTION protein variant has a unique amino acid sequence To understand H5N1, we must first establish the and will induce a specific response from the adaptive features c o m m o n to all influenza subtypes. Influenza immune system. As the expressed variants of can be classified into three categories: A, B, and hemagglutinin and neuraminidase are in constant C. The distinctions are made from differences in flux from one generation to the next, no single nucleoprotein, matrix protein, visual attributes, and vaccine can permanently inoculate a population. genomes. H5N1 itself is categorized as an influenza Vaccines must be updated to reflect the predicted type A. (Horimoto, 2005) dominant influenza strain for the upcoming season of inoculation. E n t e r i n g the scene on May 21st, 1997, a human pathogenic H5N1 strain of Avian influenza was isolated from a clinic in Hong Kong (Nature, 2005). What had typically been known as a strain of influenza afflicting only birds had suddenly made the jump to being pathogenic in humans. That singular event has triggered a tremendous amount of research activity and H5N1 has since captured the attention of international health organizations, governments, and the media. Despite this overwhelming concern, as of October 24th, 2005, only a total of 62 deaths directly attributable to H5N1 have been reported to the World Health Organization since 2003 (WHO, 2005). This is in stark contrast to the 10-25% of Canadians that are expected to contract conventional flu annually, with these cases resulting in an estimated 500-1500 influenza-related fatalities (Health Canada, 2003). What is the basis of our distress with H5N1? Is it justified? Is it all hype? Not surprisingly, the truth lies somewhere in the middle.
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November 2005 I Issue 7 Influenza A virus Neuraminidase (NA)
Matrix Protein (Ml)
Hemagglutinin (HA)
sense m R N A is exported out of the nucleus back into the cytoplasm to synthesize influenza proteins. S o m e of these proteins are re-introduced back into the nucleus to assist with viral R N A replication and vRNP assembly. Towards the end of the replication cycle, vRNPs form and leave the nucleus. The final stage then involves the progeny virion assembling and budding from the host cell's plasma m e m b r a n e completing the cycle of infection and replication (Whittaker, 2001; Knipe & Howley, 2001). INCREASED H5N1 VIRULENCE
Figure 1: Representation of an Influenza virion (a virus particle). Notable features include the surface glycoprotein spikes hemagglutinin (HA) and neuraminidase (NA). The eight g e n o m e segments promote accelerated genetic recombination of influenza (Murphy & Webster, 1996).
After fusion of the virion with the host cell takes place, e n d o s o m e uncoating results in the release of the viral g e n o m e (viral RiboNucleoProteins; vRNPs) into the cytoplasm. vRNPs are then imported into the nucleus where they are replicated. Positive-
H5N1 has been shown to exhibit much greater virulence than c o m m o n strains of influenza. As of October 26, 2005, H5N1 has displayed a fatality rate of just over 5 0 % , with the previously mentioned fatality count of 62 being out of a total 112 cases ( W H O , 2005). While this figure m a y be inflated due to incomplete H5N1 surveillance and sub-clinical infections (i.e. infections of H5N1 that do not result in symptoms), it still remains far in excess of the usual 0.06% to 0.25% fatality rate of typical influenza (CDC, 2005a).
Replication Cycle of an Influenza Virus Influenza virus Nucleus
(8) Re-import
(9)vRNA(-) _ replication (10) Assembly
""â&#x20AC;˘ (11) Formation of progeny vRNPs
Figure 2: Replication cycle of an influenza virus. (1)The influenza virion binds to sialic-acid containing receptors on the surface of the host cell and (2) fuses to form endosomes. (3) Endosome uncoating results in the release of the viral g e n o m e (viral RiboNucleoProteins; vRNPs) into the cytoplasm. vRNPs are then (4) imported into nucleus where they are (5) replicated. (6) Positive-sense m R N A is exported out of the nucleus back into the cytoplasm (7) to synthesize influenza proteins. S o m e of these proteins are (8) re-introduced back into the nucleus to assist with (9) viral R N A replication and (10) vRNP assembly. Towards the end of the replication cycle, (11) vRNPs form and (12) leave the nucleus.The final stage then involves the (13) progeny virion assembling and (14) budding from the host cell's plasma m e m b r a n e (Whittaker, 2001).
