Emerging and Endemic Pathogens NATO Science for Peace and Security Series This Series presents the results of scientific meetings supported under the NATO Programme: Science for Peace and Security (SPS) The NATO SPS Programme supports meetings in the following Key Priority areas: (1) Defence Against Terrorism; (2) Countering other Threats to Security and (3) NATO, Partner and Mediterranean Dialogue Country Priorities The types of meeting supported are generally "Advanced Study Institutes" and "Advanced Research Workshops" The NATO SPS Series collects together the results of these meetings The meetings are coorganized by scientists from NATO countries and scientists from NATO's "Partner" or "Mediterranean Dialogue" countries The observations and recommendations made at the meetings, as well as the contents of the volumes in the Series, reflect those of participants and contributors only; they should not necessarily be regarded as reflecting NATO views or policy Advanced Study Institutes (ASI) are high-level tutorial courses intended to convey the latest developments in a subject to an advanced-level audience Advanced Research Workshops (ARW) are expert meetings where an intense but informal exchange of views at the frontiers of a subject aims at identifying directions for future action Following a transformation of the programme in 2006 the Series has been re-named and re-organised Recent volumes on topics not related to security, which result from meetings supported under the programme earlier, may be found in the NATO Science Series The Series is published by IOS Press, Amsterdam, and Springer, Dordrecht, in conjunction with the NATO Public Diplomacy Division Sub-Series A B C D E Chemistry and Biology Physics and Biophysics Environmental Security Information and Communication Security Human and Societal Dynamics http://www.nato.int/science http://www.springer.com http://www.iospress.nl Series A: Chemistry and Biology Springer Springer Springer IOS Press IOS Press Emerging and Endemic Pathogens Advances in Surveillance, Detection and Identification edited by Kevin P O’Connell U.S Army Edgewood Chemical Biological Center Aberdeen Proving Ground, MD, USA Evan W Skowronski U.S Army Edgewood Chemical Biological Center Aberdeen Proving Ground, MD, USA Alexander Sulakvelidze University of Florida Gainesville, FL, USA and Lela Bakanidze National Center for Disease Control Tbilisi, Republic of Georgia Published in cooperation with NATO Public Diplomacy Division Proceedings of the NATO Advanced Research Workshop on Advances in Surveillance, Detection and Identification of Emerging and Endemic Pathogens Tbilisi, Georgia 24–26 June 2008 Library of Congress Control Number: 2010934368 ISBN 978-90-481-9639-5 (PB) ISBN 978-90-481-9636-4 (HB) ISBN 978-90-481-9637-1 (e-book) Published by Springer, P.O Box 17, 3300 AA Dordrecht, The Netherlands www.springer.com Printed on acid-free paper All Rights Reserved © Springer Science + Business Media B.V 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Preface It is a truism among biologists that an organism’s phenotype is the product of both its genotype and its environment An organism’s genotype contains the total informational potential of the individual, while its environment shapes the expression of the genotype, influences the rate of mutation and occurrence of modifications, and ultimately determines the likelihood that the genotype (or fractions thereof) will survive into the next generation In the relationship between host and pathogen, therefore, each forms a part of the environment of the other, mutually influencing the biology of both partners on scales ranging from the life history of individuals to the fate of populations or entire species Molecular biologists working on problems in pathogenesis generally think of the host organism as the pathogen’s environment and perhaps occasionally consider the pathogen as part of the host’s environment However, because “environment” can be defined at many scales, so, too, can phenotypes: if a pathogen, as a species, is considered to exist in a host, as a species, then among its phenotypes is the nature of the pandemic disease it can cause within the host community The contributors to the proceedings of this NATO Advanced Research Workshop have treated the interplay of environment and genotype in the host–pathogen relationship and its relationship to the problem of emerging infectious disease at both the macroscopic and microscopic/ molecular levels along this continuum of scale (with some human history thrown in at times for good measure) Keynote Chapter The contribution from the meeting’s keynote speaker highlights the importance of understanding the underpinnings of pathogen phenotypes at both scales The example of Vibrio cholerae