BIOTECHNOLOGY

AND INFECTIOUS
DISEASES

BIOTECHNOLOGY
AND INFECTIOUS
DISEASES
Modern Strategies for Finding,
Evading, and Defeating Wicked
Pathogens

BROOKE A. JUDE

MOMENTUM PRESS, LLC, NEW YORK

Biotechnology and Infectious Diseases: Modern Strategies for Finding,

Evading, and Defeating Wicked Pathogens
Copyright Momentum Press, LLC, 2017.
All rights reserved. No part of this publication may be reproduced, stored
in a retrieval system, or transmitted in any form or by any means
electronic, mechanical, photocopy, recording, or any otherexcept for
brief quotations, not to exceed 400 words, without the prior permission
of the publisher.
First published by Momentum Press, LLC
222 East 46th Street, New York, NY 10017
www.momentumpress.net
ISBN-13: 978-1-60650-903-6 (print)
ISBN-13: 978-1-60650-904-3 (e-book)
Momentum Press Biotechnology Collection
Cover and interior design by Exeter Premedia Services Private Ltd.,
Chennai, India
10 9 8 7 6 5 4 3 2 1
Printed in the United States of America

This book is for Craig and Catherine, who both give me the space I need
to push myself to try new things, and the support to know I can do them.
In loving memory of Ron Taylor, who always taught his students to be the
best possible versions of ourselves. Thanks go to you, RKT.

Abstract
With human populations growing in number and moving to increased
global travel, pathogens capable of causing human disease become an
ever-present threat. These infectious diseases account for and contributed
to so-called global wicked problems. Historical evidence points to
centuries-old cases of ancient disease. Medicinal treatments and preventative strategies have also been documented and repeated through time. As
disease numbers increase, cases of infection rise, and dangerous pathogens evolve, the numbers and variety of these wicked problems increase.
Current biotechnological advances can be used to design more effective
methods of detection, treatment, and prevention of infectious diseases.
This text, Biotechnology and Infectious Diseases: Modern
Strategies
for Finding, Evading, and Defeating Wicked Pathogens will document a
core set of wicked infections agents, including Vibrio cholerae, influenza,
and HIV, as a method of describing ways of cutting edge diagnostic
and therapeutic tools function. This text begins with an introduction to
the pathogenic mechanisms displayed by some of the most dangerous
diseases. Descriptions of the pathogens themselves are followed up by
such topics as use of lateral flow assays for low-level detection, application of nanosized aptamers for therapeutic drug delivery, and design
of novel
vaccines
currently being developed for emerging infections
including Ebola and Zika. New technologies are being developed as fast
as new pathogens are evolving. The key to making breakthroughs taming
these wicked infectious diseases will be in finding commonalities between
organisms and exploiting biotechnological advances.

KEYWORDS
anthrax, aptamers, bacteria, Biotechnology, cholera, CRISPR, Ebola, gene
therapies, HIV, influenza, molecular diagnostics, outbreak, pathogen,
programmable nucleases, SARS or MERS, vaccine, virosomes, virus,
wicked problems, Zika

Contents
List of Figures

xi

List of Tables

xv

List of Boxes

xvii

Acknowledgments

xix

1 Introduction to Infectious Diseases

1.1Infectious Diseases as Wicked Problems
1.2Defining the Problem of Infectious Disease
1.3 Infectious Disease Structure
1.4 Infectious Disease Environment
1.5 Wicked Pathogens
1.6Conclusion

1
1
4
5
12
12
22

2 Defining Features of an Infectious Disease Outbreak

23
2.1Characterization of an Infectious Disease Outbreak
23
2.2Early Methods of Infectious Disease Detection and
Surveillance25
3Biotechnology for Detection, Monitoring,
andSurveillance
3.1Introduction
3.2 Culture-Based Detection
3.3Culture Independent Detection of Pathogens
3.4 Oligonucleotide Detection
3.5 Nanodetection Techniques
3.6 Cellular Sensor Detectors

33
33
34
39
41
45
50

4Biotechnological Advances for Treatment of

Infectious Diseases
4.1Introduction
4.2 Treatment of Symptoms

53
53
54

x Contents

4.3 Blockade of Pathogen Spread

56
4.4 Host Genome Editing
71
4.5 Conclusion75
5 Biotechnology and Vaccine Development
5.1Introduction to Immunological Response
5.2 Vaccine Introductions
5.3 Vaccine Types and Targets
5.4 Vaccine Formulation
5.5 Vaccine Adjuvants
5.6 Route of Vaccine Delivery
5.7 Future Vaccine Developments
5.8Conclusions

77
78
79
81
85
91
93
96
96

Notes

99

References

103

Index

129

List of Figures
Figure 1.1.Methods of the Gram stain. The Gram stain protocol is
utilized to determine the structure of the bacterial cell wall.
The protocol applies a series of dyes, mordants, and washes.
(A) Gram-Negative cell wall appears as pinkin color from
the safranin stain; (B) A Gram-Positive cell wall appears
purple in color, due to the crystal violet or Grams iodine
complex in the cell wall.
7
Figure 1.2.Nucleic acid structures of viral genomes. Viruses are
classified based on the chemical makeup and structure of
their genome. (A) Class I double stranded DNA (dsDNA),
(B) Class II single stranded DNA (ssDNA), (C) ClassIII
doublestranded RNA (dsRNA), (D) Class IV single
strandedRNA (ssRNA) (+) plus sense, (E) Class V single
ssRNA (-) minus sense, (F) Class VI ssRNA reverse transcribed, (G) Class VII dsDNA reverse transcribed.
11
Figure 1.3.Pathogen reservoir and route of infection. A sampling of
fivewicked pathogens that survive outside of the host
where it causes infection. (A) Vibrio cholerae persists in
the brackish water environment bound to zooplankton and
phytoplankton. (B) HIV is believed to have resulted from
a spillover event from SIV (Simian Immunodeficiency
Virus) infection of primates. (C) The reservoir for Ebola
virus is not yet defined, however it is believed to be one of
the manyviruses that can persist in bats. (D) The multiple
strainsof influenza have a variety of reservoir species,
including avian and swine. Some viral strains can infect
cells simultaneously,providing an environment to undergo
reassortment andgenetic shift. (E) Zika virus is a type
of flaviviridae that is transmitted through insect vectors.

xii List of Figures

The virus is carried in the blood and bodily fluids from an

infectedindividual, via the insect to another susceptible
host. 
13
Figure 2.1.Kochs postulates. Kochs postulates are the standard
methodfor determining if a virus or bacteria is the
causativeagent for an infection in an organism. In this
method, four steps are: (A) Observe the suspected illness
ordisease in a patient; (B) Culture the microorganism using
appropriate media and conditions; (C) Infect a healthy host
with the microorganism, resulting in illness and symptoms
that replicate that of the original host; and (D) Reisolate
the pathogen from this subsequently infected host, and
demonstrate it is identical to that originally cultured.
27
Figure 3.1.Summary of pathogen detection methods. (A) Visual
diagnostics, including microscopy are utilized to characterize bacteria and viruses. (B) Culture-based detection
utilizes growth in media, acellular, as well as growthon
susceptible cells. (C) Nucleic acid-based detectionmethods
include PCRto detect DNA, reverse transcriptase PCR and
quantitative real time PCR to detect RNA. (D) Antigen-
baseddetection methods that examine proteins include
Western blotting, ELISA, and lateral flow devices.
35
Figure 3.2.Mechanism of action for lateral flow assays. The general
waylateral flow test function beings with (A) antibodies
embedded into an absorbent matrix. (B) The wicking pad
is placed into a sample with suspected pathogen, where
it migrates toward the antibodies. If the sample contains
the pathogen, it will have the opportunity to bind to the
antibodies. (C) The device is moved to a second reagent
thathas labeled antibody specific to the suspected pathogen.
(D) Following the binding of the second antibody tothe
suspected pathogen, a colorimetric band indicates their
presence.
46
Figure 3.3.Nanomotion sensors. In this method, (A) an AFM cantileveris inoculated with a suspected bacterial culture.
(B)If thebacteria is present and bound to the cantilever,
it is detected through motion, while (C) the loss or lack of
motioncan be detected.
49
Figure 4.1.Mechanism for disrupting infection. There are a number
of stages of the infectious cycle that are vulnerable for