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November 2005 I Issue 7 and infectivity but low case-fatality (Dawkins, 1976). This natural selection takes time as a particular virus What is it about H5N1 that causes becomes adapted to a n e w host. However, in the this increased virulence? short term, zoonotic viral transmission can render any previously acquired i m m u n e modulating adaptations The answer is by no means simple and the question is still being actively pursued by researchers. There are, ineffective and virulence can dramatically increase however, several theories explaining this increased to the detriment of the n e w host species. This viral novelty in conjunction with a lack of any pre-existing virulence. immunity in the host culminates in a highly virulent Human H5N1 Results from a Zoonotic (Animal-Topathogenic transmission, suspected to be the case Human) Transmission with H5N1 (Lederberg, 1997). Aquatic birds form the primary reservoir for influenza Potential Mechanisms of Increased Virulence (Figure 3) and yet with most strains of waterfowl, influenza is non-lethal (Horimoto, 2001). This Several mechanisms have been proposed to explain interesting observation raises a key concept in the the workings of this increased virulence. gene-centred view of evolution: given sufficient time and generations of replication, an organism is Increased Hemagglutinin Cleavage expected to evolve to maximize the number of copies of its g e n o m e globally, also known as its inclusive One proposed mechanism considers genetic fitness. This is the very concept that Richard Dawkins variability of the hemagglutinin surface glycoprotein. proposes in his influential but controversial book, The As previously mentioned, hemagglutinin (HA) is Selfish Gene (Dawkins, 1976). critical in fusing influenza with host cells. It does this by attaching to sialic-acid receptors on the surface of a host cell and undergoing a conformational change. This fuses the viral envelope and the host cell together, initiating the complex viral infection process leading to the production of more virion (Whittaker, 2001). In the late stages of influenza replication, the H A expressed on the newly created virion will bind to its host cell while being released and prevent immediate dispersion of the infection. Surrounding proteases (protein-degrading enzymes) cleave the HA, releasing the newly created virion and leaving it free to infect neighbouring cells. These proteases are Figure 3: Waterfowl are the predominant reservoirs for influenza A viruses such oftentimes body region-specific, meaning viruses will as H5N1. Contact between species can result in viral transmission and adaptation to a n e w host population. Zoonotic (Animal-To-Human) transmissions are lethal adapt their H A glycoproteins to cleave in the presence as humans most likely lack pre-existing immunity (Horimoto and Kawaoka, of certain enzymes, most commonly, those present 2001). near respiratory endothelial cells (Goto & Kawaoka, 1998). Research conducted by Goto and Kawaoka Assuming the validity of the gene-centred view of describes h o w a critical adaptation of neuraminidase evolution, w e arrive at an important conclusion: (NA) protein is able to bind and increase the amount lethality is not in the best interest of any virus for it of surrounding plasminogen proteases and thereby destroys the host the virus lives within. Without a enhance H A cleavage. As a result, the variety of tissues host, a virus can neither replicate nor propagate. which are susceptible to infection is increased (Goto & Evolutionary, natural selection would favour Kawaoka, 1998). As this mechanism was discovered to strains of viruses that possess high transmission apply to a murine (mouse) strain of influenza derived
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from H1N1 (the 1918 Spanish Flu strain), there is a PREREQUISITES FOR A P A N D E M I C remote possibility that a similar mechanism occurs with Avian Flu H5N1. At the m o m e n t , this mechanism Put simply, there are three prerequisites in order f has only been observed in laboratory strains and pandemic to emerge. not in wild-type H5N1 (Katz et al., 2000). However, should the connection be m a d e between enhanced 1. Novel: A novel pathogen emerges - one that H A cleavage and H5N1, it would be consistent with h u m a n s have no immunity against. the increased virulence and systemic (body-wide) 2. Virulent: It infects h u m a ns causing serious symptoms being observed in patients such as multiillness. organ failure and non-bacterial sepsis syndrome 3. Contagious: It spreads easily and sustainably (Beigel et al., 2005). Investigations into this a m o n g humans. possibility are ongoing. LETHALITY As amply demonstrated, H5N1 is both IS N O T IN THE H5NI Escapes Anti-Viral Cytokine novel and virulent. It is novel in the BEST INTERESTS OF ANY Responses sensethatthe h u m a n population has VIRUS FOR IT DESTROYS not been exposed in any significant Another recent theory for increased manner to H5N1 and therefore THE H O S T THE VIRUS LIVES virulence relates to findings that WITHIN. W I T H O U T A HOST, any hosts it encounters will lack describe lethal H5N1 influenza virus immunity. H5N1 has definitely A VIRUS CAN NEITHER having the ability to escape host shown to be a highly virulent strain REPLICATE NOR anti-viral cytokine responses. The key of influenza with a large case fatality PROPAGATE. g e n o m e segment implicated in this rate (CDC, 2005b). i m m u n e system evasion is NS1, the only All that remains is for H5N1 to be nonstructural protein of the influenza A virus. contagious. Therein lies the lynchpin to the NSVs primary function is "the inhibition of cellular, entire sound, fury, and commotion - for without "the anti-viral interferon (IFN) response" (Garcia-Sastre establishment of efficient and sustained human-toet al., 1998; Cox et al., 2005, pg. 658). Despite these h u m a n transmission of the virus" (CDC, 2005b, para. viral strains inducing a strong i m m u n e response, 27) all of our fears remain hypothetical. they remain resistant to any antiviral effects. In a study conducted by Seo, Hoffman, and Webster, it ASSESSING THE PANDEMIC POTENTIAL was demonstrated that the 1997 H o n g Kong strain of H5N1 is unaffected by the activity of interferons and Imagine a highly contagious airborne pathogen with a tumour necrosis factor-alpha, two cytokines released 5 0 % fatality rate and a m e a n time from onset of illness by the body's innate i m m u n e system and the first to death ranging from 9 to 23 days ( W H O , 2005; Beigel line of defence in the response against an influenza et al., 2005). That scenario is exactly what faces the infection (2002). In this theory, a mutation in the international health community should H5N1 obtain NS1 molecule of H5N1's g e n o m e that encodes two the final pandemic prerequisite of human-to-human non-structural proteins seems to confer resistance to transmission. As of October 26, 2005, reports have H5N1, a conclusion drawn from the unaffected viral been inconclusive as to whether efficient h u m a n titers (concentrations) after treatment with a variety to-human transfer has occurred. Transmissions of cytokines. Their hypothesis is that the N S g e n o m e reported have ranged from household infections to segment both resists degradation and encodes a single case of child-to-mother transmission (Beigel proteins that inactivate anti-viral proteins produced et al., 2005; Hien et al., 2004; Ungchasuk et al., 2005). by the host's defence system. Further specifics and However, the major criticism of these reports has mechanisms are currently u n k n o w n (Seo, Hoffman, & been that primary infection from avian sources or Webster, 2002). poultry sources were the likely vector of infection and in the case of the child-to-mother transmission, close
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November 2005 I Issue 7 contact between child and mother was involved. All current reports indicate that transmission through regular contact is very inefficient and has a low likelihood of transmission. Further underscoring h o w the current strain of H5N1 in south-east Asia is currently unable to transmit very effectively is a cross-sectional survey conducted on hospital workers working with H 5 N 1 case-patients in Hanoi, Vietnam. While more than 9 5 % reported exposure to more than one H5N1 casepatient, 7 2 % reported symptoms, and 2.4% fulfilled criteria for a secondary infection, not one employee had detectable antibodies for H5N1 Influenza A (Idem, 2004).
virulence of H5N1 with the transmission abilities of conventional influenza, all three prerequisites for a pandemic would be fulfilled (Horimoto & Kawaoka, 2001). This reassortment has not yet been reported and is the critical event that international health organizations are anxiously awaiting. It is the last remaining prerequisite for pandemic Avian Flu.