is considered macroscopically and genetically in an examination of the factors influencing the emergence and spread of new strains of human bacterial pathogens Citrus greening, caused by the bacterium Liberibacter asiaticus and vectored by the Asian citrus psyllid Diaphorina citri, is discussed to illustrate the effect of a vector species’ biology on disease emergence and spread An unfortunate lesson from these examples is that diseases that have already emerged and have spread rapidly may be difficult to control; however, any hope of disease control will be founded on an understanding of the genetic and molecular basis for pathogenesis and the environmental factors (including vectors) that contribute to the transmission of the microorganism Section I: Surveillance The next four chapters treat country-specific approaches, and their results, in one of the most fundamental tasks in combating emerging infectious disease: detecting and v vi PREFACE describing the incidence of disease in a geographic region By its very nature, this effort is labor-intensive in terms of fieldwork (both human and environmental) and in the subsequent laboratory analysis of samples In the Balkans, the Caucasus, and the Central Asian republics, like elsewhere in the developed and developing worlds, surveillance work ranges from the basic (trapping and culturing from members of a reservoir species) to the complex (use of sensitive laboratory molecular methods, such as PCR) and the application of the resulting data to forestalling and controlling the outbreak of endemic diseases Akimbayev et al., from Kazakhstan, and Gurbanov and Akhmedova, from Azerbaijan, provide a description of surveillance efforts in recent years that highlight the human and economic factors that influence disease transmission From the Republic of Georgia, Bakanidze et al provide a historical perspective that demonstrates the role that militaries have played in the development of public health methods and practices, born of necessity: throughout history, armies over time have lost more soldiers to disease than to violence Complementing the paper by Bakanidze and colleagues, the chapter by Zhgenti et al reports on the use of modern molecular biological techniques to differentiate closely related strains of pathogenic bacteria isolated from both environmental and clinical samples in Georgia and throughout the Caucasus Stikova describes a syndrome-based, nationwide effort deployed in the Republic of Macedonia to report priority communicable diseases that is complementary to the routine surveillance system that reports individual confirmed disease cases This system, called ALERT, aided in forecasting and detecting the start of the influenza season The goal of surveillance always has been actionable information that would allow public health workers to forestall the spread of disease “Classical” surveillance and epidemiologic reporting as described in these first four chapters, however, now also provides data that are being analyzed by advanced computational and geographic methods known collectively as Geographical Information Systems Blackburn rounds out Section I by describing new tools that enable the fusion of climatologic, geographic, and epidemiologic data with concepts in ecological niche theory to construct models that may predict the future incidence, prevalence, and transmission of Bacillus anthracis, but the methods are generalizable to other diseases Section II: Molecular Analysis and Tools At the scale of the bacterium and bacterial genome, the contributors to this section each provide an example of how cutting-edge molecular biological methods are being applied to answer key questions in the study of emerging infectious disease How did the pathogens we observe in the world come to their present state? Technical challenges abound in the analysis of biological specimens for evidence of ancient infections Aboudharam et al describe the development of dental pulp as a target material for isolation of bacterial DNA and the diagnosis of ancient bacteremias, including Yersinia pestis infections Key to their methodology is the development of single-use primer pairs for the detection and amplification of ancient target sequences in a method they term “suicide PCR.” What determines the severity of disease a pathogen may cause? Perry et al demonstrate the utility of comparative genomics in identifying a putative hemagglutinin gene (“Region E”) that is present in Brucella melitensis 16M and absent in Brucella PREFACE vii abortus The data suggest that “Region E” has a host-specific influence on virulence, and the authors speculate that expressing the hemagglutinin in certain Brucella strains may improve their performance as vaccines What genome-wide adaptations predispose