List of Figures xiii

therapeutic action, including (A) entry of the cell by the

pathogen, (B) the replication of the genome, (C) translation
of the RNAinto pathogen proteins, and (D) release of the
pathogen.
57
Figure 4.2.Mechanism for aptamer selection. To discover highly
specific nucleic acid aptamers, (A) a nucleic acid library of
random sequences is prepared. (B) The nucleic acids fold
into secondary structures. (C) The library is probed with the
target protein, allowing those with a high affinity to bind to
the protein, and (D) the unbound proteins can be washed
away. (E) The aptamer specific nucleic acid can then be
amplified for further use.
60
Figure 4.3.Mechanism for transgenic therapeutic and vaccine protein
production. Depicted is two different uses for the plant
Nicotimiana benthimiana for therapeutic purposes. (A) A
transgenic virus can infect the plant, and produce antibodies
in response to the expressed viral proteins. In the example
given, this strategy was utilized to produce the Zmapp,
for Ebola therapeutic. (B) Influenza proteins can be introduced to the plant via Agrobacterium tumifaciens, a natural
infectious agent for plants. These proteins are then able to
bepurified and used for vaccine production.
62
Figure 4.4.Method for RNAi viral silencing. In (A), the normal
methodfor an RNA virus infection includes cell entry,
genome replication, and translation of RNA into virion
proteins. The genomes and virions are assembled and
released, completing the infectious cycle. In (B), when an
RNAi expressing virus is coinfected with an infectious
virus, the RNAi sequence is complementary to the infectiouspathogen, and when the RNA dimerizes, the RNAi
pathway triggered, and the final virion proteins and mature
virus is unable to be produced.
68
Figure 5.1.Vaccine design strategies. Vaccines are based on methods
for providing antigenic determinants to the host to illicit
an immune response. (A) The wild type virus provides the
robust and typical immune response that could provide
protection. (B) Whole viral vaccine constructs include
whole inactivated virtues, incapable of replication due
to heat or chemical killing, or live virus capable of replication, but unable to cause the pathogenic effects. (C)In

xiv List of Figures

this third category, virus subunits are provided outside of

the context of a complete viral structure, including viral
proteins, nucleicacid, or viral-like peptides. (D)Finally,
viral antigenscan be manipulated to be presented in
a noncanonical way to illicit an authentic and robust
immunogenicresponse,such as recombinant viral subunits,
immunogenic antigens expressed on a nonpathogenic virus,
or bacterial cell e xpressing antigenic proteins.
82
Figure 5.2.Mechanism for vaccine antigen production in plants. This
strategy was developed to optimize and increase production of viral antigens for vaccines. In the strategy described
earlier, tobacco plants are infected with recombinant viral
vectors. This plant tissue can be harvested to purify large
amounts of antigen. This recombinant antigen was determined to produce a protective response in a murine model
system.90

List of Tables
Table 1.1. Describing infectious diseases as a wicked problem

Table 1.2.Hurdles encountered in infectious outbreak, and evolution

ofsolutions

Table 1.3. Features of infectious diseases

15

Table 3.1. Summary of pathogen detection methods

51

Table 5.1. Immunoglobulin classes

94

List of Boxes
Box 1.1. The Mechanics of the Gram Stain

Box 1.2. Describing Pathogen Genomes

Box 2.1. How Super-Spreaders Can Impact an Outbreak

24

Box 2.2. Kochs PostulatesOld and New Methods

26

Box 2.3. Resurrection of Infections Yields Modern Insight

28

Box 2.4. SARS, MERSPredicting Future Coronaviral Outbreaks? 30

Box 2.5. The Eradication (and Return) of Polio?

30

Box 4.1. Aptamers and Their Use in SELEX Technology

58

Box 4.2. Griffithsin-Algae Versus Virus Versus Human

61

Box 4.3. Flemings Lucky Find

66

Box 5.1. The Cold Chain Reliance: Enemy to Rural Vaccine Delivery 83

Acknowledgments
The careful editing of this manuscript is due, in full, to my editor and
friend Stephanie Stockwell, as well as the careful eyes and suggestions of
Craig Jude, Mindy Nye, and Matthew Deady, who all added immensely
to this work. Figure construction and completion is also credited to Craig
Jude. My family deserves recognition for being so understanding as this
large scale project took up much of my time. Finally, I want to thank Mike
Tibbetts and Phil Pardi for their general encouragement, which was much
appreciated throughout this process.

CHAPTER 1

Introduction to Infectious
Diseases
1.1INFECTIOUS DISEASES AS WICKED
PROBLEMS
There are wicked problems facing the field of infectious disease. The
term wicked problems was defined by Rittel and Webber in 1973 when
comparing and contrasting the issues encountered in social policy and
ethics (Rittel and Webber 1973). These are problems that multiple groups
identify as needing solutions, however there are no linear solutions that
meet all needs. All solutions have potential ramifications on the others
and often come with benefits for some of the various groups overlapping
in goals, while also in conflict with others. Wicked problems are defined
by 10 features1 that describe why the solutions to these problems are so
difficult to obtain.
This term has been previously used in literature to discuss sociological problems, ethical dilemmas, and environmental issues such as
climate change (Rittel and Webber 1973; Lazarus 2009). Recently the
global problem of infectious disease has been examined through this lens,
and the Rittel and Webber definitions can be applied to the aspects of
solving problems in infectious disease research (Table 1.1). Specifically,
studies have examined if hypervirulent viruses should be constructed for
research purposes, putting the public at risk of infection (Koblentz 2014),
or if implementation of human immunodeficiency virus (HIV) prevention strategies cross ethical boundaries that undermine their effectiveness
(Lavery 2016). For example, in case of eradication of a particular infectious disease, there are complex and overlapping interactions between
local policy maker and environmental advocate, between advocate and
microbial researcher, between researcher and drug company, between drug

5.Attempted solutions to wicked problems

will leave traces on landscape, and impact
future attempts at solutions
6.There is no way to know if all possible
solutions have been considered

3.Solutions are not correct or incorrect, but a

gradation of good to bad
4.There is not clear and immediate way to
test if the solution is correct

2.No clear finishing point, no idea when

problem is solved

Tenets defining wicked problems

1. No definitive formulation

How work on infectious diseases conform to this definition

Organisms that cause infectious diseases comprise many groups, sizes, shapes,
structures, methods of virulence, methods of survival
Infectious disease outbreaks can be managed, and numbers of infections reduced,
but reemergence remains a possibility, as does evolution of the pathogen to
evade solutions
Strategies, such as quarantines or vaccination designs, have clear strengths in
addition to weakness in their execution and effectiveness
Infectious disease work often impacts communities for years after the infection is
presumed defeated; as an example, downstream effects in infrastructure due to
redirection of funds, or potential environmental impacts of therapeutic efforts
Efforts that reduce pathogen load in the environment or in a host can also cause
collateral damage; this damage can disrupt, for example, ratios of community
members within a population, or alter the normal microbial balance in a host
Time is often of the essence when treating or preventing an infectious outbreak,
and there are few ways to pilot potential solutions to help predict downstream
effects

Table 1.1. Describing infectious diseases as a wicked problem

2 BIOTECHNOLOGY AND INFECTIOUS DISEASES

 9.Wicked problems can be described in a

variety of ways, and the solutions will
change according to these descriptions
10.Those who work on wicked problems
become implicit in the consequences the
solutions may cause

8.Every wicked problem can be a symptom

of another wicked problem

7.Each wicked problem is unique from

every other

Due to the complexity of communities, both external or landscape and host or

microbial, potential solutions depend on each specific set of details, and may
differ between communities, and individuals within the communities
Outbreaks of infectious diseases are often linked to other infections, as well as
other large scale environmental or sociological problems (e.g., Vibrio cholerae
infections due to hurricane damage, rising waters, climate change)
Infectious diseases impact communities in a multitude of ways, depending on if
the individuals charged with solving the wicked problem is the family, community, health care establishment, government, and so on
Workers charged with developing solutions to infectious diseases are also
responsible to consider the overall health of the patient, the cost of the care, the
feasibility of the therapeutic, and the impact on other facets of society

Introduction to Infectious Diseases3

4 BIOTECHNOLOGY AND INFECTIOUS DISEASES

company and p hysician, between physician and patient, and between host
cells and evolving virus. All of these factions participate in the landscape
of wicked problems. All community members working on these problems
have vested interests in finding solutions, however most solutions are not
universally acceptable.
Although like all wicked problems, those problems brought on by
infectious disease prove difficult to solve, there is real hope that they can
be managed or tamed. This taming has been executed historically through
careful examination of the infectious methods of infectious disease,
and develops strategies for detection, treatment, and prevention. With
the advent of biotechnological advances, these solution strategies have
become more effective, and will hopefully evolve as quickly as the pathogens they are fighting.

