**1.3.3 Bacterial colonization and virus infection in the intestines**

Another interesting possibility is that the age at infection makes a difference in the pathology. In a similar fashion, it has been shown that colonization of the gut and intestines in early infancy by bacteria plays a role in the development of the adaptive immune system and structural development of the gut (Strachan, 1989). It is well known that due to improved hygiene some viral infections that would normally occur in early life are encountered for the first time at a later stage. For example, mononucleosis is associated with late infection of Epstein-Barr virus (EBV) (Pohl, 2009). Mononucleosis is rare in third world countries. Similarly, hepatitis A and B are less likely to cause disease if exposed to at an early age, and chickenpox (caused by varicella-zoster virus) is more severe in adults. Apparently, late infections typically give rise to a more severe pathology and concomitant increased activation of the immune system. The increased activation of the immune system may dispose for the establishment of an autoimmune condition. This hypothesis would explain the apparent conflict in data indicating that viral infections may confer protection and susceptibility. The MIDIA study offers a unique possibility to test this hypothesis.

### **1.4 Viral infections as triggers of type 1 diabetes**

Viral infections have long been considered as triggers of T1D, and there are several lines of evidence implying virus infections in utero or early life in the aetiology of T1D . The high frequency of T1D in children with congenital rubella syndrome was the first indication of a viral involvement, and hinted towards the importance of the intra-uterine environment (Menser et al., 1978). Intra-uterine rubella infection is now rare in Scandinavia due to vaccination, but the incidence of T1D is high and continues to rise. Mumps and measles were also suspected of playing a role in T1D (Vuorinen et al., 1992), and a plateau in T1D incidence in Finland was also noted after measles, mumps and rubella vaccine was introduced (Hyöty et al., 1993). Measles vaccination was also suggested to be protective in a Swedish study (Dahlquist et al., 1991). Another interesting observation is that acute viral infections can be associated with disease onset (Elfaitouri et al., 2007; Frisk et al., 1992; Osame et al., 2007). There is also a seasonal correlation between periods of viral infections

Genetic Testing of Newborns for Type 1 Diabetes Susceptibility – The MIDIA Study 343

study shows no difference in positivity for human parechovirus infections in stool samples (Tapia et al., 2010). Ljungan virus has earlier been shown to cause diabetes-like condition in rodents (Niklasson et al., 2006), but was not identified in samples from children in our studies (Tapia et al., 2008, Tapia et al., 2010). HPeV1 has been reported to show no association with T1D (Tauriainen et al., 2007), but there is no data on the other types of HPeVs. However, sequencing should be done to see if there is any difference in strains, and to test for all the new human Parechoviruses. Human parechoviruses seem to be asymptomatic, common viruses in childhood, and data on their epidemiology (and of other

The genus Cardiovirus consists of 2 species, encephalomyocarditis virus and theliovirus (Liang et al., 2008). Until recently only rodent cardioviruses have been known. Encephalomyocarditis virus has been shown to induce diabetes in mice, and thelioviruses have been associated with myocarditis, and MS like symptoms in mice (Chiu et al., 2008). Recently, human thelioviruses, termed Saffold virus 1-8, have been discovered (Drexler et al., 2008). There is little known about them, but a serological study shows that SAFV3 are ubiquitous and cause infection early in life (Zoll et al., 2009), but are apparently asymptomatic. Being a recently discovered virus, only a few studies have detected it with molecular methods (Abed & Boivin, 2008; Jones et al., 2007; Day, 2009; Coulson et al., 2002), so making assumptions about its epidemiology may be premature. Being in the picornaviridae family, and being closely related to rodent pathogens suggest they might be unknown human pathogens, and they should be studied both to get a clear picture of infections in early childhood and if there is any association with T1D in humans. The MIDIA

The family reovirus consists of six genera. Two of these genera, rotavirus and reovirus, have been shown to infect -cells, and are of interest to the project. They are double-stranded RNA viruses, also known to be common and ubiquitous (Honeyman et al., 1998). Rotaviruses are the single most important cause of severe diarrheal illness in infancy in both the developed and undeveloped world. They are spread through the fecal-oral route. Studies in mice have shown infection of -cells (Comins et al., 2008) indicating molecular mimicry (Toniolo et al., 1980). These viruses are being studied by a post doc. in the MIDIA

The name reovirus is a derivation of respiratory enteric orphan viruses, acknowledging that they can infect the respiratory and gastrointestinal system, but are not associated with any known disease (Wetzel et al., 2006). They are generally regarded as benign, but have also been associated with symptoms. In mice, they have been shown to infect -cells, but have also been shown to delay overt diabetes in mice. Screening longitudinally collected stool samples for these viruses will show whether they are associated with any disease in human

Our results show that infections in early life are much more prevalent and asymptomatic than previously known, but also highlight the need for more studies on viral infections in

common asymptomatic viruses) will be used to test the hygiene hypothesis.

study offers an excellent opportunity to study these viruses.

project, and we will pool our data to study the infections in childhood.

infants, and also be used to test the hygiene hypothesis.