Two DOWN, ONE TO GO
From the epidemiological information present, it is clear that a grave threat exists should H5N1 acquire pandemic status. M a n y comparisons have been m a d e between the Avian Bird Flu and the 1918 Spanish Flu pandemic that claimed countless lives (Horimoto How will H5N1 acquire the ability to efficiently transmit & Kawaoka, 2001; Seo et al., 2002; Unchusak et al., from human-to-human? 2005). In popular media, it is often stated that w e are "overdue" for a pandemic (Piller, 2005). Such claims Influenza ordinarily acquires mutations through imply disasters follow s o m e preordained schedule - a two processes: antigenic drift and antigenic misleading impression. All that is occurring is simply shift. Of the two processes, antigenic drift is the viral microevolution, following no set timeline and more gradual whereby natural selection pressures having very few certainties. applied to influenza strains select for advantageous It is at this unique point in time that w e hemagglutinin (HA) and neuraminidase (NA) genes. can be both calm and anxious. W e can take solace A single point mutation can result in sufficient drift in the knowledge that on the global stage, H 5 N 1 to confer a benefit to the influenza virus. However, still has a minor effect on loss of life in comparison it remains unlikely that efficient human-to-human to conventional influenza. At the s a m e time, the transmission will evolve from such gradual processes excellent surveillance efforts and cooperation of (Cox et al, 2005). the World Health Organization, the Centers for Antigenic shift is a change of a significantly Disease Control and Prevention, and national health greater order whereby R N A segments from two agencies throughout the world must continue and different viral strains are reassorted. As previously be adequately funded. Public attention m a y w a x mentioned, the eight genetic segments in which and w a n e with the w h i m s of popular media, but influenza A's g e n o m e is composed allow for it is ultimately the long-term efforts of scientists, exchanges between viral strains. Multiple scenarios epidemiologists, and health policy makers that will have been proposed whereby reassortment might determine our preparedness and response. M take place between conventional h u m a n influenza, which has efficient human-to-human transmission A special thank-you goes to Dr. Karen Mossman, Dr. Brian Lichty and Avian Bird Flu H5N1, which does not. There are and Dr. Martin Stampfli for their time, patience, and guidance in two ideal hosts for this exchange. A n H5N1 infected the virology and immunology of this article. I would also like to h u m a n could also simultaneously be infected with thank Alexander Caudarella and Andrew Schepmyer for their supportive research contributions and discussions. conventional influenza. With both viruses in a c o m m o n organism, reassortment can occur. Alternatively, reassortment could take place in an intermediary organism such as swine under conditions of dual infection. In either exchange of genetic components, should a combination be created that retains the
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West Nile Virus
Joseph Catapano
S i n c e its first North American case in 1999, West Nile Virus ( W N V ) has quickly established itself as an endemic disease of epidemic proportions (Gerber etal., 2004). While the m o d e of migration from its native Africa is not understood, the virus that was first discovered in 1937 has n o w spread to Europe, Mexico, the Caribbean and is predicted to hit South America next year (Gould et al., 2004). W N V is carried by mosquitoes and birds, and through bites, is transmitted to humans. It cannot be passed between people without blood contact though there have been cases reported of people becoming infected Mosquito vector Culex pipiens complex
through breast feeding, blood transfusions, as well as transplants, which necessitates better screening at blood donor clinics (Granwehr et al., 2004). There is currently no vaccine available, but there are m a ny prospective vaccines undergoing clinical trials with results to be published shortly. Generally, the young and healthy are not at risk for developing serious illness or neurological disorders associated with W N V ; however, the elderly and immunocompromised are at a significant risk for developing complications. Regardless, measures should be taken to avoid mosquito bites and control W N V (Granwehr et al., 2004).
Incidental infections Other mammals
Rarer, non vector transmission n utero Breast milk Occupational exposure Blood transfusion Organ transplant
Vertebrate reservoirs
a^*___ukiuusE Figure 1: Typical West Nile Virus (WNV) life cycle (Gould et al., 2004).
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November 2005 I Issue 7 While humans are considered dead-end hosts, several cases have occurred where the virus WNV is a single-stranded RNA flavivirus that, oncewas transmitted between h u m a n s through breast in a host's blood stream, is able to replicate within milk, organ transplantation and blood transfusions. the endoplasmic reticulum of vascular endothelial Blood transfusions and transplants are the most cells (Gerber et al., 2004; Brandt et al., 2004). W N V is worrisome m o d e s of transmission as only 2 0 % of maintained by a mosquito-bird-mosquito life cycle individuals exhibit symptoms, and the majority of (Figure 1). Although this cycle is broken w h e n other WNV-infected donors will be unaware of their disease vertebrates are infected, the virus does not replicate status (Granwehr et al., 2004). Between August 2002 enough to be transmitted further and the dead-end and January 2003, 20 cases of W N V transmission through blood transfusion were reported, forcing host becomes seriously ill (Gerber et al., 2004). The virus was originally endemic in Africa and the Food and Drug Administration to screen donors parts of Asia, but has quickly spread since the 1990's, from April through November - the peak mosquito introduced into n e w ecosystems through epidemics season (Granwehr et al., 2004). Even donors with in Romania and North America (Gerber et al., 2004). It WNV-like symptoms were deferred for 28 days, and was originally hypothesized that the virus would die those with symptoms after donating had to report to in the winter with most of the mosquito population. both the Centers for Disease Control and Prevention W h e n the virus resurfaced in 2000, scientists found and the blood collection agency. The Food and Drug that it was harboured in the blood of Celux mosquitoes Association also insists on conducting nucleic acid (Gould et al., 2004). The migration of birds continues tests before accepting donated blood to screen to bring W N V further south. The number of infected asymptomatic donors. birds increased tenfold between 2001 and 2002, and then again doubled in 2003 (Figure 2; Granwehr et al., SIGNS AND SYMPTOMS 2004). Many subtypes of W N V have emerged since the outbreak in 1999, and the virus seems to be evolving Few infected individuals exhibit symptoms, and of throughout North America.This poses a severe threat, those, less than 1 % develop neurological disease because the adaptations might render the virus more (Brandt et al., 2004). Symptom s range from a slight virulent. fever to severe neurological disease. Symptom s EPIDEMIOLOGY
5-i
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9858 4156
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03 U
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48 19
:
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2000
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Year Figure 2: Human cases of West Nile Virus infection in the USA, 1999-2003. (Granwehr et al., 2004)
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neurological disorders: encepahalitis, meningitis and acute flaccid paralysis (Figure 3; Granwehr et al., 2004). Encephalitis and meningitis are both lifeWest Nile Meningitis threatening diseases caused by an inflammation of Clinical signs of meningeal inflammation, including nuchal rigidity, the brain and its lining, respectively. Acute flaccid Kerrig's or Brudzinski's sign, or photophobia. Other evidence of acute paralysis resulting in asymmetric limb loss is less infection, including one or more of: fever (>38° C) or hypothermia Clinical Criteria for assessment of patients with suspected West Nile Virus infection
(<35° C); cerebrospinal-fluid pleiocytosis (=5x109 leucocytes per L); peripheral leucocyte count > 10x10 9 per L; neuroimaging findings consistent with acute meningeal inflammation.