1.2DEFINING THE PROBLEM OF INFECTIOUS

DISEASE
Our global community allows for individuals to live in world where far
afield populations routinely interact, and share infectious diseases. Individual infections cause many possible symptoms, as well as an equally wide
range of severity, dependent on both the host (organism being infected,
such as an animal or plant) and the agent (the cause of the infection).
Infections are typically caused by microbes, organisms that are detectably only by microscopy, specifically, bacteria2 and viruses.3 Microbes
are transmitted from host to host by multiple means, including contamination of food and water sources, via bodily fluids, and through the air. As
infectious agents, and more specifically, individuals harboring infectious
agents, make their way to travel hubscrossing oceans on airliners, and
through large global venues and gatheringstheir potential of reaching a
large number of potential hosts increases. Luckily, as the reach of infections has grown, so too have the number of technologies for monitoring,
treating, and preventing life-threatening pandemics.4
Society has gone through a number of transitions in ways of dealing with pathogenic illness (Table 1.2). Over time, the way an infectious
outbreak5 is identified and managed has changed. Defining an outbreak,
assessing its extent, isolating the causative agent, and determining
methods for treatment and prevention have all undergone major transitions through biotechnology improvement (Table 1.2). For example,
simple observation of symptoms as a means of diagnosis has given way to
culture-based methods of detection, now an essential tool for identification

Introduction to Infectious Diseases5

Table 1.2. Hurdles encountered in infectious outbreak, and evolution of

solutions
Infectious disease
problem

Traditional solution

Biotechnology-based
solution

Recognition of an
outbreak

Observation by imme- Global observation

diate community
possible

Assessing outbreak
extent

Word of mouth

Global health
organization
communication

Identifying infectious
agent

Visualization using
culture based
methods; Kochs
postulates

Detection of nucleic
acid, protein, cellular
signatures of potential pathogen

Treatment and
methods

Treatment of
symptoms

Development and
use of specific and
designed antimicrobial agents

Halting outbreak
spread

Quarantine; improved Development and

hygiene
design of vaccines
and antimicrobials

of infectious agents. Likewise, treatment of individuals suffering from a

microbial infection was often mediated only palliative measures. These
methods are giving way to targeted therapeutics to disarm harmful pathogens. A preferred strategy to study human infections uses model systems,
organisms in research that are susceptible to infection by the pathogen.
Common model systems include animal models (e.g., mice, zebrafish,
fruit flies) and cultured cells. These models allow for testing modes of
pathogen transmission, researching methods for disruption of the infectious cycle, and development of vaccine strategies. All of these model
systems based experiments are made possible through biotechnological
advances.

1.3INFECTIOUS DISEASE STRUCTURE

Key to all methods of pathogen detection, treatment, and prevention is
understanding of the basic principles of how the microorganism is structured, and how it can effectively infect and cause disease in a host. Many

6 BIOTECHNOLOGY AND INFECTIOUS DISEASES

microorganisms persist in symbiotic6 relationships with their host. In

host or microbial relationships, there are bacterial communities termed
as commensals. Commensals are capable of, and inhabit host tissues and
surfaces, providing benefits to the host. These microbial communities are
referred to as microbiomes, and often provide key nutrients and ward off
harmful pathogen invaders. Pathogens in the same environment would be
capable of causing detrimental outcomes to the host, such as cell death
(Hooper and Gordon 2001). The physical and structural characteristics
between commensals and pathogens are often not distinct, and are more
related to the intricate relationship between the host and the collection of
microbial inhabitants present.
Pathogens differ from nonpathogenic and normal bacterial and viral
flora due to their ability to cause disease; the result of cellular and tissue
damage and perhaps death in a susceptible organism. For the cycle of infection to occur, the pathogenic agent must find a permissive host or cells,
replicate, and have the capacity for replication and spread. In the process
of infecting cells, tissue damage and cell death cause physical symptoms.
Infectious diseases are primarily caused by either bacterial or viral
microorganisms. Bacteria make up one arm of the rRNA-based phylogenetic tree of life.7 Grouped with the closely related microorganisms,
archaea, in the three-domain system, bacteria comprise the group termed
prokaryotes. Prokaryotes are single-celled organisms and have a number of distinctive characteristics that separate them from multicellular
eukaryotic organisms. Bacteria, specifically, are defined by their lack of
a nucleus, where eukaryotic nucleic acid is typically sequestered and lack
other membrane bound organelles. There are two major classifications
of bacteria, termed Gram-Negative and Gram-Positive (Box 1.1). These
distinctions are defined by different architecture in their cell membrane
structures and provide a gold standard diagnostic tool to begin to identify
a potential pathogen.
Box 1.1. The Mechanics of the Gram Stain
The Gram Stain technique was published in the late 1800s, and named
after H.C. Gram. Gram was a pioneering scientist who developed the
technique, which is still today a routine and essential first step in the
identification and diagnosis of cultured bacterial pathogens worldwide. Gram stains consist of a series of dyes (crystal violet, safranin),
mordants8 (Grams iodine), and washes (ethanol, water). The procedure allows for the differentiation of the two classes of bacterial cells
based on cell wall structure, as the membrane structures typical of

Introduction to Infectious Diseases7

Gram-Positive and Gram-Negative bacteria differentially retain dyes

during the procedure (depicted in Figure 1.1). In the standard protocol,
bacterial cells are applied and dried on a glass microscope slide. The
bacterial cells are heat fixed to the slide, and purple crystal violet stain
is applied. This staining step is followed with a water wash, and the
A
1. Crystal violet 2. Wash H2O
Purple-stain cell wall

3. Grams Iodine

4. Alcohol

5. Wash H2O

Cytoplasm

Gram-negative
1. Crystal violet 2. Wash H2O
Purple-stain cell wall

3. Grams Iodine

4. Alcohol

6. Safranin
Pink Counterstain

5. Wash H2O

6. Safranin
Pink Counterstain

Cytoplasm

Gram-positive

Peptide cross links

Membrane
Cell wall
(NAG-NAM)

Figure 1.1. Methods of the Gram stain. The Gram stain protocol is
utilized to determine the structure of the bacterial cell wall. The protocol
applies a series of dyes, mordants, and washes. (A) Gram-Negative
cell wall appears as pink in color from the safranin stain; (B) A Gram-
Positive cell wall appears purple in color, due to the c rystal violet or
Grams iodine complex in the cell wall.

(Continued)

8 BIOTECHNOLOGY AND INFECTIOUS DISEASES

Grams iodine mordant is added to trap the crystal violet that could
access the cell wall. A second water wash is followed up by an alcohol-
decolorizing step to remove only loosely associated violet dye. The
final step applies a pink safranin stain to serve as a counter stain for any
cells fully decolorized by the alcohol step. Gram-Positive cells, with a
thick peptidoglycan layer on the outermost surface, are heavily stained
with trapped crystal violet (Weissenbck et al. 2010), looking purple in
color under microscopic examination. Gram-Negative cells are characterized by having two concentric phospholipid bilayer membranes,
with a much thinner peptidoglycan structure situated between these
membranes. On the outer leaflet of the outer membrane decoration,
lipopolysaccharide (LPS) coats the surface. These Gram-
Negative
cells are not stained purple through this technique, but rather take up
the safranin counterstain and appear pink in color. This differential
staining allows for visualization of the shape of the cell (e.g., spherical
or coccoid, rod or bacillus), size of the cell, and the configuration (e.g.,
grouping in characteristic clusters). While this information is key for
the identification of microbes, Gram status (and cell shape, size, and
configuration) is not indicative of pathogenicity.

