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EMC virus-induced diabetes in mice
There are two variants of EMC-M virus; EMC-B and EMC-D which were isolated by a method known as plaque purification. The EMC-M virus induces a syndrome like diabetes, where there is hypoinsulinaemia, hyperglycaemia, glycosuria, polydipsia and polyphagia in those strains of mice that are genetically susceptible. The EMC-B virus does not lead to diabetes in the animals that are
infected by this virus whereas the EMC-D virus lead to diabetes in 90% of the animals it infected. These two variants are distinguished from one another by their nucleotide sequences. There was a difference of 14 nucleotide sequences between them both. Only one amino acid however is found to be important in diabetes occurrence involving the EMC virus when further investigations were carried out. This amino acid is alanine which is at position 776 on the polyprotein. When this amino acid is exchanged for threonine at this same nucleotide position there was no diabetes occurrence. However, in exchanging threonine for alanine on the polyprotein there was diabetogenicity. This nucleotide position is critical because it lies in the region of the capsid protein VP1 which is very hydrophilic such that when alanine is exchanged for threonine the region becomes less hydrophilic and there is a greater efficiency of attachment between the virus and the beta cells.

Only certain strains of inbred mice develop diabetes when infected with EMC-M virus. (Jun and Yoon 2001, 274). The EMC virus replicates in the beta cells leading to the recruitment of macrophages in the pancreatic islets. Macrophages when activated secrete cytokines such as IL-1, TNF-? and nitric oxide which results in destruction of beta cells leading to diabetes in mice. (Jun and Yoon 2001, 273) Additional studies make mention of the recruitment of activated macrophages being the initial step and was followed by T cells, B cells and natural killer cells infiltrating the site. The mechanism involved in macrophage activation is still unknown. The mechanism in which the mediators released by macrophages destroy beta cells is still uncertain. See appendix D for the mechanism of action of the EMC virus.

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KRV induced diabetes
The CD4+ T cells in rats are divided into Th1 like CD4+ T cells and Th2 like CD4+ T cells. Before infection there is a balance between these two CD4+ cells, and macrophages and CD8+ T cells are at rest.

When rats become infected with KRV, macrophages become activated and secretes IL-12 which activates the Th1 cells. The Th1 like T cells secrete IL-2; which activates CD8+ T cells, and IFN-?. This leads to an increase in the Th1 cells and CD8+ T cells. Beta cells are killed as a result of the activation of these two cells (Jun and Yoon 2001, 281).

In addition to viruses causing diabetes, they can also prevent diabetes. In animal models of rat and mice, the viruses Lymphocytic choriomeningitis virus (LCMV) and mouse hepatitis virus (MHV) prevents type 1 diabetes from developing. Autoimmune diabetes was found to be prevented in diabetes prone rats as well as NOD mice. Later studies found that the protection against T1D was due to an interferon gamma induced protein-10 chemokine gradient. However more recent studies found that protection was due to the expression of two receptors; invariant T cell and natural killer cell receptors on invariant natural killer T cells which is a population of lymphocytes. These T
cells are able to prevent T1D from developing spontaneously by recruiting and activating plasmacytoid dendritic cells to the pancreas. These dendritic cells control the replication of the virus thus inhibiting destruction of the pancreatic tissue, by producing IFN-alpha in large quantities. The dendritic cells then migrate to the lymph nodes of the pancreas and produces TGF-?. There the TGF-? is responsible for converting the naïve T cells to Tregs which move to the pancreas and produces TGF-?. The TGF-? suppresses anti-LCMV and anti-islet T cell response to thus inhibit pancreatic islet destruction and type 1 diabetes (Ghazarian, Diana, Simoni, Beaudoin and Lehuen 2012, 244). See appendix E for the mechanism of action of KRV.

The progression of Type I Diabetes in Human and Mouse models
The group of viruses highly thought to cause type 1 diabetes in humans is the Coxsachievirus B serotypes (CVBs) 1-6 which are members of the enterovirus family. Insulitis and islet cell damage have been found in infants that died of Coxsachievirus infection (Laitinen et al. 2013). Research from case reports and small patient series suggest that CVBs may include diabetogenic serotypes. However other viruses such as certain echovirus serotypes have also been linked to type 1 diabetes (Laitinen et al 2013; Tauriainen et al 2011; Al-Hello et al 2008). A study showed that CVB1 infections peaked in the few months before autoantibodies appeared. This corresponds to the rise in the frequency of enterovirus RNA in mice serum that has previously been observed along with islet antibody induction in enterovirus-infected mice (Laitinen et al 2013; Oikarinen et al 2013; Oikarinen et al 2011). Studies on patients with type 1 diabetes found the presence of enterovirus (EV) RNA and EV proteins in the pancreatic islets but the serotype of the involved enteroviruses has not been identified (Oikarinen et al 2013; Richardson et al. 2013; Richardson et al. 2009; Ylipaasto et al. 2004).

