Scientific Research and Essay Vol. 4 (2), pp. 042-058, February, 2009

Available online at http://www.academicjournals.org/SRE

ISSN 1992-2248 © 2009 Academic Journals

Review

Department of Biotechnology College of Egineering and Technology, IFTM campus, Delhi Road, Moradabad 244001,

U.P., India.

Department of Biochemistry, Bundelkhand University, Jhansi, U.P., India.

Department of Biotechnology and Microbiology, College of Applied Sciences, IFTM Campus, Lodhipur-Rajput, Delhi

Road, Moradabad 244 001, U.P., India.

associated with potential beta cell toxicity. Beta cells are weak in antioxidative defense, which makes

them hypersensitive to oxidative stress. Acknowledging the inhibitory potential of in vivo conditions

scientists have developed two models resembling a slowly progressing coxsackievirus infection first,

by restricting the production of viral progeny with a selective inhibitor of viral RNA replication and

second, by means of lowering the multiplicity of infection. Hydrogen peroxide has been established as

the oxidative stressor. Recent studies reflect that a productive CVB-infection results in lytic beta cell

death. When pharmacologically restricted by guanidine-HCl, the viability increases dramatically through

decreased necrosis and associates with simultaneous stimulation of apoptotic death. In summary, the

review introduces potential mechanistic models for enterovirus infections in beta cells.

based on genetic predisposition triggered by an environ-

mental factor e.g. virus infection. The effector stage,

insulitis, is characterized by gradual invasion of macro-

phages, T-cells and the produced cytokines and oxidative

radicals into the islets (Rasilainen et al., 2004). The pre-

*Corresponding author. E-mail: [email protected].

factor; NO, nitric oxide; NOD mouse, spontaneously diabetic

non-obese; ROS, reactive oxygen species; SOD, super oxide

dismutase; TNF – , tumor necrosis factor alpha.

formation of islet cell autoantibodies even years before

the development of diabetic symptoms. Enterovirus infec-

tions are epidemiologically linked to the pathogenesis of

T1D (Rasilainen et al., 2004). Beta cells exhibit poor

intracellular antioxidative capacity, which renders them

vulnerable to oxidative stress. Thus, both enterovirus

infections and oxdative stress are considered to repre-

sent particularly potential triggers and/or accelerators of

beta cell destruction.

It is generally known that the amount of viral particles

needed to establish a local infection is low and all viruses

invading a physiological environment face natural resis-

tance by factors of the hosting immune system (Rasilai-

nen et al., 2002). Considering that coxsackievirus infec-

tions most often remain subclinical in vivo, the prog-

mental models of slowly progressing enterovirus infection

and reports regarding the patterns of mechanisms of

virus induced beta cell damage. Further, this scientific re-

port includes: characterizing the mechanisms of oxidative

beta cell injury; counteracting it by means of antioxidative

agents and studying the effects of virus infection on

intracellular redox balance.

and Lernmark, 1998). The resulting insulin deficiency

leads to a chronic metabolic derangement associated

with significant secondary morbidity. According to the tra-

ditional opinion consistent within most autoimmune

diseases, both a genetically predisposed individual and a

suitable environmental trigger are needed for the des-

tructive process, in the case of T1D against beta cells, to

begin. Nowadays, the group of human leukocyte antigen

(HLA) genes is considered to account for the major ge-

netic risk and virus infections to represent prime environ-

neously diabetic non-obese (NOD) mice, whose

pathogen-nesis mimics human type I diabetes (Anita et

al., 2006). Most probably both cytotoxic (CD8+) and

helper (CD4+) T-cells are required for both the primary

attack and the later overt destruction, respectively, to pro-

gress (Bendelac et al., 1987; Hanafusa et al., 1988;

Jarpe et al., 1990; Jarpe et al., 1991; Miller et al., 1988;

Sumida et al., 1994; Wicker et al., 1994; Wong et al.,

1996). Besides, a pool of antigen presenting cells (APC)

is indispensable in the vicinity of pancreatic islets to intro-

factor alpha (TNF-), are thought to play a primary role in

activating signal transduction pathways leading to beta

cell dysfunction and apoptosis (Delaney et al., 1997;

Mandrup, 1996; Rabinovitch, 1998). The direct killing

through the granzyme-perforin pathway by cytotoxic T

cells is also believed to be a key mechanism ultimately

leading to apoptotic beta cell death.