Bacterial pathogens have a number of genotypic (relating to the

encoded instructional information) and phenotypic (related to the actual
expression of genes) features that allow them to successfully infect an
organism. The genomes of bacterial cells are encoded on circular, double-
stranded DNA moieties (Box 1.2). Most bacteria have one chromosome;
however, a small number, including some pathogens, have two chromosomes. Genes on the chromosomes are often arranged in operonsthat
is, genes encoding products related to the same task are situated next to
each other and are produced sequentially. Bacteria also can encode and
carry information on smaller circular sequences of double-stranded DNA,
termed plasmids. Plasmids often carry genes that encode toxins, antibiotic
resistance mechanisms, or other structures that can enhance virulence.
Plasmids, unlike chromosomes, are not essential for cellular life, and cells
can survive if plasmids are lost. Some bacterial cells, especially those that
are aquatic, can move through their environments by producing flagellar
structures, that are powered by motors (H+ or Na+ driven) that rotate, rotating the attached flagellum. Like multicellular organisms, bacteria have
developed sensory systems to recognize and respond to their environment. The most critical observation that bacterial cells need to make is to
recognize the population makeup of surrounding microbial communities.

Introduction to Infectious Diseases9

Using networks of pathways known as quorum sensing, b acteria produce,

sense, and respond to small molecules as a method of determining the surrounding microbial community. In this manner, the single bacterial cells
within the community can behave as a group, allowing for collaboration
for success in high-energy activities; biofilm9 production, motility, and
toxin production. These uniquely bacterial characteristics provide potential targets for diagnostics and therapeutics.
Viruses, the other (sub)microscopic class of infectious and wicked
diseases are more difficult to neatly divide based on structural and genetic
characteristics. Unlike bacterial cells, and all other life forms, viruses are
not bound by encoding their genetic material on double-stranded DNA
chromosomes. All viruses have nucleic acid based genome(s) (Figure 1.2),
protected by a proteinaceous capsid coat structure. To minimize the size
of the genome, many of the viral structural components are geometrically
based. Even the simplest viral capsid structure becomes elaborate by taking a single protein capsid subunit and constructing a complex repeating
proteinaceous shell.
Viral life cycle requires entry into host cells for basic molecular tasks,
including replication of genome, transcription and translation, and production of progeny viruses. Many viruses not only have a proteinaceous
capsid, but may also have a lipid membrane-based envelope, derived
from the host cell membrane structure. Viral proteins on the capsid and
envelope provide the ligand necessary for host cell recognition, docking, and entry (Smith et al. 2004). The overall classification, structural,
and pathogenic mechanisms of viruses are much more varied and wide
ranging than bacteria.

Box 1.2. Describing Pathogen Genomes

The genomes of pathogens provide the instructions needed to allow
for faithful replication of the organism. Typical structure and location
for eukaryotic genomes are double-stranded linear chromosomes comprised of deoxyribonucleic acid (DNA) contained within a membrane
bound nucleus. In contrast, pathogens of bacterial and viral origins
have related, but much more varied genomic structures. Along with
shape (circular vs. linear), chromosomes differ in material, DNA or
ribonucleic acid (RNA). The orientation characteristic refers to the
relationship of the nucleic acid strands to their ability to encode material to be transcribed (instructions for building RNA) or translated
(Continued)

10 BIOTECHNOLOGY AND INFECTIOUS DISEASES

(instructions for building protein). Possible orientations include double stranded (ds) or single stranded (ss). In the case of RNA-based
genomes, orientations include also plus sense (+) or minus sense (-).
Plus sense is capable of direct translation by the ribosome, the cellular
machine charged with translating RNA into protein, while minus sense
requires first to be transcribed by a virally encoded RNA dependent
RNA polymerase. Typical configurations are as follows.
Bacterial genomes
Chromosome10
Plasmid
Viral Genomes

Material
DNA
DNA
Material
DNA
RNA
RNA
RNA

Shape
Circular
Circular
Shape
Linear
Linear
Linear
Linear

Orientation
Double stranded
Double stranded
Orientation
Double stranded
Double stranded
(+) sense
(-) sense

Whether viruses are alive or not is often debated. The case for viruses
being nonliving entities is based primarily on the fact that viruses are
unable to replicate without the assistance of a permissive host cell. Most
viruses are much smaller than bacterial cellsranging between 0.02 and
0.3 Mand have correspondingly small genomes. Viruses can get by
with limited genetic material due to their heavy dependence on host cells
for most vital functions, including all steps of protein production. Viruses
are academically organized in a schema called the Baltimore Classification, which distinguishes the viruses into seven groups, based on the
chemical structure of the genome (e.g., DNA vs. RNA, single stranded vs.
double stranded, plus sense vs. minus sense) (Figure 1.2).
Viral genomes house genes that encode the structural proteins (e.g.,
capsid) as well as any genes necessary to convert the genomic material into
a format that is readily used by the native host cell (e.g., encoding unique
viral enzymes like reverse transcriptase, integrase, RNA polymerase).
Capsid structures are often economical in geometric design, allowing for
a minimal (or even single) protein structure to assemble in a repeated fashion to create a large enough shell to contain essential genetic material and
proteins. Viruses typically also have surface-exposed proteins that provide
the ligand for specific receptor mediated tethering to cells they are capable
of infecting. These ligands can be incorporated into the capsid structure
(e.g., adenoviruses) or can be part of the plasma membrane-based envelope that surrounds some viruses (e.g., influenza).

Introduction to Infectious Diseases11

Viral Class/Nucleic Acid

Class I
dsDNA

H H
B

Class II
ssDNA
H

Class III
dsRNA
OH OH
Class IV
ssRNA (+)

D
OH
E
OH
F
OH

Class V
ssRNA ()

Class VI
ssRNA (RT)

H H

Class VII
dsDNA (RT)
H H

OH

Figure 1.2. Nucleic acid structures of viral genomes. Viruses are classified
based on the chemical makeup and structure of their genome. (A) Class I double
stranded DNA (dsDNA), (B) Class II single stranded DNA (ssDNA), (C) Class
III double stranded RNA (dsRNA), (D) Class IV single stranded RNA (ssRNA)
(+) plus sense, (E) Class V single ssRNA (-) minus sense, (F) Class VI ssRNA
reverse transcribed, (G) Class VII dsDNA reverse transcribed.

The life cycle of a virus includes successful interaction between

the virus and host cell, delivery of nucleic acid and proteins to the host
cell cytoplasm, and halting of normal cellular replication, transcription,
and translation in favor of viral processes. Following the production of
genome copies and required viral proteins, viruses are assembled and
released from the host cell, completing the infectious cycle.