Innate Immunity of Virus-Induced Type 1 Diabetes
The CVBs enter islet pancreatic cells through their Coxsachie and adenovirus receptor (CAR), which is the major receptor for CVBs (Laitinen et al., 2013). Once inside the cells, the virus undergoes the viral replication cycle. It releases its single stranded RNA (ssRNA) which is replicated forming a double stranded RNA (dsRNA) intermediate using host cell machinery.

This dsRNA is a pathogen associated molecular pattern (PAMP) and is a strong trigger for pattern recognition receptors (PRRs) such as Retinoic acid- inducible gene-1-like receptors (RLRS) and Toll like receptors (TLRs). The RLR MDA5 recognises dsRNA from the picornavirus family which includes enteroviruses. When viral DNA binds to MDA5 this triggers NF-?B (Nuclear Factor Kappa light chain enhancer of B cells), IRF3-7 (IFN regulatory factors 3 and 7) and STAT (Signal Transducer and Activator of Transcription proteins) 1/2. Type 1 interferons (IFN-1? and ?) and chemokines are produced by the signaling cascade (Todd 2010).

Both dsRNA and ssRNA stimulate the endosomal TLRs 3 and 7. TLR3 binds short dsRNA segments. It recognises the dsRNA imitator polyinosinic:polycytidylic (poly I:C). This molecule may either protect or induce and increase the severity of T1D depending on the dose in mouse studies. A study using mice expressing lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) found that IFN-? is produced by TLR3 stimulation after LCMV infection.TLR7 binds ssRNA and was also shown to produce IFN-? after LCMV infection. In NOD (non-diabetic obese) mice TLR7 stimulation in conjunction with CD40 activation of dendrictic cells (DCs) is capable of activating diabetogenic Cytotoxic T lymphocytes (CTLs) in pancreatic lymph nodes to induce autoimmunity (Morse and Horwitz 2017; Lang et al. 2005; Lee et al. 2011.

This IFN-? production increases the expression of MHC class 1 receptors on the surface of the ?-cells of the pancreas. The type 1 IFNs produced lead to an inflamed, antiviral state. They trigger the Protein Kinase R (PKR) pathway to prevent viral replication and activate cellular immunity such as Natural killer (NK) cells and cytotoxicity and phagocytosis of macrophages.

Adaptive Immunity of Virus-Induced Type 1 Diabetes

The MHC class I proteins present the antigen to CD8+ T cells which become CTLs upon contact with their recognised antigen. They destroy the infected islet cells along with NK cells. The CTLs and NK cells release IFN-?. IFN-? has the effect of possibly upregulating MHC class I and II receptors, enabling macrophage activation, and increasing leukocyte entry into tissues by inducing chemokines and adhesion molecules(Walker and Herrath 2015; Schroder et al. 2004).
IFN-? binds to Interferon gamma receptors (IFNGRs) and initiates a signalling pathway that may be the cause of ? cell death by apoptosis (Walker and Herrath 2015). A virus-infuced model showed that IFN-? was required for diabetes (Walker and Herrath 2015; von Herrath and Oldstone 1997). IFN-? promotes an IgG class switch in B cells and promotes Th1 cells while inhibiting Th2 cells. See appendix F for the signalling cascades in ? cells upon CVB infection.

Antigen Presenting Cells (APCs) with HLA class II receptors bind peptides for T lymphocyte recognition. The autoantigens are preproinsulin (PPI) the most important, insulinoma-associated antigen 2 (I-A2), glutamic acid decarboxylase (GAD) and zinc transporter (ZnT8).
CD4+ cells bind these HLA class II in the periphery and islets and are stimulated to proliferate forming helper T cell subsets (Todd 2010). Evidence has shown that CD4+ and CD8+ likely both play a role in the immune response that drives type I diabetes (T1D). The T helper subsets formed by HLA class II binding are responsible for CD8 T cell activation (Walker and Herrath, 2015; Todd 2010; Bevan 2004).