c o m m o n (CDC, 2004). TREATMENT
West Nile encephalitis
Once an individual is infected with WNV, the current practice is to assist the patient in fighting off the infection in the form of respiratory support, m a n a g e m e n t of cerebral edema, and prevention of secondary bacterial infection (Gerber et al., 2004). Ribavirin, gamma-globulins, and steroids have all been suggested as treatment, but none have been subjected to clinical trials. In addition, interferon (an immuno-globulin) and ribavirin also demonstrate anti-WNV properties in vitro. However, the use of Acute flaccid paralysis ribavirin in managing the W N V in Israel showed Acute onset of limb weakness with clear progression over 48 h. At increased mortality (Brandt et al., 2004). least two of:asymmetry of weakness; areflexia/hporeflexia of affected Immunoglobulins yield the most promising limb(s); absence of pain, paraesthesia, or numbness in affected limb(s); cerebrospinal-fluid pleiocytosis (=5x10 6 leucocytes per L) results. The US National Institute of Health recently and raised protein concentrations (=5x10 6 mg/L); electrodiagnostic sponsored a study to find effective ways of studies consistent with an anterior-horn-cell process; spinal-cord combating W N V with immunoglobulin therapy. MRI documenting abnormal increased signal in the anterior grey matter. Immunoglobulins serve as antibodies in the blood and are able to neutralize certain surface proteins Figure 3: Clinical criteria for assessment of patients with suspected WNV on the premembrane of the virus (Granwehr et al., infection (Granwehr et al., 2004). 2004). Intravenous immunoglobulins have been typically develop after an incubation period of 2 to 14 shown to decrease W N V populations in hamsters w h e n administered within 24 hours of infection. days (Brandt et al., 2004). West Nile Fever is the term Unfortunately, use of immunoglobulins would used to describe symptomatic infection without any require active screening of people at risk and the neurological disease. West Nile Fever is associated late onset of W M V symptoms - typically beyond with malaise, nausea, vomiting, eye pain, headache, 24 hours - constrains the efficacy of this method of malagias and rashes, and lasts between 3 to 6 days treatment (Gould et al., 2004). Currently, the available (Granwehr et al., 2004). If the virus crosses the blood vaccines are ineffective for the general population. brain barrier, further neurological diseases arise and An equine vaccine m a d e available in 2001 shows may result in death. These severe cases commonly little effectiveness in humans, but there has since occur in the immunocompromised and the elderly been s o m e progress. The inactivated Japanese due to their decreased immunological capabilities encephalitis virus and the yellow fever virus are (Granwehr et al., 2004). Hypertension is identified being tested. However, these vaccines only reduce as a potential risk factor, as the endothelial walls the severity of W N V infection. deteriorate over time and allow the serum efficient Scientists believe that there are two access to areas of the brain (Granwehr etal., 2004). W N V viruses currently being studied that carry hope infection of the central nervous system leads to three for future W N V vaccines. The first vaccine uses Encephalopathy (depressed or altered level of consciousness, lethargy, or personality change lasting at least 24 h. Other evidence of C N S inflammation, including two or more of: fever (>38° C) or hypothermia (<35° C);cerebrospinal-fluid pleiocytosis (=5x109 leucocytes per L); peripheral leucocyte count > 10x10 9 per L; neuroimaging findings consistent with acute inflammation (with or without involvement of the meninges) or acute demyelination; presence of focal neurological deficit; evidence of meningeal inflammation; electroencephalography findings consistent with encephalitis; seizures, either n e w onset or exacerbation of previously controlled.