12 BIOTECHNOLOGY AND INFECTIOUS DISEASES

1.4INFECTIOUS DISEASE ENVIRONMENT

Within environments, the presence of pathogens is dependent on the
needs of the pathogenic organism, as well as the permissivity (ability to
survive, or move to and replicate in a particular environment) of the environmental niche. There are four major categories when considering these
pathogens or niches interactions: ability of the pathogen to (1) persist in
the environment, (2) transit via arthropod carriers, (3) persist within a
living reservoir and (4) actively disseminate from an infected host. Most
pathogens that persist in an environment (without the need of a host cell)
are bacterial in nature, as viruses require a host cell for propagation.
Pathogens that are moved from susceptible host to another susceptible
host via an arthropod carrier are a form of metazoonosis. In this case the
carrier harbors pathogen with no overt symptoms of disease, and zoonosis is defined as being able transmit pathogen from vertebrate animal to
human (e.g., zoonotic mosquito-borne flaviviruses, Zika, Dengue, West
Nile, Chickungunya (Weissenbck et al. 2010)) (Figure 1.3). Adistinct
cohort to the first two are pathogens that are in a living reservoir (e.g.,
ducks and H5N1 (Kim et al. 2009), bats and severe acute respiratory syndrome (SARS), Ebola (Field and Field 2009; Jayme et al. 2015)). Finally,
in terms of what humans are most likely to come in contact with are
pathogens actively replicating and disseminating from one human host
to new susceptible hosts (e.g., sexual transmission of HIV, aerosolized
transmission of SARS, inhalation anthrax infection). The overall number
of pathogens capable of causing infections worldwide is large. The metrics of contagion, morbidity and mortality figures11 for many pathogens
are concerning in terms of ecological management, in terms of wildlife
and arthropod population numbers, and prevention of future outbreaks.
By examining current pathogenic outbreaks of representative pathogens,
discussion of novel techniques for surveillance, and planning for adaption of current treatment strategies for newly evolved molecular motifs, a
more connected and mobile society can also be nimbler when it comes to
fighting infectious disease.

1.5 WICKED PATHOGENS

Identifying and quantifying the cohort of pathogens capable of infectious
disease present worldwide is at once a task that is insurmountable, and
yet one that is attempted by researchers and clinicians on a daily basis.
These pathogens, which can fall under the definition of wicked problems
to solve, take the form of both bacteria and viruses, and reside in all four

Introduction to Infectious Diseases13

A
Zika
Vibrio cholerae

Ingestion

Mosquito
Bodily fluids

Aerosols

Bodily fluids

Influenza

Avian

HIV I
HIV II

Constant
recombination

Zoonosis
Care of patients
Bodily fluids

Swine

SIV

Ebola

Circular DNA

Bats

Single Strand
RNA

Figure 1.3. Pathogen reservoir and route of infection. A sampling of five wicked
pathogens that survive outside of the host where it causes infection. (A) Vibrio
cholerae persists in the brackish water environment bound to zooplankton and
phytoplankton. (B) HIV is believed to have resulted from a spillover event from
SIV (Simian Immunodeficiency Virus) infection of primates. (C) The reservoir
for Ebola virus is not yet defined, however it is believed to be one of the many
viruses that can persist in bats. (D) The multiple strains of influenza have a
variety of reservoir species, including avian and swine. Some viral strains can
infect cells simultaneously, providing an environment to undergo reassortment
and genetic shift. (E) Zika virus is a type of flaviviridae that is transmitted
through insect vectors. The virus is carried in the blood and bodily fluids from an
infected individual, via the insect to another susceptible host.

pathogen niches described in the previous section (Section 1.4). To protect

the public from health threats, most complete knowledge of pathogens
must be gathered. This includes information of microbial structure and

14 BIOTECHNOLOGY AND INFECTIOUS DISEASES

identity, evidence of microbial, and predicting of pathogen movement are

all utilized to examine the presence of microbes with pathogenic potential (Tapper 2006). The number of distinct pathogenic threats worldwide
are too numerous to detail each at length, however specific examples of
wicked pathogens are excellent representatives of the current outbreak,
and may serve as excellent case studies and predictions of what may be
problematic in the future.
1.5.1 INFECTIOUS OR PATHOGENIC BACTERIA
Pathogenic bacterial species have the potential to spread and cause disease
worldwide. One major advantage that bacterial species have as a wicked
problem is their ability to survive in environmental reservoirs, eliminating the possibility of compete eradication. Bacterial species readily can
exchange genetic information, using conjugation, transduction, or transformation.12 These evolutionary shifts provide opportunity for developing
strains that can evade antibiotic treatment, and ones that become more
virulent in populations. Moreover, bacterial cells that are environmental
and nonpathogenic can convert to a pathogenic, wicked infectious disease,
transformed over time. The following examples provide descriptions of
two major wicked bacterial strains, one aquatic dwelling, another found
in soil. Both examples have been major modern scourges, and have the
potential to remain problematic, requiring constant vigilance and the aid
of biotechnological advances (Table 1.3).
1.5.1A VIBRIO CHOLERAE
The bacterial species Vibrio cholerae is a motile curved bacillus, having
a Gram-negative cell wall structure. Its 4 Mb of genetic content encoded
on two double-stranded DNA circular chromosomes (Table 1.3). This
genome encodes for over 3,500 genes necessary for survival and virulence. The Vibrio cholerae species is defined by the features of the outer
membrane. The major feature of Gram-negative bacteria is the LPS that
decorate the surface of the bacterial cells, as well as across the surface of
the flagella. Each sugar structure configuration of the LPS is encoded by
the individual genome, and is referred to as a serogroup. To date, over 200
different serogroups of Vibrio cholerae have been identified to be living
and persisting in the aquatic environment (Hirsch et al. 1995). However,
only two serogroups (O1 and O139) have been associated with widespread

Virus: Group V

Virus: Group VI 9.75 kb

Virus: Group IV 2732 kb

Virus: Group V

Virus: Group IV 10 kb

Influenza A

HIV

SARS and
MERS

Ebola

Zika

1819 kb

() sense linear
ssRNA
(+) ssRNA

(+) ssRNA

2 (+) sense linear

ssRNA

circular, 2 plasmids
(pXO1189 kb and
pXO296 kb)
Total13.5 kb 8 segments of ()
sense ssRNA

5.2 mb

Gram-positive
bacteria

Bacillus
anthracis

Genome size
Nucleic acid
4 mb
Circular, two
chromosomes

Classification
Gram-negative
bacteria

Pathogen name
Vibrio cholerae

Table 1.3. Features of infectious diseases

E dimer arranged in
smooth icosahedral
shape

Membrane or envelope
Gram-negative curved
bacillus, cell wall or
flagella covered in LPS
Gram-positive bacillus;
D-glutamate capsule
layer
Lipid membrane with
HA, NA and M2 transmembrane channel
Lipid membrane with
gp120 and gp 41
envelope proteins
Lipid envelope decorated with spike
glycoprotein
Lipid envelope with GP1

NP; vp35; vp40; GP1;

GP2; sGP; vp30; vp24
C, pr, M, E, NS1, NS2A,
NS2B, NS3, NS4A,
NS4B, NS5

PB2; PB1, N40, PB1-F2;

PA; HA; NP; NA; M1,
M2, M42, NS1, NEP
GAGMa, CA, NC; POL
RT IN; vif; vpr; tat; rev;
ENVSU, TM; nef
2, HE, S, 4, 5, E, M, N

Approx. 5,000

Proteins
Approx. 3,500

Introduction to Infectious Diseases15

16 BIOTECHNOLOGY AND INFECTIOUS DISEASES

disease. Epidemic serogroups of V. cholerae are not only unique in surface

structure, but also in the expression of virulence factors. To cause disease,
V. cholerae infection is initiated through attachment to the small intestinal surface by factors, including a carbohydrate binding ligand, GbpA
to N-acetyl glucosamine (GlcNAc) on host cells (Kirn, Jude, and Taylor
2005; Jude et al. 2009). Surface attachment is followed by the induction
of virulence factors, including the toxin coregulated pilus (TCP) that
meditates microcolony formation (Kirn et al. 2000) and production of
toxins, including cholera toxin (CT).
V. cholerae persists in brackish water; often bound to chitin on plankton and copepods (Faye et al. 2014). Environmental abundance naturally
fluctuates with weather patterns and climate (Lipp, Huq, and Colwell
2002). Due to its persistence in the aquatic environment, this is not a
pathogen that can be eradicated worldwide. Thus, methods for monitoring the presence of V. cholerae are crucial for maintaining public health.
Accurate monitoring can reveal the epidemic threat of the strains present
and estimate the number potential infectious cases. This monitoring provides clear insight for development of prevention and treatment protocols.
V. cholerae is representative of a bacterial pathogen capable of causing
waterborne illness both in a seasonal predicable fashion, in addition to
causing disease following a disturbance or disaster-related (e.g., earthquake, hurricane) event.
1.5.1B BACILLUS ANTHRACIS
The Gram-positive bacillus-shaped bacterium that is the cause of anthrax
is Bacillus anthracis. B. anthracis is also a persistent member of environmental microbial communities and niches, present in soil in a resistant spore form, germinating only when environmental conditions are
precisely favorable (Van Ness 1971). B. anthracis encodes its genetic
information on a single, circular chromosome 5.2 Mb in length. It also
carries two virulence plasmids called pXO1 and pXO2, which encodes
virulence factors and toxins essential for the optimal infectious process.
B. anthracis can successfully replicate and disseminate when carried by
a permissive host, and can infect permissive animals species (e.g., cattle)
by such routes as inhalation, ingestion, or skin wounds (Mock and Fouet
2001). Recent evidence suggests that B. anthracis may live in a vegetative biofilm state within the environment, with lifestyle behavioral shifts
triggered when infected by a virus (bacteriophage) (Schuch et al. 2009).
Rapid growth of cells in a permissive environment causes expression of