a pathogen to cause severe disease? Rakin examines the contributions of both gain-of-function genetic changes (via lateral gene transfer) and negative selection (favoring what is termed pathoadaptive mutations) in the evolution of pathogenic bacteria His analysis points out the importance of single-nucleotide polymorphisms that, besides being markers for strain identification, can have significant effects on the functions of virulence and pathogenicity genes The implication of these results is apparent: in a selective environment or host, mutations can occur that lead to a sudden emergence of a virulent bacterial strain What tools are available for practical studies when containment is not available or practical, but safety must be maintained? Researchers have long used non-pathogenic surrogates, or “simulants” in place of pathogens and protein toxins for reasons of convenience, safety, reduction of expense, and speed of work Such simulants have included benign enteric bacterial species, bacteriophage (especially MS2), and proteins such as ovalbumin Ouellette et al review here information that suggest that baculoviruses, long used in organic agriculture and widely regarded as having no ill effects on humans, animals or plants, may serve as a new class of simulants for some viral pathogens How recent advances in sequencing affect the genetic analysis of pathogens? Molecular biologists are relying on the rapidly decreasing cost per base of DNA sequencing to support the continuing effort to detect and identify the genes (as is discussed by Perry et al.) or gene variants (as in Rakin) that influence bacterial pathogenicity and virulence Khan briefly reviews the procession from Sanger dideoxy sequencing (and the dye-coupled PCR-driven variant) to so-called next-generation sequencing (NGS) methods NGS methods have a much higher throughput than the Sanger methods but with generally smaller average read lengths Concurrent increases in computational power allow the rapid querying of databases for bacterial identification However, although faster computation also speeds contig formation from unique sequences, short read lengths can result in more contigs that require more effort to assemble into finished whole bacterial genomes Fortunately, complementary technologies such as whole genome optical restriction mapping are emerging that very rapidly provide the scaffolding data needed to match the increased rate at which NGS produces contigs Bacterial genome sequencing that 15–20 years ago required years of effort now takes weeks The rate at which sequencing technology is accelerating has been compared with Moore’s Law in computing power, except that the rate of improvement for sequencing has proven to be steeper than the drop in the cost of memory and clock cycles over time The “next” in NGS likely is ready to become dated in use as single molecule sequencing methods are being commercialized by at least two companies and faster methods still are certain to follow No sequencing technology currently is employed widely outside of laboratories or core facilities However, entrepreneurs are fervently seeking the right combinations of technology and business models that will put NGS (and beyond) into the hands of nonlaboratory end users (clinicians, epidemiologists, law enforcement, and first responders) The eventual goal is to provide a user with an encyclopedic understanding viii PREFACE of the DNA sequences present in a sample, breaking the barrier that currently separates sensitivity plus specificity from speed of analysis The possibility of a technology that will permit fast, accurate, complete data from genuinely unknown samples (unlike PCR) may at last be on the horizon Acknowledgements We gratefully acknowledge the assistance of the staff members of the Edgewood Chemical Biological Center, the Georgian National Center for Disease Control, and the University of Florida for their contributions to the success of this Advanced Research Workshop, whose speakers contributed to this volume We also thank the members of the staff of ARW Secretariate for their tireless help with the logistical details of the conference In particular, we thank Geoff Doyle of SAIC, for his outstanding organizational skills, and Rebecca Bryan for fellowship and her good humor as well as expert assistance with innumerable tasks during the conference in Tbilisi In the production of the conference proceedings, the NATO Science Series staff, in particular Ms Wil Bruins, provided invaluable advice and assistance Lastly, we thank Jean McHale for the many hours spent editing, formatting and compiling the papers included in this book Without her expert help the publishing of this book of conference proceedings would not have been possible ix ENVIRONMENTAL