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November 2005 I Issue 7 a live-attenuated yellow fever 17D virus, termed even after washing. Finally, use of bacterial and insect Chimerivax, as a backbone. The vaccine is a hybrid growth inhibitors can decrease the overall mosquito of the premembrane and envelope protein genes population in the community, thereby decreasing of W N V and the non-structural genes of the 17D the number of bites. The most effective insecticides virus. Chimerivax was previously used to develop cause d a m a g e to the environment and will invariably vaccines for yellow and dengue fevers (Granwehr kill other insects that are beneficial or crucial to the et al., 2004). The vaccine prepares the body to deal ecosystem (Brandt et al., 2004). with the infection before it occurs by exposing the i m m u n e system to the shell of the W N V virus. Upon CONCLUSION second exposure, the i m m u n e system will produce the appropriate antibodies for the virus. The second Since arriving in North America, WNV has spread virus being used in developing a W N V vaccine is a to the Pacific Coast, Canada, Northern Mexico, live dengue-4-chimeric virus containing the prM and and Jamaica. Although h u m a n cases have yet to E protein genes of W N V . This vaccine lowers mouse be reported in these areas, steps must be taken to neurovirulence and neuroinvasiveness of wild-type ensure that the absence of this virus is maintained W M V , and even protects the vaccinated organism (Granwehr et al., 2004). W N V has great potential to do from second exposure to W M V (Granwehr et al., harm in the elderly population, to blood transfusion 2004). recipients and to transplant recipients. It is time to realize that W N V has b e c o m e endemic in North PREVENTION America with migrating bird populations that return from the South, further spreading the disease after Since vaccinations for WNV are not currently available, every winter season. While certain vaccines m a y soon the only method of W N V prevention is to avoid be developed and m a d e available in hospitals, it is mosquito bites. Even before applying insecticides imperativethatcommunitiestakeaction in preventing or other chemicals in mosquito-infested areas, the mosquito bites by adopting measures recommended most effective w a y of eliminating large populations by health experts. M of mosquitoes is to remove their breeding grounds - standing water. Cleaning out drain pipes, filling in standing water, and removing containers that m a y collect rainwater will greatly reduce the number of mosquitos (Gerber et al., 2004). In addition, securing screens around the house, using bed netting, applying mosquito repellent, and wearing low-exposure clothing outdoors are all effective ways of preventing mosquito bites (Brandt et al., 2004). The most effective mosquito repellents contain a substance known as DEET. The concentration of DEET can range anywhere from 10-30%, with higher concentrations being more effective. However, DEET has been linked with adverse effects and should only be applied sparingly and be kept away from cut skin. To prevent ingestion of DEET by children, their hands should be washed immediately afteroutdooractivities (Gerber et al., 2004). Clothing treated with Pyrethroid, another class of insect repellent compounds, also reduces the rate of mosquito bites in a similar manner through mosquito repulsion and remains on clothing
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Avian Influenza Pandemi : Fight or Flight?
R o m y Cho
L a t e l y , the buzzing about Avian Influenza in the media has been getting louder. Striking images of dead poultry and ducks fill not only our televisions, but our minds. With all this commotion, w e cannot help but look d o w n at the chicken on our dinner plates and wonder if w e are really safe. What is being done to protect us from another deadly influenza pandemic? The key weapo n against any viral pathogen is a vaccine. Unfortunately, there is currently no h u m a n vaccine available against the Avian Flu, and the task of developing one remains daunting ( W H O , 2005). The flu virus has the capacity to mutate at high rates, and it takes at least four months to grow and maturate vaccines (Park, 2005). By the time one is developed and produced, the virus m a y have already mutated to another virulent form. Several countries are currently preparing a prototype H5N1 vaccine. However, until the exact strain of H5N1 emerges and a pandemic is declared, the final vaccines will not be available (CBC News Online, 2005). That is not to say there is no hope against the Avian Flu - several drugs exist to combat the disease. Tamiflu (oseltamivir) and Relenza (zanamivir) are two such drugs under the class of neuraminidase inhibitors ( W H O , 2005). They are designed to inhibit the active site of neuraminidase, an enzyme that protrudes from the surface of the influenza virus. These drugs ultimately stop viral replication, inhibiting exchange of newly formed viral particles between cells (Moscona, 2005). They show great potential for
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preventing influenza infections and reducing the severity and duration of flu symptoms. In the event of a pandemic, they will play an important role in limiting sickness and death. Trials are desperately needed in order to assess whether these drugs work better alone or in conjunction with one another (CBC News Online, 2005). For m a x i m u m effectiveness, Tamiflu and Relenza should be administered within 48 hours of infection. They m a y also improve prospects of survival in h u m a n infections if administered early, but clinical data for this is limited ( W H O , 2005).
Figure 1: With the rise in cases human infections and deaths, these Vietnamese w o m e n protect themselves from the Avian Flu (US Department of State, 2005).
M 2 inhibitors, such as amantadine and rimantadine, are an older class of antiviral drugs also available for use ( W H O , 2005). Located in the viral envelope, M 2 proteins play a crucial role in uncoating
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November 2005 I Issue 7 the virus and exposing its contents to the host cell's cytoplasm. Inhibitors sterically block this process by binding to transmembrane proteins of the host cell (National Institute of Allergy and Infectious Diseases, 2004). Unfortunately, the M 2 gene is susceptible to frequent mutations and drug resistance develops rapidly, limiting their effectiveness in a pandemic. The M 2 inhibitor, amantadine, also sometimes has rare but disturbing side effects such as psychotic episodes (Abbott, 2005). DRUG RESISTANCE IN HUMANS The development of drug resistance is a realistic concern, especially since only a handful of medications exist to treat Avian Flu. A recent case in Vietnam retrieved ten viral clones from a patient sample. W h e n tested with Tamiflu, sixclones were highly resistant (IC50>763nM), three clones were slightly resistant (7.1 nM<IC50<12.5 n M ) and one clone was highly sensitive to the drug (IC50=0.6nM). IC50 is the concentration of a drug needed to inhibit viral replication by 5 0 % (Mai Le et al., 2005). More drugs are required to inhibit resistant viruses, and thus IC50 values increase with resistance. Tamiflu-sensitive viruses generally have an IC50 between 0.1-10 n M (Mai Le et al., 2005). Furthermore, the patient in Vietnam had been taking care of her 21-year old brother, w h o had also been infected with the H5N1 strain of the Avian Flu. The brother's viral samples contained a neuraminidase gene that was identical to the patient's clone 7 virus. The patient's lack of interaction with poultry, and the timing between infections of the two siblings raised the possibility of viral transmission from human-toh u m a n (Mai Le et al., 2005).
to m a n a g e a pandemic. Roche Pharmaceuticals is currently the sole producer of Tamiflu. They have recently quadrupled production and licensed several generic drug companies to produce the drug (Abbott, 2005). Roche has also m a d e a donation of 3 million doses to the World Health Organization ( W H O , 2005). However, a supply bottleneck persists with Roche's monopoly. At current rates of production, it would require over a decade to produce enough Tamiflu to treat 2 0 % of the world's population. Tamiflu production is a complex and time-consuming process and is difficult to transfer to other facilities. The option of overriding the Roche patent to produce a generic version of the drug m a y be exercised if there was ever a shortage during a pandemic (CBC N e w s Online, 2005). Recent studies using mathematical models suggest that drugs m a y prevent a pandemic if extensively used around main outbreak centers with strict quarantine and other non-medical measures (Weir, 2005).