Introduction to Infectious Diseases17

virulence factors for infection, while serving as a mode of replication for

subsequent reinfection of other hosts (Mock and Fouet 2001). Widespread
infections are rare, however occasional cutaneous, gastrointestinal, or
inhalation anthrax cases occur when there is incidental contact of a susceptible human host with contaminated soil or animal hides (Greenfield
and Bronze 2003). Symptoms of infection are mediated by the effects of
three virulence factors; protective antigen, lethal factor, and edema factor
(Mock and Fouet 2001). However, the disseminating nature of anthrax
spores, and the severity of inhalation infection by B. anthracis in humans
make it a potential bioterrorism threat, a notion validated in the 2001 U.S.
mail-propagated attacks (Inglesby et al. 2002). Mechanisms and updated
techniques for surveillance and detection of B. anthracis remain of crucial
importance to public safety.
1.5.2 INFECTIOUS VIRUSES
Many global-threatening wicked pathogens belong to the viral classification, dependent on host cells for propagation and spread. The molecular structure, shape, and characteristics of viruses are widely varied. This
wide variation adds to the difficulty in developing accurate methods for
detection, treatment, and prevention. The unique partners of interaction
between viral capsid proteins and host receptor proteins provide likely
candidates for antiviral drug development. These structures are also prone
to evolutionary changes that make these true wicked infectious diseases.
Since viruses are not capable of surviving outside of a host cell, methods
of direct culture and amplification of viruses is only possible through inoculation of permissive cell lines. Researchers must preliminarily establish
these alternative ways to identify and quantify specific viral presence in
an environment. Similar to bacterial pathogens, however, viral pathogens
can persist in permissive hosts, and can transit from a zoonotic (animal
infection) to a human pathogen, in a term referred to as spillover (Childs
2007; Childs and Gordon 2009).
1.5.2AINFLUENZA
Influenza belongs to Baltimore group V; defined as enveloped, negative
sense, single-stranded RNA, segmented (multiple individual lengths of
genome) viruses of approximately 10 kb in length. Influenza viruses
encode for approximately 13 proteins (Table 1.3), and can infect host cells

18 BIOTECHNOLOGY AND INFECTIOUS DISEASES

that directly correspond to the molecular and antigenic nature of the viral
surface receptors neuraminidase (NA) and hemagglutinin (HA). Ultimately, influenza variants are categorized by the identity of the NA and
HA features (e.g., H1N1, H5N1), with 16 HA and 9 NA variants identified
thus far (Ma et al. 2009).
To enter into a permissible host cell, the viral HA protein interacts
with the cellular receptors sialic acid (Skehel and Wiley 2000; Smith etal.
2004), and the lectins DC-SIGN and L-SIGN (Londrigan et al. 2011) on
the surface of alveolar lung epithelial cells. Lectins are carbohydrate-
binding proteins that adhere specifically to sugar structures. They are well
known for their ability to mediate pathogen-host cell interactions, initiating the infectious process. In humans, infection symptoms include fever
and respiratory tract involvement. Currently, a multivalent annual vaccine
is produced, however due to the rapid evolution and change of the virus,
more stable and effective treatments and preventative measures are currently being sought.
While capable of causing acute infection in humans, some variants of
influenza can persist in avian and swine species, where the route of transmission is fecal or oral, as opposed to respiratory (Graham et al. 2008).
It is possible for two variants of influenza to simultaneously infect the
same cell. Moreover, some organisms are able to host multiple variants
of influenza simultaneously (Russell and Webster 2005). This coinfection
of hosts and cells can lead to the segmented and reassortable influenza
genomic to produce highly varied chimeric viral variants. The resulting virus can have widely different antigenic determinants as well as
host altered permissivity and infectiousness. Current research examines
whether host species provide a bias toward specific reassortment events.
Additionally, it is possible host identity plays a significant role in which
antigens are prevalent in a community. These facts underscore the need
to focus surveillance efforts on not just patients, but reservoir or carriers
(Watson et al. 2015; Pinsent et al. 2016).
1.5.2BHIV
HIV is an enveloped retrovirus belonging to Baltimore group VI. HIV
encodes its genome on 2 (+) sense, linear RNA fragments, encoding 10
structural, enzymatic, and regulatory proteins. Interestingly, and leading to
its classification as a retrovirus, this genome undergoes reverse transcription via a unique viral RNA dependent DNA polymerase, termed reverse
transcriptase (Baltimore 1970; Mizutani, Boettiger, and Temin 1970). Once

Introduction to Infectious Diseases19

reverse transcribed, a second viral enzyme, integrase, inserts a double-

stranded DNA fragment of the viral genome into the double stranded DNA
host genome. HIV is the causative agent of Acquired Immune Deficiency
Syndrome (AIDS), and is a viral pathogen linked to a zoonotic spillover
event from the zoonotic virus of African nonhuman primate species, SIV
(reviewed in Hirsch et al. 1995).
Transmitted via bodily fluid (i.e., blood (Simon, Ho, and Abdool
Karim 2006), sexual contact, and potentially breast milk (Magoni et al.
2005)), HIV binds to permissive cells expressing CD4 and appropriate
chemokine receptors. Other ligands for cell entry include specific lectins.
HIV utilizes the lectin DC-SIGN and others (Shattock and Moore 2003),
providing potential therapeutic targets. Upon membrane fusion, the
genomic content is reverse transcribed into double-stranded DNA by a
virally encoded enzyme, reverse transcriptase (reviewed in Turner and
Summers 1999) and integrated into the host genomic DNA. A patient
infected with HIV typically progresses by a destruction of the Tlymphocyte cell13 population. This lack of immune cell protection, and subsequent
lack of immune response to infection, culminates in AIDS (Lloyd 1996;
Simon, Ho, and Karim 2006).
1.5.2C SARS AND MERS
SARS and Middle Eastern Respiratory Virus (MERS) are caused by similarly structured corona viruses, featuring an envelope, a matrix capsid, and
a single-stranded (+) RNA genome. Recent potential zoonotic spillover
events in the China (Field and Field 2009) and the Middle East (Banik,
Khandaker, and Rashid 2015) have resulted in newly emerging viral respiratory infections. Transmitted through aerosolized droplets, this class of
viruses has been documented to cause various levels of pathogenicity in
patients (Hilgenfeld and Peiris 2013). However, information and practices
developed in responding to the more acute outbreak of SARS allowed for
clear evaluation, preparation, and rapid response following detection of
MERS (Hilgenfeld and Peiris 2013).
1.5.2DEBOLA
The recent largest and most widespread epidemic of Ebola in 2014 captured headlines and worldwide concern, based on both the human-tohuman spread, as well as concern from the previously high d ocumented