**1.4.3 Cardiovirus** 

**1.4.4 Reovirus** 

**1.4.5 Other viruses** 

and onset of T1D (Jun & Yoon, 2003; Richer & Horwitz, 2003). Several viruses have been implicated as having an association with T1D, amongst them members of the picornaviridae and other viruses.

#### **1.4.1 Mechanisms proposed for viral triggering of autoimmunity**

There is unfortunately limited data on viral infections in young children (and infection load in early life), but there are several proposed mechanisms for how viruses might be associated with T1D. Viruses can activate polyclonal cells and trigger production of autoantibodies (Hiemstra et al., 2001), viruses can directly infect and lyse cells, viral antigens might mimic self-antigens, inflammatory responses stemming from viral infections might trigger autoimmunity (Horwitz et al., 1998), or as predicted in the hygiene hypothesis, viruses might be needed for proper maturation and regulation of the immune response.

#### **1.4.2 Picornavirus**

Picornaviruses are small RNA viruses that replicate mainly in the gut, and are spread by the fecal-oral route. The family has several well-known human and animal pathogens, but also many viruses without any known pathology. Human picornaviruses are known to be mostly asymptomatic and are common in infancy, with a prevalence of 10-12% in stool samples for human enterovirus (Cinek et al., 2006), human parechovirus (Tapia et al., 2008) and cardiovirus (Blinkova et al., 2009). The picornaviridae family currently consists of 8 current genera and 4 proposed genera.

The enterovirus genus consists of ten species, with six of them having human hosts (human enteroviruses A-D, rhinovirus A-B). Human enteroviruses are the most promising candidates, with two case-control studies that have shown an association between maternal enterovirus infection during pregnancy or enterovirus infection in children and risk of T1D (Dahlquist et al., 1995; Viskari et al., 2005). There are also studies that showed no association (Richer & Horwitz, 2009). In particular, Coxsackievirus (a member of human enterovirus B) is suspected of having a role in the development of autoimmunity (reviewed in Graves et al., 2003). In addition, enteroviruses have been shown to be more present in the sera (Elfving et al., 2008; Oikarinen et al., 2008), small intestine (Richardson et al., 2009) and pancreatic islets (Harvala & Simmonds, 2009) of recently diagnosed T1D patients (Clements et al., 1995; Andreoletti et al., 1997). A recent study by our group suggest that there is less enterovirus infections among children with high genetic risk for T1D compared with control children, although the difference is not statistically significant due to the low number of children presently tested (Tapia et al., 2011). Moreover, there appear to be a higher prevalence of enterovirus infections during early life in children who do not develop autoimmunity later, suggesting that enteroviral infections confer a protective effect against the development of autoimmunity (Wolthers et al., 2008). The parechovirus genus consists of two species, the murine virus Ljungan virus (LV) and human parechovirus (HPeV). Human parechovirus 1 and 2 have been known since the 1960s, and were originally classified with the enteroviruses as echovirus 22 and 23. Several new parechoviruses have recently been reported, with HPeV3-8 being described from 2004 to 2009 and HPeV9-14 recently announced. They are common in children, uncommon in adults and are present worldwide. Our previous data show that human parechoviruses are present in approximately 12% of stool samples from infants without causing symptoms (Tapia et al., 2008). There are, however, studies linking them to several serious conditions. Our most recent case-control study shows no difference in positivity for human parechovirus infections in stool samples (Tapia et al., 2010). Ljungan virus has earlier been shown to cause diabetes-like condition in rodents (Niklasson et al., 2006), but was not identified in samples from children in our studies (Tapia et al., 2008, Tapia et al., 2010). HPeV1 has been reported to show no association with T1D (Tauriainen et al., 2007), but there is no data on the other types of HPeVs. However, sequencing should be done to see if there is any difference in strains, and to test for all the new human Parechoviruses. Human parechoviruses seem to be asymptomatic, common viruses in childhood, and data on their epidemiology (and of other common asymptomatic viruses) will be used to test the hygiene hypothesis.