DRUG DILEMMAS Figure 2: Avian Flu vaccine development is a pressing issue (Tribune, 2005).
Knowing that this biological concern is no longer limited to avian populations, m a n y developed countries are beginning to stockpile medications ( W H O , 2005). Online pharmacies have been receiving a flood of orders - a ten-fold increase in the past month - with each company n o w receiving 20 orders a day (CBC N e w s Online, 2005). Present levels of global drug production fall short of the quantities required
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The prices of drugs are too expensive for m a n y countries, particularly developing countries, which tend to suffer a lack of resources during pandemics (Marshall, 2005). O n e pill is needed to protect against the virus for one day, and based on past influenza pandemics, the first wave in a given area is believed to last up to 100 days. Knowing this, an individual
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November 2005 I Issue 7 awarded to ID Biomedical Corporation (Quebec City), and they are hoping to begin testing on people next year (CTV N e w s Online, 2005). The goal is to develop a virus seed bank so that a vaccine can quickly be created in the event that a h u m a n strain of the virus emerges. Influenza vaccines are traditionally How Is CANADA DOING? grown in fertilized chicken eggs ( W H O , 2005). This cost-effective method uses approximately one or The WHO has also been urging all countries to two eggs to produce one dose of vaccine. However, develop plans in preparation for a possible outbreak. Currently, only 50 countries have divised a protocol, this production method m a y be problematic as the a m o n g which Britain and Canada have obtained legal H5N1 strain is lethal to the embryo, killing it before status for their plans (Abbott, 2005). It has always been the virus can be harvested. The promising use of difficult to even persuade developed and wealthy reverse transcriptase to produce a version of the virus countries to stockpile on medications, especially that is non-lethal to the embryo is currently being w h e n the drugs m a y not be used during the term of investigated ( W H O , 2005).This process involves using the current government (Abbott, 2005). The drugs reverse transcriptase to convert viral R N A into D N A also have a short five-year shelf life, and widespread (Neumann et al., 2005). Naturally, the influenza virus use could m a k e situations worse by creating resistant does not encode reverse transcriptase nor does it go strains (CBC N e w s Online, 2005). However, with the through a D N A intermediate. Mutations can then be 2004 Avian Flu crisis in British Columbia, where an introduced into the cloned D N A and w h e n this D N A H7 influenza virus was detected on a farm, Canada is is reconverted back into the R N A state, the mutations beginning to see the urgency of the situation (Public pass on and occur in the g e n o m e of the viral RNA. In Health Agency of Canada, 2005). That crisis clearly thefuture, there will likely bean increasing preference demonstrated h o w novel strains of influenza, albeit to generate influenza vaccine strains through reverse genetics as it is less cumbersome than traditional not H5N1, could emerge in Canada. Canada was one of the first countries to methods (Neumann et al., 2005). order Tamiflu. It has purchased 23 million doses to protect essential workers and treat those w h o were already infected (CBC N e w s Online, 2005). Through the Canadian Pandemic Influenza Plan developed in 1988, guidelines are outlined for coordinated efforts between all levels of government, public healthcare officials, and emergency workers, and describes their roles and responsibilities in surveillance and responding to a pandemic (Public Health Agency of Canada, 2005). Health Canada's National Microbiology Laboratory in Winnipeg also works with laboratories across the country to identify and monitor emerging Figure 3:The shortage of Tamiflu and other antiviral medications arouses global influenza strains (CTV N e w s Online, 2005). According anxiety (http://www.roche.com.tw/medicine/Tamiflu.gif). to the W H O , Canada is on the right track. With the measures it has taken through its Pandemic Influenza Although we have yet to see the global effects Plan, it should be well-prepared in the event an Avian of Avian Flu, the pandemic virus still poses a great threat. The numerous unprepared countries, limited Flu pandemic occurs (CBC N e w s Online, 2005). In addition, Canada is one of the few countries supply of vaccines, and drug production difficulties to have obtained the genetically-modified seed strain m a y limit our ability to contain H5N1 should a of H5N1 for vaccine development (CBC N e w s Online, pandemic arise. Adequately addressing these issues 2005). The contract to produce the vaccine has been should be a priority for international health policy, ffl would need to stockpile 100 pills and start taking them the m o m e n t the virus circulated within the relevant region - a burdensome cost to m a n y thirdworld countries (CBC N e w s Online, 2005).
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Looking for something to do? Have you really been "meducated"? Try the following multiple choice questions and submit your answers online at www.meducator.org. Participants with the highest scores will automatically be entered into a draw for the Meducator prize.