20 BIOTECHNOLOGY AND INFECTIOUS DISEASES

fatality rates for Ebola infections (Baize et al. 2014). Ebola is classified as belonging to Baltimore Class V, with a () sense linear ssRNA
genome that is packaged in a filamentous shaped proteinaceous viral
capsid. The genome encodes eight proteins, and its gamma-lipid envelope, decorated with viral GP1 protein, interacts in a lectin-based interaction with cell surface receptors DC-SIGN (same as used in HIV)
and NPC1 (Niemann Pick Disease type C1 protein) (Nyakatura, Frei,
and Lai 2015; Dolnik et al. 2008). There is a broad range of susceptible cells, including alveolar macrophages, hepatocytes, and dendritic
cells (Martines et al. 2015). This leads to disease manifestation with a
wide range of symptoms and mechanisms of spread. Ebola symptoms
are classically described as hemorrhagic disease, however the most
recent outbreak in 2014 proved to have less hemorrhagic symptoms in
patients. Other symptoms include rash, joint pain, impaired function of
the liver and kidney, and fluid loss based hypovolemic shock (Mendoza,
Qiu, and Kobinger 2016). No vaccines are currently available, however
therapeutics and preventative measures are in all stages of development
(Martnez-Romero and Garca-Sastre 2015; De Clercq 2015; Mendoza,
Qiu, and Kobinger 2016).
Ebola is presumed to have a reservoir in bat populations (Jayme
et al. 2015), similar to other viruses including SARS and MERS (Field
and Field 2009; Han et al. 2015). There is also evidence of transmission from pigs to nonhuman primates (Weingartl et al. 2012), as well as
transmission between guinea pigs, in model system studies (G. Wong
et al. 2015). Recent concern even included Ebola as a potential foodborne pathogen, with the virus able to be passed on via ingestion of
contaminated food (Bausch and Bausch 2011). For patients suspected
of an Ebola infection, samples of saliva and blood are primary sources
of infectious particles (Bausch et al. 2007), and to a lesser extent, skin,
stool (Bausch et al. 2007), semen (Mate et al. 2015), and in ocular tissue
(Chancellor et al. 2016).

1.5.2EZIKA
Currently the topic of viral concern worldwide, the genome of the Zika
virus (Baltimore Class IV) is a 10 kb (+) sense single-stranded RNA, that
encodes for 11 proteins. Zika is an insect vector-borne flaviavirus, transmitted by the Aedes species of mosquitos (Focosi, Maggi, and P
istello
2016). Although vector-mediated transmission is the primary route of

Introduction to Infectious Diseases21

Zika infection, infection via sexual contact is also possible (McCarthy

2016; Hills et al. 2016). The virus gains entry into susceptible human epidermal epithelial cells, via viral E protein interaction with cell receptors
including DC-SIGN (same lectin used in Ebola and HIV), AXL, Tyro,
TIM-1, and utilizes clathrin-mediated entry, and a version of autophagous entry to gain access to the cell (Hamel et al. 2015). Zika virus was
first recognized and characterized in 1947 in a Ugandan macaque (Dick,
Kitchen, and Haddow 1952; Dick 1952). Zika virus is closely related to
Dengue, but also has similarities to West Nile Virus as well as Chickungunya (Cao-Lormeau, Roche, and Teissier 2014). Humans are the primary
host for Zika viral infections, however nonhuman primates may also be
susceptible to infection (Musso and Gubler 2016).
Zika cases experienced a resurgence in French Polynesia in 2013
2014 and in South and Latin America in 2015 (Fauci and Morens 2016).
Researchers are monitoring the 20152016 outbreak for similarities to
other outbreaks including vector-borne viruses such as West Nile, Dengue,
and Chickungunya. These viruses can infect concurrently, and suddenly
(Villamil-Gmez et al. 2016). There is precedence of arborviruses
coinfecting patients often in tandem with other vector-borne infectious
agents, including Dengue (Dupont-Rouzeyrol et al. 2015).
Although rarely fatal, and often asymptomatic, Zika infections
often present with fever and rash. Of more concern are cases of Guillen
Barre Syndrome (GBS), affecting the nervous system, that have been
linked to Zika infections (Oehler et al. 2014), similar to the prognosis
documented in previous Chickungunya virus outbreaks (Oehler et al.
2015). Other recent pathologies have been observed on the fetuses
of infected women, including fetal developmental symptoms such as
brain encephaly, microencephaly, following birth to mothers with noted
viral infections occurring over the course of their pregnancy (Oliveira
Melo et al. 2016). Year 2015 represented the first linkage of fetal brain
development symptoms with Zika viral infection in pregnant women
(Oliveira Melo et al. 2016). Further characterization of the virus at the
molecular level (Faye et al. 2014) may lead to the characterizing of the
2015 strain as being more virulent, or it remains possible that previous
sequelae from earlier pathogenic infections had not been well documented. This is most relevant when examining the most recent finding
that the Zika virus is capable of infecting adult mouse neural stem cells,
and this infection can result in a cell death and lowered cell numbers
(Li et al. 2016). This finding could point to potential neural symptoms
in adults and infants.

22 BIOTECHNOLOGY AND INFECTIOUS DISEASES

1.6CONCLUSION
All wicked pathogens described here serve as examples of infectious
disease threats. These pathogens are both bacterial and viral, and differ
in many parameters. Some of the pathogens are environmental (Vibrio
cholerae, Bacillus anthracis), while others utilize organisms to transmit from case to case (Zika). Pathogens also utilize several methods for
gaining access to the cells they are attempting to infect, and causing the
virulent effects of a disease state. Interestingly, some highly dissimilar
pathogens use identical mechanism for infection, as Zika, Ebola, and HIV
all make use of the lectin DC-SIGN for cell entry. Understanding all the
ways that cells can enter and infect cells make it possible to recognize
strategies in emerging pathogens, and more readily adapt and develop
detection methods and therapeutics.

Index
A
Adaptive immune system, 77, 78
Adjuvant, 88, 9293. See also
Vaccine adjuvant
(AFM) cantilevers. See Atomic
force microscopy cantilevers
Agrobacterium tumifaciens, 6263
Alum adjuvanted vaccines, 92
Anthrax, 16, 17
Antibacterial agents, 6466
Antibodies, 78
Antibody response, 80
Anticoagulant factors, 56
Antigenic determinants, 89
Antigen presenting cells (APC),
80
Antigens, vaccine, 80
APC. See Antigen presenting cells
Aptamers, 5860
Archaea, 6
Atomic force microscopy (AFM)
cantilevers, 4950
Autoinducers, 65
B
Bacillus anthracis, 1617
Bacteria, 4
Bacterial vaccines, 83
Bacteriophage, 66
B cell receptor, 78
Bioavailablity, 69

Biotechnology, 8891, 97
Bone marrow (B cells), 80
C
Capsid coat structure, 9
Carbohydrate binding agents, 59
Carbohydrate-protein interactions,
5962
Cellular sensor detectors, 5051
Chinese hamster ovary (CHO)
cells, 63
Chitosan, 70
Cholera
detection, 26
vaccine, 83
Cold chain, 77, 83
Commensals, 6
CpG adjuvant, 88
CpG motifs, 93
CRISPR/Cas-based editing,
7374
Culture-based detection, 3435
characterization of pathogens,
3839
examination, 3738
media and culturing conditions,
3637
propagation-based viral
detection, 38
Culture independent detection
ELISA, 4041

130Index

IFA, 41
slide agglutination assays, 3940
D
DC-SIGN, 5960
DNA-based assay, 43
DNA-based vaccines, 8485
DNA replication, 65
Dukoral, 83
E
Ebola, 1920, 55
EBOTab, 63
ELISA. See Enzyme linked
immunosorbant assay
ELSD. See Evaporative light
scattering detector
Enzyme linked immunosorbant
assay (ELISA), 29, 4041
Escape variants, 63
Eukaryotic organisms, 6
Evaporative light scattering
detector (ELSD), 49
F
Flemings Lucky Find, 6667
Flumist, 95
G
Genome editing, 7172
CRISPR/Cas-based editing,
7374
delivery, 74
TALEN editing, 73
treating of pathogenic infections,
74
ZFN editing, 7273
Genomes of pathogens, 910
Glycans, 59
Glycofullerene, 61
Gram-Negative bacteria, 68
Gram-Positive bacteria, 68
Gram Stain technique, 68
Griffithsin (GRFT), 61