#### **1.4.3 Cardiovirus**

342 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

and onset of T1D (Jun & Yoon, 2003; Richer & Horwitz, 2003). Several viruses have been implicated as having an association with T1D, amongst them members of the picornaviridae

There is unfortunately limited data on viral infections in young children (and infection load in early life), but there are several proposed mechanisms for how viruses might be associated with T1D. Viruses can activate polyclonal cells and trigger production of autoantibodies (Hiemstra et al., 2001), viruses can directly infect and lyse cells, viral antigens might mimic self-antigens, inflammatory responses stemming from viral infections might trigger autoimmunity (Horwitz et al., 1998), or as predicted in the hygiene hypothesis, viruses might be needed for proper maturation and regulation of the immune response.

Picornaviruses are small RNA viruses that replicate mainly in the gut, and are spread by the fecal-oral route. The family has several well-known human and animal pathogens, but also many viruses without any known pathology. Human picornaviruses are known to be mostly asymptomatic and are common in infancy, with a prevalence of 10-12% in stool samples for human enterovirus (Cinek et al., 2006), human parechovirus (Tapia et al., 2008) and cardiovirus (Blinkova et al., 2009). The picornaviridae family currently consists of 8

The enterovirus genus consists of ten species, with six of them having human hosts (human enteroviruses A-D, rhinovirus A-B). Human enteroviruses are the most promising candidates, with two case-control studies that have shown an association between maternal enterovirus infection during pregnancy or enterovirus infection in children and risk of T1D (Dahlquist et al., 1995; Viskari et al., 2005). There are also studies that showed no association (Richer & Horwitz, 2009). In particular, Coxsackievirus (a member of human enterovirus B) is suspected of having a role in the development of autoimmunity (reviewed in Graves et al., 2003). In addition, enteroviruses have been shown to be more present in the sera (Elfving et al., 2008; Oikarinen et al., 2008), small intestine (Richardson et al., 2009) and pancreatic islets (Harvala & Simmonds, 2009) of recently diagnosed T1D patients (Clements et al., 1995; Andreoletti et al., 1997). A recent study by our group suggest that there is less enterovirus infections among children with high genetic risk for T1D compared with control children, although the difference is not statistically significant due to the low number of children presently tested (Tapia et al., 2011). Moreover, there appear to be a higher prevalence of enterovirus infections during early life in children who do not develop autoimmunity later, suggesting that enteroviral infections confer a protective effect against the development of autoimmunity (Wolthers et al., 2008). The parechovirus genus consists of two species, the murine virus Ljungan virus (LV) and human parechovirus (HPeV). Human parechovirus 1 and 2 have been known since the 1960s, and were originally classified with the enteroviruses as echovirus 22 and 23. Several new parechoviruses have recently been reported, with HPeV3-8 being described from 2004 to 2009 and HPeV9-14 recently announced. They are common in children, uncommon in adults and are present worldwide. Our previous data show that human parechoviruses are present in approximately 12% of stool samples from infants without causing symptoms (Tapia et al., 2008). There are, however, studies linking them to several serious conditions. Our most recent case-control

**1.4.1 Mechanisms proposed for viral triggering of autoimmunity** 

and other viruses.

**1.4.2 Picornavirus** 

current genera and 4 proposed genera.

The genus Cardiovirus consists of 2 species, encephalomyocarditis virus and theliovirus (Liang et al., 2008). Until recently only rodent cardioviruses have been known. Encephalomyocarditis virus has been shown to induce diabetes in mice, and thelioviruses have been associated with myocarditis, and MS like symptoms in mice (Chiu et al., 2008). Recently, human thelioviruses, termed Saffold virus 1-8, have been discovered (Drexler et al., 2008). There is little known about them, but a serological study shows that SAFV3 are ubiquitous and cause infection early in life (Zoll et al., 2009), but are apparently asymptomatic. Being a recently discovered virus, only a few studies have detected it with molecular methods (Abed & Boivin, 2008; Jones et al., 2007; Day, 2009; Coulson et al., 2002), so making assumptions about its epidemiology may be premature. Being in the picornaviridae family, and being closely related to rodent pathogens suggest they might be unknown human pathogens, and they should be studied both to get a clear picture of infections in early childhood and if there is any association with T1D in humans. The MIDIA study offers an excellent opportunity to study these viruses.

#### **1.4.4 Reovirus**

The family reovirus consists of six genera. Two of these genera, rotavirus and reovirus, have been shown to infect -cells, and are of interest to the project. They are double-stranded RNA viruses, also known to be common and ubiquitous (Honeyman et al., 1998). Rotaviruses are the single most important cause of severe diarrheal illness in infancy in both the developed and undeveloped world. They are spread through the fecal-oral route. Studies in mice have shown infection of -cells (Comins et al., 2008) indicating molecular mimicry (Toniolo et al., 1980). These viruses are being studied by a post doc. in the MIDIA project, and we will pool our data to study the infections in childhood.