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Brent Mollon - Revisiting Smallpox
Jaron Chong - Avian Flu H5N1
7. What rate are the three prerequisites for a viral pandemic? 1. The form of smallpox infection that is known for a 30% human fatality is associated with what virus? I. Origination in host population. a) Cowpox II. Novel introduction to target population. b) Vaccinia III. Evasion of immunological responses. c) Variola minor IV. Contagious spread within target population. d) Variola major V. Virulence and high rate of case-fatality e) Monkeypox I, II, 2. The Center for Disease Control plans to take which actions in thea)event of III a smallpox outbreak? b) I, IV, V c) II, III, V a) Vaccinate high-risk individuals d) II, IV, V b) Utilize ring-vaccination to contain the spread of the virus e) III, IVV c) Vaccinate the entire population of a state distant from the site of outbreak 8. Does infection with H5N1 necessarily result in a high probability of d) All of the above fatality? e) A and B only
Elena Igwe - Synthesizing AMPs 3. How do AMPs kill bacteria? a) Through enzymatic breakdown of the bacterial plasmid b) By disrupting the integrity of the bacterial cell m e m b r a n e c) By inhibiting binaryfissiona m o n g bacterial cells d) By inserting into a m e m b r a n e protein to induce cell lysis 4. Select the true statement.
a) Yes. Previous human cases have demonstrated a high case-fatality rate. b)Yes.H5N1 can result in 1 0 0 % fatality in poultry populations. c) No. T h e apparent high case-fatality rate m a y b e a n artifact of subclinical cases of H 5 N 1 not being detected. d)AandB. e) None of the above
Romy Cho - Avian Influenza Pandemic
a) AMPs are in the same class as defensins and interferons. b) A M P s are anionic, hydrophobic and globular c) Overuse of A M P s can lead to bacterial resistance, similar to antibiotics d) A M P s are easy to synthesize but the process is costly
9. Select the true statements regarding vaccinations.
a) Immunoglobulins are capable of destroying the WNV b) W M V symptoms include nausea, headache, and rashes c) The incubation period for W N V before an infected individual shows symptoms is around or 24 hours d) W N V has been spreading south with the help of birds migrating in the winter
10. T h e current drug, Tamiflu, used to treat the Avian Flu:
I. In hopes of producing a viable vaccine, reverse transcriptase might b e able to create a non-lethal copy of the viral g e n o m e . II. Vaccine research can b e conducted using a genetically modified seed strain of the H 5 N 1 virus. III. Vaccines can b e produced quickly u p o n higher d e m a n d . Joseph Catapano - West Nile Virus IV. T h e m a i n difficultly with producing a n Avian Flu vaccine is 5. The groups at greatest risk for developing fatal symptoms as a result of because its complex structure. W M V infection are: V. Vaccines for the virus will b e outdated quickly d u e to the rate at which the virus is able to confer resistance. a) Elderly and newborns b) Newborns and pregnant w o m e n a) I, III, IV c) Immunocomprimised and newborns b)l,ll,V d) Elderly and immunocomprimised c) I, II, III d)l, II 6. Select the false statement.
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a) Binds and dismantles the viral caspid b) Binds to the active neuraminidase site o n the targeted host cell c) Binds to the active neuraminidase site o n the surface of the virus d) Binds to all receptors o n the host cells to block the virus
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November 2005 Issue 7
htm Rosengard, A.M., Liu, Y„ Nie, Z. & Jimenez, R. (2002). Variola virus i m m u n e evasion design: Expression of a highly efficient inhibitor of h u m a n complement. Proceedings of the National Academ y of Sciences of Arita, I. (2005). Smallpox vaccine and its stockpile in 2005. The Lancetthe United States of America, 99, 8808-8813. Rosenthal, S.R., Merchlinsky, M., Kleppinger, C, & Goldenthal, K.L. (2001). Infectious Diseases, 5(10), 647-652. Developing n e w smallpox vaccines. Retrieved October 19, 2005 BBC News; U K edition (2005). W H O agrees to smallpox research from http://www.cdc.gov/ncidod/EID/vol7no6/rosenthal.htm [Electronic Version]. Retrieved October 19, 2005 from http://news. Tumpey TM., Basler, C.F., Aguilar, P.V., Zeng, H., Solorzano, A., Swayne, bbc.co.uk/1/hi/health/4568097.stm D.E. et al. (2005). Characterization of the reconstructed 1918 Spanish Centers for Disease Control and Prevention. (2004a). Smallpox disease overview. Retrieved October 19, 2005 from http://www.bt.cdc.gov/ influenza pandemic virus. Science, 310, 77-80. Weiss M.M., Weiss, P.D., Mathisen, G. & Guze, P. (2004). Rethinking agent/smallpox/overview/disease-facts.asp Centers for Disease Control and Prevention. (2004b). Smallpox vaccine Smallpox. Clinical Infectious Diseases, 39, 1668-1673. overview. Retrieved Oct 10, 2005 from http://www.bt.cdc.gov/ W H O (1980). The global eradication of smallpox: Final report of the agent/smallpox/vaccination/facts.asp global commission for the certification of smallpox eradication. In Centers for Disease Control and Prevention. (2004c). What C D C is doing History of International Public Health, No. 4, Geneva: World Health to protect the public from smallpox? Retrieved October 19, 2005 Organization. from http://www.bt.cdc.gov/agent/smallpox/prep/cdc-prep.asp Yang, H., Kim, S.