H
Herd immunity, 89
High performance liquid
chromatography (HPLC), 48
HIV. See Human
immunodeficiency virus
Host genome, 7172
CRISPR/Cas-based editing,
7374
delivery, 74
TALEN editing, 73
treating of pathogenic infections,
74
ZFN editing, 7273
HPLC. See High performance
liquid chromatography
Human immunodeficiency virus
(HIV), 1819
Humoral response, 80
I
IFA. See Indirect
immunofluorescence assay
Immunologic memory, 79
Immunostimulating compounds
(ISCOMS), 92
Indirect immunofluorescence assay
(IFA), 41
Infectious disease outbreak
characterization, 2325
influenza pandemic, 2829
Kochs postulates, 2628
polio, 3031
SARS and MERS, 30
super-spreaders, 2425
Infectious diseases
Bacillus anthracis, 1617
Ebola, 1920
environment, 12
features, 15
HIV, 1819
influenza, 1718
problems, 45
SARS and MERS, 19

Index 131

structures, 511
Vibrio cholerae, 1416
wicked problems, 14
Zika, 2021
Influenza, 1718
Influenza pandemic, 2829
Innate immune system, 78
Inoculum, 79
ISCOMS. See Immunostimulating
compounds
K
Kochs postulates, 2628
L
Lateral flow assays, 4548
Lectins, 19, 59
Ligands, 57
Lipopolysaccharide (LPS), 8
Liposomes, 70
LPS. See Lipopolysaccharide
M
Matrix-assisted laser desorption or
ionization time-of-flight mass
spectrometry (MALDI-TOF), 48
MDR. See Multidrug resistant
strains
Memory B cell, 78
MERS. See Middle Eastern
Respiratory Virus
Metazoonosis, 12
Microbes, 4
Microbiomes, 6
Microneedles, 84
Middle Eastern Respiratory Virus
(MERS), 19, 30
MLVA. See Multiple locus VNTR
analysis
Model systems, 5
Molecular beacons, 45
Monoclonal antibodies (mAB), 62
Multidrug resistant strains (MDR),
65

Multiple locus VNTR analysis

(MLVA), 43
Multiplex PCR, 4243
Multivalent, 88
N
N-acetylglucosamine (NAG) cell
wall, 64
NALT. See Nose associated
lymphoid tissue
Nanoantibiotics, 7071
Nanodetection techniques
chemical signatures, 4849
lateral flow assays, 4548
nanomotion sensors, 4950
Nanomotion sensors, 4950
Nanoparticles, vaccine
formulation, 8788
NHEJ. See Nonhomologous end
joining mechanism
Nicotimiana benthimiana, 6263
Nonhomologous end joining
mechanism (NHEJ), 72
Nose associated lymphoid tissue
(NALT), 95
Nucleic acid hybridization, 45
O
Oligonucleotide detection, 4142
molecular beacons, 45
nucleic acid hybridization, 45
PCR, 4243
RT-PCR, 4345
Oligonucleotide vaccine, 8485
OMPs. See Outer membrane
proteins
Outbreak, infectious, 45
Outer membrane proteins (OMPs),
87
Outer membrane vesicles, 87
P
Pandemics, 4
Pathogen, 1214

132Index

Pathogen detection, 51
cellular sensor detectors, 5051
culture-based detection, 3435
characterization of pathogens,
3839
examination, 3738
media and culturing
conditions, 3637
propagation-based viral
detection, 38
culture independent detection
ELISA, 4041
IFA, 41
slide agglutination assays,
3940
nanodetection techniques
chemical signatures, 4849
lateral flow assays, 4548
nanomotion sensors, 4950
oligonucleotide detection, 4142
molecular beacons, 45
nucleic acid hybridization, 45
PCR, 4243
RT-PCR, 4345
Pathogen genomes, 910
Pathogenic bacteria
Bacillus anthracis, 1617
Vibrio cholerae, 1416
Pathogen treatment
antibacterial agents, 6466
antibody cocktails, 6263
carbohydrate-protein
interactions, 5962
disrupting infection mechanism,
5657
drug delivery systems, 6970
examination, 5354
host genome, 7172
CRISPR/Cas-based editing,
7374
genome editing, 7475
TALEN editing, 73
ZFN editing, 7273
nano antimicrobials, 7071

nucleic acid function, 6364

patterns and similarities, 54
RNAi, 6769
symptoms, 5456
therapeutic development, 5758
PCR. See Polymerase chain
reaction
Penicillium notatum, 66
Permissivity, 12
PFGE. See Pulse field gel
electrophoresis
Phosphorodiamidate morpholino
oligomers (PMOs), 67
Phylogenetic tree of life, 6
PMOs. See Phosphorodiamidate
morpholino oligomers
Polio, 3031
Polyclonal antibodies (pAB), 63
Polymerase chain reaction (PCR),
4243
Primers, 42
Programmable nucleases, 72, 73
Prokaryotes, 6
Pulse field gel electrophoresis
(PFGE), 41
Q
Quorum sensing, 65
R
Random amplification of
polymorphic DNA (RAPD), 43
Rapid influenza antigen diagnostic
(RIDT) assays, 47
Relative risk or risk ratio (RR), 24
Reverse transcriptase PCR
(RT-PCR), 4345
Reverse transcription inhibitors
(RTI), 63
RGENs. See RNA-guided
engineered nucleases
Ribosome, 10
RIDT assays. See Rapid influenza
antigen diagnostic assays

Index 133

RNA-guided engineered nucleases

(RGENs), 73
RNAi silencing, 6769
RNA replication, 64
RR. See Relative risk or risk ratio
RT-PCR. See Reverse transcriptase
PCR (RT-PCR)
RTI. See Reverse transcription
inhibitors
S
Salmonella typhi, 24
SARS. See Severe acute
respiratory syndrome
SELEX technology, 5859
Self-replicating RNA vaccines,
8485
Severe acute respiratory syndrome
(SARS), 19, 30
Slide agglutination assays, 3940
Smallpox, vaccination, 79
Sol gel, 71
Spanish Flu, 28
Spillover, potential, 28, 30
Subunit vaccines, 84
Super-spreaders, 2425
Surveillance, 33
Symbiotic relationships, 6
T
TALENs. See Transcription
activator-like effector nucleases
T cells, 78
TCI. See Transcutaneous
immunization
Thymus (T cells), 80
Time of flight (TOF), 48
TLR. See Toll-like receptor
T lymphocyte cell, 19
TOF. See Time of flight
Toll-like receptor (TLR), 92
Toxoids, 84
Transcription activator-like
effector (TALE) nucleases
(TALENs), 72, 73

Transcutaneous immunization
(TCI), 95
Tunicamycin, 64
Typhoid Mary, 2425
V
Vaccine
anatomical response, 80
definition, 77
design strategies, 81, 82
history, 7980
oligonucleotide, 8485
subunit, 84
whole cell, 81, 83
Zika, 96
Vaccine adjuvant
aluminum salts, 92
bacterial agonists, 9293
definition, 91
ISCOMS, 92
lipid, 93
metals, 93
Vaccine delivery
immunoglobulin, 94
injection route, 9495
nasal route, 9596
oral, 94
transcutaneous route, 95
Vaccine formulation
biotechnology, 8891
definition, 85
nanoparticles, 8788
viral vectors, 8586
virosomes, 8687
VLPs, 86
Variable number of tandem repeats
(VNTR), 43
Variolation, 79
Vibrio cholerae, 1416
Viral genomes, 1011
Viral-like particles (VLPs), 86
Virosomes, 70, 8687
Virus
Ebola, 1920
HIV, 1819

134Index

influenza, 1718
SARS and MERS, 19
Zika, 2021
VNTR. See Variable number of
tandem repeats
W
WHO. See World Health
Organization
Whole cell vaccines, 81, 83
Wicked problems, 14

World Health Organization

(WHO), 23
Z
Zika, 2021, 96
Zinc fingered nucleases (ZFNs),
7273
Zinc-finger protein (ZFP),
72
ZMapp, 62
Zoonosis, 12