INFLUENCES ON THE RELATIVE STABILITY 139 suspensions of 60% n-propyl-alcohol, 70% isopropyl-alcohol, or 80% ethyl-alcohol A formulation consisting of 0.2% paracetic acid (PAA) and 80% ethanol (v/v) completely inactivated VACV after a 30 s exposure [119] In addition, Kramer et al [75] studied the virucidal effects from an alcohol based formulation including 55% ethanol (w/w) with 10% (w/w) propan-1-ol, 5.9% (w/w) propan-1.2-diol, 5.7% (w/w) butan-1.3-diol and 0.75% phosphoric acid In suspension tests, VACV inactivated after a 30 s exposure 5.1.8 Solvent/Detergent Treatment Treatments including combinations of solvents/detergents have been widely used in the healthcare industry to inactivate viruses within plasma or blood products Cocktails of VACV (10% v/v) and Tri-(n-butyl)-phosphate (TNBP)/Tween 80 in solutions of antihaemophilic factor incubated at 28°C for h reduced virus titer by log10 [90] The observed resistance of VACV to TNBP/Tween 80 treatment supported earlier findings [92] Comparatively, combinations of VACV (10% v/v) and TNBP/cholate in solutions of intravenous immunoglobulin completely inactivated virus >1 h [90] The healthcare industry also commonly includes treatments using caprylic acid salts in plasma products Suspensions of VACV (10% v/v) in Gamunex® intermediate solution containing 10% sodium caprylate resulted in complete virus inactivation within when incubated at 22°C in 20 mM caprylate [90] 5.1.9 Surface Disinfectants The virucidal activity of monopercitric acid (MPCA) was determined by suspension tests against both nonenveloped and enveloped viruses including VACV [120] The study showed that viruses were 99.9% inactivated by a 0.5% concentration within 30 s demonstrating MPCA as a suitable candidate for a disinfectant Sugimoto and Toyoshima [107] reported on the inactivation of VACV by Nα-Cocoyl-L-Arginine Ethyl Ester, DLPyroglutamic Acid Salt after a 30 exposure at room temperature At all tested concentrations (0.025%, 0.05%, 0.1%, and 0.25%), there was greater than 90% inactivation of VACV Ferrier et al [29] studied the virucidal effects of a non-corrosive commercial disinfectant whose composition combines a suite of viral inactivating agents including quaternary ammonium, aldehydes, alcohol and detergent The disinfectant Sanytex® was assessed in the presence of protein in both suspension and surface tests against VACV Suspension assays showed 1% concentrations of Sanytex effectively inactivating (log reduction >4) VACV in the presence of mg/mL protein In parallel, the authors tested the virucidal activity of sodium hypochlorite in suspension assay and reported 0.525% concentration of active chlorine inactivating VACV (log reduction >4) with 10 mg/mL protein present In surface tests, Sanytex was less effective requiring higher concentrations and longer exposure times for viral reduction The virus was effectively inactivated after 15 exposure by 10% detergent concentration and after 10 by a 30% concentration of detergent containing 10 mg/mL protein concentration Overall, the study demonstrated Sanytex as a highly effective disinfectant for use in laboratories and clinical facilities 140 G.D OUELLETTE, P.E BUCKLEY, AND K.P O’CONNELL In evaluating the virucidal efficacy of three surface disinfectants (quaternary ammonium, phenolic, and iodophor) on a simulated hard-environmental surface with a 10 exposure at 20°C, it was observed that the quaternary ammonium compound was the most effective at inactivating VACV (ca 4.5 Log10 reduction) [31] Both iodophor and phenolic, however, produced similar results with an inactivation of 3.9 Log10 and 3.7 Log10 respectively As illustrated above, various disinfectants are used for inactivation of pox viruses in laboratory, clinical and hospital facilities However, these are generally not suitable for the home environment, posing both health and environmental hazards Butcher and Ulaeto [15] assessed common household disinfectants on VACV and reported that a household, chloroxylenol-based disinfectant completely inactivated VACV at ambient room temperature 5.1.10 Miscellaneous With recent concerns over the use of smallpox virus as a biological weapon, broad poxinhibitory agents have been investigated ST-246 (4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6, 6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol-2(1H)-yl)-benzamide) was identified as a potential chemotherapeutic agent for use against smallpox virus [121] In cell culture, ST-246 was demonstrated a specific inhibitor of poxvirus replication in two separate VACV assays: (1), in a cytopathic effect assay, an EC50 for inhibition was recorded at 0.01 μM, and; (2), a lack of extracellular virus formation in a virus yield assay after exposure to μM ST-246 Similarly, plaque formation