Finland has the highest incidence of T1D in under 15-

year-old children worldwide. It has gradually increased

during the last 43 years from 12/100000 (1953) to

thus being 3.4 % between 1965 and 1996 (Somersalo,

1955; Tuomilehto et al., 1999). The incidence is not only

geographically, but also seasonally distributed peaking in

winter (Levy-Marchal et al., 1995). Finland and some

other Nordic high-incidence countries have also shown

gender-impact with a slight male predominance (Padaiga

et al., 1997). Approximately 10% of newly diagnosed T1D

including HLA DP, DQ and DR (Campbell and Trowsdale,

1993; Davies et al., 1994;). These genes encode hetero-

dimeric proteins expressed on APC-cells and have a

function in presenting antigenic peptides to CD4+ T cells.

HLA class II genes have a high degree of polymorphism

and marked linkage disequilibrium that potentiate the risk

to form certain high-risk allele-pairs. Furthermore, these

susceptibility alleles may then choose potential autoreac-

tive T-cells during thymic sorting, and enhance the dis-

ease probability. Many recent studies have demonstrated

a cell to die. The specific pathway chosen depends large-

ly on the trigger, but the ultimate result may vary accord-

ing to the cellular capacity of defense and the net status

of the host (age, inflammation etc.). A general divi- sion is

traditionally done by the resulting morphological charac-

teristics: apoptotic, pyknotic, necrotic (Figure 1).

always associated with specific pathology. It is an unre-

gulated process and follows no specific pattern. The sa-

lient features include cytoplasmic and mitochondrial

pendent on perforin-built channels on target cell mem-

brane allowing granzyme entry and caspase activation

(Garcia-Sanz et al., 1987; Kagi et al., 1994; Yoon and

Jun, 1999).

Beside the well-known apoptotic and necrotic path-

ways, a pyknotic form of cell death has also been differ-

rentiated. It morphologically resembles apoptosis with an

intact plasma membrane, but is characterized by con-

densed but intact chromatin, unlike the apoptotic frag-

mented DNA, demarcated at the margins of the nucleus

phenomenon has also been observed during an apoptotic

process, which due to a sudden ATP depletion or some

other disability was interrupted and turned into secondary

necrosis (Hirsch et al., 1997; McCarthy et al., 1997).

Enteroviruses

Picornaviruses are a diverse group of small, non-enve-

loped animal viruses with single-stranded RNA genome

developed world (Rotbart, 2002). Picornaviruses were

previously classified according to their physicochemical

properties (particle density, pH-sensitivity) and serolo-

Enteroviral genome and viral replication cycle

In order to multiply, the virus must first bind to its specific

receptor on the surface of a target cell. Several receptors

have been demonstrated for enteroviruses: coxsackiea-

denovirus receptor (CAR) is recognized by CVB 1-6 and

CAR (Chehadeh et al., 2000). After contacting the recep-

tor, the virus should successfully enter the cell, which

commonly requires some conformational changes of the

viral capsid. During entry or immediately afterwards, the

viral genomic material is released into the host’s cyto-

plasm in a process called uncoating (Figure 2). Once

released in the cytoplasm, the viral genomic RNA, about

7500 nucleotides in length, functions directly as a tem-

plate for the production of viral proteins. The single open

reading frame encodes a large precursor poly-protein.

This precursor includes two proteolytic sequences (2A

and 3C), which cleave the precursor into intermediate

product is the virus RNA-dependent RNA polymerase

(3D). It duplicate the genomic RNA into a negative sense

strand, which then acts as a template for positive -strand

RNA synthesis. Finally, the newly pro-duced genomic

material is packaged into the procapsids formed by

twelve pentameric structures, each of which contains five

protomers (VP0-VP3-VP1 –heterotrimers) (Korant, 1973).

During this RNA-encapsidation, the VP0-protein is further

cleaved into VP2 and VP4 (maturation cleavage; relevant

As suggested earlier, the outcome of an enterovirus

infection may vary between aggressive fulminant and

acute subclinical (Ramsingh, 1997b; Yoon et al., 1979).

In suitable conditions persistence may develop (Adachi et

al., 1996; Chehadeh et al., 2000; Conaldi et al., 1997a;

1997b Klingel et al., 1992; Tam and Messner, 1999). In

general, a viral invasion into an organism supplied with a

properly functioning immune system always attracts it to

respond. The innate arm of immunity composed of natu-

tein tyrosine kinases (JAK 1, TYK2) which further act to

stimulate STAT-, Crk- and IRS- pathways (Ahmad et al.,

1997; Darnell et al., 1994; Uddin et al., 2000). These

routes finally lead to the induction of anti-viral interferon

stimulated genes (ISG) (Bose and Banerjee, 2003).