The name reovirus is a derivation of respiratory enteric orphan viruses, acknowledging that they can infect the respiratory and gastrointestinal system, but are not associated with any known disease (Wetzel et al., 2006). They are generally regarded as benign, but have also been associated with symptoms. In mice, they have been shown to infect -cells, but have also been shown to delay overt diabetes in mice. Screening longitudinally collected stool samples for these viruses will show whether they are associated with any disease in human infants, and also be used to test the hygiene hypothesis.

#### **1.4.5 Other viruses**

Our results show that infections in early life are much more prevalent and asymptomatic than previously known, but also highlight the need for more studies on viral infections in

Genetic Testing of Newborns for Type 1 Diabetes Susceptibility – The MIDIA Study 345

significant effect on symptoms of anxiety or other mental health disorders as result of the testing, though a few mothers did seem to react strongly. Previous studies were conducted in a setting in which the mothers were asked questions about it in connection with the genetic testing project. The MIDIA study was designed differently. When completing the questionnaire the mothers were not aware that their answers were going to be used for any particular comparisons, though they were rightfully informed that the personal data would be used for multiple research purposes. Thus, our results were not affected by reporting bias associated with maternal attitudes towards genetic risk information or other factors motivating to under- or over-report poor mental health. Since 50% of mothers who got their child tested for genetic high-risk for T1D also participated in the Norwegian Mother and Child Cohort (MoBa) study, all data used came from MoBa. In MoBa data was available both from the 30th week of pregnancy and when the child was 6 months of age. These data therefore permit to answer the main question to what extent receiving information about a

young child having high risk for T1D changes maternal well being and health.

The MIDIA study is a longitudinal cohort study with inclusion of children with the highrisk HLA genotype (DRB1\*04:01-DQA1\*03-DQB1\*03:02/DRB1\*03-DQA1\*05-DQB1\*02), with follow-up from three months of age up to 15 years of age. Recruitment to MIDIA started in small scale in the summer of 2001, covered the whole country of Norway from March 2006 (60,000 births per year) and was stopped in December 2007 since it was suddenly found to be against the Norwegian Biotechnology Law. Both approvals from the Regional Medical Committee and the Norwegian Data Inspectorate had been given before recruitment to MIDIA started. In December 2007 close to 48,000 children were recruited to MIDIA. Of those 1,047 were identified with the high-risk genotype. Approval from the government was given for further follow-up of those already identified with the high-risk genotype. At the end of March 2011, 19 of these children had got Type 1 Diabetes, 33 were confirmed positive for two or three autoantibodies and 24 for one. A total of 4,829 blood samples, 18,275 stool

samples and 4,412 questionnaires are presently available for analysis in the cohort.

A questionnaire summarizing weekly diaries was filled out at 3, 6, 9 and 12 months of age. Blood samples were taken at the same intervals. After this period, a questionnaire and a blood sample are asked for annually (Stene et al., 2007). For more information on MIDIA, see www.fhi.no/midia. In The Norwegian Mother and Child Cohort Study (MoBa), questionnaires have been asked for at 17th, 22nd and 30th week of pregnancy, and when the child is 6 and 18 months old as well as then the child get 3, 5, 7 years of age (Magnus et al., 2006) . Blood samples were asked for at 17th week of pregnancy and at the time of delivery from the mother and cord blood was taken from the baby (Rønningen et al., 2006). For more

The incoming blood samples in the MIDIA study are immediately tested at the Hormone Laboratory, Aker Hospital, for diabetes associated autoantibodies as marker of -cell autoimmunity, autoantibodies against insulin, anti-glutamic acid decarboxylase (GAD), and against the protein tyrosine kinase related protein IA-2 (Petersen et al., 1994; Bingley et al., 2001). High titres of one autoantibody or titres above the cut-off for two or three

**2. Material and methods** 

**2.1 Research design and subjects** 

information on MoBa, see www.fhi.no/morogbarn.

**2.2 Outcome measurements** 

children. There are several viruses that are considered common in childhood that should be studied to test the hygiene hypothesis, and viruses that have been implicated with T1D, or human strains of animal viruses associated with T1D, should be studied. In addition, newly discovered viruses will also be evaluated as candidates for testing. However, these viruses will have a lower priority than the candidates listed above.