Th1 cells produce IFN-?. Th2 cells may also play a role in ? cell death as low IL-10 production may be predisposing (Todd, 2010; Barrett et al. 2009, Kurreeman et al. 2004, Walker and Herrath, 2015). Peripheral blood T cells analysis from newly diagnosed individuals and control individuals found that persons with type I diabetes were more likely to have T cells that are antigen specific producing IFN-? alone, or an IFN-? and IL-10 response. Non-diabetic subjects produced IL-10 alone (Walker and Herrath 2015; Arif et al. 2004). The increase in IFN-? mRNA detected in newly diabetic individuals blood samples compared with at-risk individuals was also seen in increased IL-4 and IL-10 (Walker and Herrath, 2015; Han et al. 2011). It is not yet known whether Th1, Th2 or both are the cause of ? cell destruction (Walker and Herrath, 2015)

Th17 cells produce IL-21 which enhances Th17 cell differentiation. IL-21 is a pleiotropic cytokine; it is necessary for CD8+ T cells to have IL-21 receptors (IL-21Rs) for diabetes to develop in mouse models.In CD4+ T cells IL-21 promotes cell survival and resistance to Treg cells.CD4+ T cell activation ability is increased in macrophages. B cells are promoted to plasma cells.Pancreatic dendritic cells require IL-21R signals to acquire MHC class II receptors and to migrate to draining lymph nodes (Rodriguez-Calvo and Herrath, 2015). T cells that produce IL-21 also produce high levels of IFN-? and TNF-? (Rodriguez-Calvo and Herrath, 2015). See appendices G and H for the pleiotropic effects of IL-21 and the cytokines produced by T cells respectively.

Development of Type 1 Diabetes
Autoantibodies produced by plasma cells such as islet cell antibodies (ICA) and antibodies to insulin (IAA) cause an attack on the ? cells by autoreactive T cells. These T cells infiltrate the islets, a key feature of TID, causing insulitis (Laitinen et al., 2013, Walker and Herrath, 2015, Rodriguez and Herrath, 2015). The death of ? cells continues as the virus has either “hit and run”, or is evading the immune response “hit and stay”. In a hit and run strategy the virus has already infected cells and been removed from the body and therefore traces are not detectable.
In the hit and stay strategy, multiple same genus viral strains or different viruses that trigger common pathways might be involved. Multiple viral hits from different viruses may be required to sufficiently impair ?-cell function. Some viral infections may have a protective role against autoimmunity (Rodriquez-Calvo and Herrath 2015; Chapman et al. 2012).

Recent studies have proven that enteroviruses, more specifically, coxsackieviruses can exist in the pancreas of NOD mice as terminally deleted forms with slower replication and no evident cytopathy for up to 35 days after immunisation (Rodriguez-Calvo and Herrath 2015; Tracy et al. 2015). Some Coxsachievirus virus serotypes such as CVB1 and CVB6 have been found to have weak antibody responses (Oikarinen et al. 2013).

A study by Beeck and Eizirik 2016, found that the ? cells do not have lack less efficient antiviral mechanisms than ? cells and so they are not able to clear viral infections without undergoing apoptosis. Owing to this, chronically infected ? cells become a target for autoimmune responses in type 1 diabetes. The increased ? cell death is caused by the accumulative effect of antigen presentation by dendritic cells and macrophages to CD4+ cells by MHC class II receptors. These CD4+ cells license CD8+ cells for activation which are thought to be the indirect killers of ? cells. The antibodies secreted by homed plasma cells for many months aid in the killing.

A combination of genetic factors and environmental factors (such as viral infection) contribute to the development of type 1 diabetes. It has been found that combinations of variations of the MHC class II receptors HLA-DQB1 gene, HLA-DQA1 genes and the HLA-DRB1 gene contribute to the development of type 1 diabetes. Persons most at risk of developing type diabetes have at their DR locus one copy of DR3 hapotype and one copy of DR4 haplotype in each cell (“HLA-DQB1 Gene – Genetics Home Reference” 2018). CTLs that recognize a part of the peptidase cleavage site and N-terminal signal peptide of PPI, bound to the HLA-A?0201 molecule have been characterized (Tom 2010; Skowera et al., 2008).

A recent study showed that marks of dedifferentiation in ? cells were seen following PolyI:C infection. This was independent of ? cell death, which confirms other recent data that enterorvirus infection leads to a loss of insulin production. The study proposed that viral infection within islets, could induce dedifferentiation in an autonomous paracrine fashion (Oshima et al. 2018; Gallagher et al. 2014).

Why study type 1 diabetes? Type 1 diabetes is an autoimmune disease in which pancreatic beta cells are destroyed by activated CD4+ and CD8+ T cells. The number of affected persons inflicted is expected to be 88.8 million by the year 2035. Reportedly there are a number of viruses that are associated with type 1 diabetes and animal models have been used to study the association of viruses with type 1 diabetes. How does type 1 diabetes develop? The mechanism of induction of type 1 diabetes in humans involves both the innate and adaptive arm of the immune system. What are current therapies used to treat the disease? Therapy currently involves the lifelong administration of insulin by injection and insulin pumps. What are the possibilities for future research? Once the causative virus has been identified a cure for type 1 diabetes will be on the horizon. A vaccine can also be developed.

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