-K., Kim, M., Reche, P.A., Morehead, T.J., D a m o n , I.K., Centers for Disease Control and Prevention. (2005). Smallpox. Retrieved Welsh, R.M. & Reinherz, E.L. (2005). Antiviral chemotherapy facilitates October 19, 2005 from http://www.bt.cdc.gov/agent/smallpox/ control of poxvirus infections through inhibition of cellular signal index.asp transduction. J. Clin. Invest, 115, 379-387. Constantin, CM., Martinelli, A.M., Bonney, E.A. & Strickland, O.L (2003). Smallpox: an update for nurses. Biological Research for Nursing, 4 Elena Igwe - Synthesizing Human Antimicrobial Peptides: Harmful (4), 282-294. or Helpful? Ellis, J. (1999). Germ welfare is next atom b o m b . In: The Toronto Star. Retrieved October 19, 2005 from http://pqasb.pqarchiver.com/ Bals, R. (2000). Epithelial antimicrobial peptides in host defence agai thestar/434280151 .html?did=434280151 &FMT=ABS&FMTS=FT&da infection. Resp Res., 1,141-150. te=May+14%2C+1999&author=John+Ellis&pub=The+Record&des Bals, R. & Wilson, J.M. (2003). Cathelicidins-a family of multifunctional c=Germ+warfare+is+next+atom+bomb. antimicrobial peptides. CMLS, 60, 711 -720. Enserink, M. (2005). W H A gives yellow light for variola studies. Science, Bel, G. & Gouyon, P.H. (2003). Arming the enemy: the evolution of 308,1235. resistance to self-proteins. Microbiology, 149,1367-1375. Fenner, R, Henderson, D. A. & Arita, I. (1988). Smallpox and its eradication. Ehrenstein, G. & Lecar, H. (1977). Electrically gated ionic channels in lipid Geneva: World Health Organization. bilayers. Q Rev Biophys, 10, 1 -34. FIRSTConsult. (2005). Bioterrorism - Smallpox. Retrieved October 19, Gordon, Y.J., Romanowski, E.G. & McDermott, A.M. (2005). A review of 2005 from http://www.firstconsult.com/home/framework/fs_main. antimicrobial peptides and their therapeutic potential as antihtm?id=01055785&type=ref&page=2&target=ahid_1980799 infective drugs. Current Eye Research, 30, 505-515. Flint, J.S., Enquist, L.W., Racaniello, V.R. & Skalka, A.M. (2004). Principles Guina, T., Yi, E.C., W a n g H., Hackett M. & Miller S.S. (2000). A Pho-P of Virology: molecular biology, pathogenesis, and control of animal regulated outer m e m b r a n e protease of Samonella enterica serovar viruses. (2nd ed.) Washington, DC: A S M Press. typhimurium promotes resistance to alpha-helical antimicrobial Friedman, H.M.& Isaacs, S.N. (2004). Smallpox. In B.D.Rose (Ed.), UpToDate peptides. Journal of Bacteriology, 182,4077-4086. (version 13.1), M A : Wellesley. Guthmiller, J.M., Vargas, K. G„ Srkantha R., Schomberg, LL, Weistroffer, Greenberg, R.N., Kennedy, J.S., Clanton, D.J., Plummer, E.A., Hague, P.I., McCray, P.B., Jr. & Tack, B.F. (2001). Susceptibilities of oral L, Cruz, J., Ennis, F.A., Blackwelder, W.C. & Hopkins, R.J. (2005). bacteria and yeast to mammalian cathelicidins. Antimicrob Agents Safety and immunogenicity of n e w cell-cultured smallpox vaccine Chemother, 45, 3216-3219. compared with calf-lymph derived vaccine: a blind, single-centre, Hancock, R.E.W. (1999). Host defence (cationic) peptides. W h a t is their randomised controlled trial. Lancet, 365,398-409. future clinical potential? Drugs, 57,469-473. Hammarlund, E., Lweis, M.W., Hansen S.G., Strelow, L.I., Nelson, J.A., Hultmark, D. (2003). Drosophilia immunity: paths and patterns. Curr Sexton, G.J., Hanifin, J.M. & Slifka, M.K. (2003). Duration of antiviral Opin Immunol., 15,2-9. immunity after smallpox vaccination. Nature Medicine, 9, 1131Miyakawa, Y, Ratnakar, P., Rao, A.G., Costello, M.L, Mathieu-Costello, O , 1137. Lehrer, R.I. & Catanzaro, A. (1996). In-vitro activity of the antimicrobial Hong, J. (2003). NBC29 health segments. Retrieved Nov 5, 2005 from peptides h u m a n and rabbit defensins and porcine leukocyte http://www.cecats.com/topics/smallpox.html. protegrin against Mycobacterium tuberculosis. Infect Immunol 64 Janeway, C.A., Travers, P., Walport, M. & Shlomchik, M J . (2005). 926-932. ' ' Immunobiology; the i m m u n e system in health and disease. (6th ed.) Lehree, R. I. & Ganz, T. (2002). Defensins of vertebrate animals. Curr Opin N e w York, NY: Garland Science Publishing. Immunol., 14,96-102. Massung, R.F., Esposito, J.J., Liu, L.-l., Qi, J., Utterback, T.R., Knight, J.C. et Paquette, D.W., Simpson, D.M., Friden, P., Braman, V. & Williams, R.C. al. (1993). Potential virulence determinants in terminal regions of (2002). Safety and clinical effects of topical histatin gels in h u m a n s variola smallpox virus genome. Nature, 366, 748-751. with experimental gingivitis. J Clin Periodontol, 29,1051-1058. Massung, R.F., Liu, L.-L, Qi, J., Knight, J.C, Yuran, T.E., Kerlavage, A.R. et al. Peschel, A. (2002). H o w do bacteria resist h u m a n antimicrobial peptides? (1994). Analysis of the complete g e n o m e of smallpox variola major Trends in Microbiology, 10,179-186. virus strain Bangladesh-1975. Virology, 201, 215-240. Pouny, Y., Rapaport, D„ Mor, A., Nicolas, P. & Shai, Y. (1992). Interaction of McFadden, G. (2005). Poxvirus tropism. Nature Reviews Microbiology antimicrobial dermaseptin and its fluorescently labeled analogues 3,201-213. with phospholipid membranes. Biochemistry, 31,12416-12423 Recommendations of the Advisory Committee on Immunization Powers, J.P.S. & Hancock, R.E.W. (2003).The relationship between peptide Practices. (2001). Vaccinia (smallpox) vaccine. Retrieved Oct 13,2005 structure and antibacterial activity. Peptides, 24,1681-1691 from http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5010a1. Reddy, K.V.R., Yedery, R.D. & Aranha, C (2004). Antimicrobial peptides:
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Niranjan Vijayakanthan, M o h a m m a d Zubairi, Hamilton Candundo, Brent Mollon, Gregory Agate - Revisiting Smallpox: Is a N o w ' D e a d ' Virus Still a Threat?
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