Interferons are also able to induce the production of

MHC-molecules on virus-infected cells and on antigen

presenting cells required for CD4+ T-cell action. Further-

more, they may initiate or contribute to the production of

antigen specific antibodies and thus activate the second

line of antiviral host defense, the adaptive or cell-media-

ted arm of immunity (Kadowaki et al., 2000). Recent

observation in human islets concluded CVB-infection to

stimulate INF- production selectively in beta cells, which

by inhibiting the efficiency of viral replication prevents the

initiation of beta cell autoimmunity (Chakrabarti

dissemination and persistence often involve induction of

apoptosis through either perforin-granzyme or death

receptor mediated pathways (Ashkenazi and Dixit, 1998;

Froelich et al., 1998). IFNs are reported to sensitize infec-

ted cells to apoptosis (Balachandran et al., 2000; Tanaka

et al., 1998). As and when beta cells are stimulated with

IFNs in combination with double-stranded viral RNA or

the cytokine IL-1ß, a synergistic apoptotic effect is

observed (Liu et al., 2002). To defend them-selves, many

ed by several different viruses (Katze, 1995; Komatsu et

al., 2000; Munoz-Jordan et al., 2003; Ronco et al., 1998).

Also cleavage of p21(ras) GTPase-activating protein

(RasGAP) and activation of the MAPK family members

ERK 1/2, registered essential for CVB3 replication, could

be affected (Huber et al., 1999a; Luo et al., 2002; Opav-

sky et al., 2002). After stimulation of the innate arm of

immunity a cell-mediated immune response is launched.

ponse by cytokines and the pathogen itself, present anti-

gen to naive T-cells, which start to develop differential

markers. Specifically the Th1 type CD4+ T-cells secrete

proinflammatory cytokines, which enhance virus-specific

host cell lysis by CD8+ T-cells and stimulate the MCH

upregulation on APC cells, that further helps in activating

antibody production by B-cells. Other interleukines

cells, like T-cells, to be more susceptible to virus and to

efficiently produce viral progeny (Liu et al., 2000b; Molina

et al., 1992). Quiescent cells (in G0 phase) on the other

hand do not support viral multiplication, but may harbor

infective viruses thus creating persistence (Feuer et al.,

2002). These theories may partially explain the individual

susceptibility of cells to infection. Some studies further

indicate several viruses capable of forcing the host cell

into a favored phase of cell cycle (Op De Beeck and

Caillet-Fauquet, 1997; Swanton and Jones, 2001). Viral

kie A viruses typically induce diseases with mucosal and

skin lesions (e.g. herpangina, hand, foot, and mouth dis-

ease) (Bendig and Fleming, 1996; Itagaki et al., 1983;

Seddon and Duff, 1971). In addition to the milder dis-

more severe and possibly chronic diseases including

meningitis, myopericarditis, epidemic pleurodynia and

T1D (Beck et al., 1990; Gauntt and Huber, 2003; Muir

and van Loon, 1997; Rotbart, 1995; Tracy et al., 2000).

They are together with echoviruses responsible for se-

vere neonatal viral infections (Chiou et al., 1998; Sawyer,

after early CVB and rubella exposures (Hyöty et al.,

1995; Lönnrot et al., 1998, 2000; Salminen et al., 2003).

Some mini case reports show evidence for intrauterine

viral exposure, a maternal enterovirus infection, as a risk

factor for future development of T1D (Dahlquist et al.,

1995, Hyöty et al., 1995). In a larger birth cohort study

(Funchtenbusch et al., 2001) contradictory results were

obtained. A recent survey reveals that while the incidence

of T1D has doubled within the last 40 years in Finland,

the incidence of enterovirus infections has simultaneously

absence of protecting maternal antibodies.

increase-ing number of patients with type I diabetes and

supports the role of enteroviruses as possible triggers.

Coxsackie B viruses have long been associated with the

pathogenesis of T1D (Andreoletti et al., 1997; Banatvala

et al., 1985; Yoon, 1990). Groundbreaking findings were

made in 1979. When the autopsy specimens of a 10-

year-old patient with diabetes showed lymphocytic infiltra-

tion of the islets and beta cell necrosis which lead to the

T1D is an immune-mediated disease in which a specific

immune response to islet beta cells is induced. In the

animal model of T1D, the NOD mouse, diabetes may be

cell types (Barco et al., 2000; Girard et al., 1999;

Goldstaub et al., 2000; Lopez-Guerrero et al., 2000;

Tolskaya et al., 1995).