was completely inhibited in cowpox virus exposed to ST-246 In orally administered ST-246 assays, formation of VACV induced tail lesions was inhibited in mice infected with live VACV via tail vein The same study also identified ST-246 as a broad virucidal chemical against several orthopoxviruses In recent studies, 4(3H)-Quinazolinone derivatives have been reported to possess both antimicrobial and antiallergic properties [25] In a cell culture study, 6-Bromo-2phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone exhibited antiviral activity against VACV in E6SM cell culture at a concentration of 1.92 μg/mL 5.2 Baculovirus The effects of antiviral agents and disinfectants on baculoviruses have chiefly focused on their use in the laboratory for sterilizing insect diet preparations, or decontaminating work areas for the prevention of virus transmission during experiments [59, 115] Earlier studies demonstrated antiviral activity from formaldehyde incorporated into diets [59, 49, 115] Formalin (0.04%) reduced viral activity when added to suspensions of HzSNPV and TnNPV or incorporated into insect diets containing viruses [59] Comparably, formalin solution (10%) has also been used to surface-sterilize eggs to prevent baculovirus infections [87] Sodium hypochlorite at various concentrations has also been used effectively as an egg surface sterilant [44, 47, 115] Ignoffo and Dutky [47] reported treatments of 0.5%, and 0.05% sodium hypochlorite for 1–30 completely inactivated TnNPV The cations strontium, ferrous and ferric chloride are also inhibitory to virus activity of the Lymantria dispar Nuclear Polyhedrosis Virus (LdNPV) [96] ENVIRONMENTAL INFLUENCES ON THE RELATIVE STABILITY 141 Several reports have identified virucidal chemicals operating by denaturing the polyhedral protein matrix of viruses Sodium carbonate (0.005M Na2CO3) induced significant swelling of HzSNPV polyhedra after incubation [3]; based on these findings, it was concluded viral polyhedra would dissolve over time Consistent with these observations, the inclusion body protein of insects was reported to dissolve several minutes after being exposed to 0.01M Na2CO3 [59] Sodium carbonate solutions dissolved inclusion bodies from HzSNPV and deactivated 99.9% of the virus [97] Furthermore, magnesium (0.003 M MgSO4) reduced infectivity by five- to sixfold and a sevenfold decrease of infectivity was reported for β-mercaptoethanol (8 M; pH 7.6) Discussion and Conclusion The ability to draw reference correlations, based on repeatable studies, between baculovirus simulants and poxviruses is a requirement for the practical use of baculoviruses for testing decontamination regimes for pathogenic viruses Overall, the research focus on virion persistence for each group has had orthogonal goals Poxvirus research has focused entirely on virus elimination (save for the viability and efficacy of vaccines stored and transported inside vials) On the other hand, baculovirus researchers have embraced the goal, in some instances, of increasing the persistance of virions to extend their usefulness as natural insecticides in agriculture These divergent research foci, and the diversity of methods and conditions employed, have resulted in many datasets that are difficult to compare with one another However, some common themes can be gleaned from a broad examination of the data We discuss below six broad physical and biochemical commonalities shared between VACV and baculoviruses These similarities suggest that one or more baculoviruses may serve as effective simulants for poxviruses for purposes of studying virus decontamination and detection • • Effect of pH In general, pox and baculoviruses are viable under similar pH ranges Near pH 7, both VACV and baculoviruses remain stable, and from pH 4–8, these viruses show good short-term persistence [3, 48, 91] Both VACV and baculoviruses rapidly inactivate below pH or above pH 11 Significant reduction in virus activity was reported for HzSNPV at pH 1.2 [48] with 88% virus inactivation after 24 h exposure at pH [36] and immediate inactivation of VACV was observed at pH 2.5 [6] Strong alkaline pH (>10) conditions are documented to dissolve the inclusion body protein in baculoviruses and inactivate the viruses [68, 97] Pox viruses are reported less sensitive to extreme pH ranges due to a low lipid content [91]; however, like baculoviruses, exposure to alkaline ranges above pH 11, results in rapid virus inactivation [6] Effect of moisture or humidity Based on a paucity of research, the most constructive generalization is to state VACV and baculoviruses are susceptible to inactivation under similar conditions of high moisture and RH A greater loss of VACV viability was observed in aerosols at 80% RH when compared to lower RH conditions