Considering development of T1D, virus infections are

regarded as the leading environmental triggers and apop-

tosis the leading form of beta cell death (Eizirik and

Mandrup-Poulsen, 2001). Their possible association is

Virus infections generally induce an inflammatory reac-

tion mediated in part by proinflammatory cytokines. In

addition to their direct proapoptotic potential, cytokines

may also launch the expression of MHC class I mole-

cules in beta cells followed by their appearance on plas-

ma membranes, which exposes beta cells to T cell me-

diated death through Fas or perforin dependent pathways

(Kim et al., 2002; Seewaldt et al., 2000).

Molecular mimicry

the inhibitory neurotransmitter gamma amino butyric acid

(GABA), is one of the important islet cell auto-anti-gens in

T1D. The sequences of GAD65 and the non-structural

protein 2C of CVB-like enteroviruses were shown to

share a similar motif (PEVKEK) and to bind the same

groove in a MHC-molecule in an exactly similar position

(Kaufman et al., 1992). In later studies with synthetic pep-

tides, the binding was restricted to HLA-DR3 molecule

(Vreugdenhil et al., 1998). Furthermore, GAD65-reactive

T-cells are present and circulating in both T1D patients

dictory observations have also been made (Atkinson and

Maclaren, 1994; Horwitz et al., 1998; Marttila et al., 2001;

Schloot et al., 2001). Accordingly, molecular mimicry

could also exist between GAD and cytomegalovirus on T-

cell level (Roep et al., 2002).

died by Horwitz et al. (1999). Their recent report con-

cluded that the primary role of an enterovirus infection

(pancreatrophic CVB4) in the pathogenesis of T1D is to

specifically infect and damage beta cells leading to

release of sequestered islet antigen and stimulation of a

local inflammatory response. The intra-pancreatic antigen

presenting cells introduce the antigen to the population of

driving force of virus induced T1D, since a non-specific

cytokine-attack did not precipitate T1D in mice with a

diabetogenic T cell repertoire, while exposure to the beta-

cell toxin streptozotozin was able to do it (Horwitz et al.,

2002). CVB infections have previously been reported to

lead to necrotic cell death in both rodent and human exo-

crine pancreatic tissue (Arnesjo et al., 1976; Lansdown,

1976; Ozsvar et al., 1992; Ramsingh, 1997a; Vella et al.,

1992). The inflammatory mediators produced during this

infective process have been speculated to activate the

1982; Maeda et al., 1999; Siraki et al., 2002). The most

common intracellular ROS molecules include superoxide

anion (O

), hydrogen peroxide (H

O

cal (-OH) (Fridovich, 1998; DiGuiseppi and Fridovich,

1984). ROS react with biological molecules such as pro-

teins, lipids, carbohydrates and DNA threatening their

integrity and exposing the organism to toxic, mutagenic

and carcinogenic assaults (Stadtman and Levine, 2000;

Steinberg, 1997; Marnett, 2000). Low molecular weight

1992; Lo and Cruz, 1995; Ohba et al., 1994; Sundaresan

et al., 1995). They also function in microbial killing during

monly, overproduction of ROS is triggered by nonphysio-

logical states like inflammation or exogenous toxins,

which stimulate phagocytic cells to produce ROS. In a

variety of disease processes associated with either acute

or chronic inflammation, increased oxidative stress ap-

pears to be involved. Depending on the surrounding

milieu, ROS molecules may also react together. Speci-

fically, in a reaction between O

and NO highly cytotoxic

with H

or Cu

/Fe

forming either highly reactive

HOCl (hypochlorus acid) or a hydroxyl radical, respect-

tively (Babior, 2000).

Highlighting the dual role of ROS in both physiological

and disease states, recent data show concentration de-

pendent stimulation of either cell growth or death. Mode-

rate levels of pro-oxidants may promote mitogenic stimuli

(Burdon and Rice-Evans, 1989) e.g. by affecting kinase

mechanisms often involve exposing to or triggering the

mitochondrial membrane permeability transition which

leads to the release of apoptogenic factors and thus

stimulation of the downstream apoptotic cascade (Arm-

strong and Jones,2002; Armstrong et al., 2002; Costan-

tini et al., 1996;. Datta et al., 2002; Dypbukt et al., 1994;

Fleury et al., 2002; Petronilli et al., 1994; Ueda et al.,

2002; Wei and Lee, 2002) Also, HIV-infection mediated

redox-active factors, that also support expression of viral

genes and pro-inflammatory cytokines (Perl et al., 2002).

p53, independent of the activating factor, may further

stimulate ROS production and induce apoptosis (Sawada

et al., 2001; Li et al., 1999).

inhibit apoptosis and recent observations on antiapoptotic

molecules with antioxidative properties further argue for a