Pathogens might affect behavior of infected reservoir hosts and hence their trappability, which could bias population estimates of pathogen prevalence. In this study, we used snap-trapping data on Puumala orthohantavirus (PUUV)-infected (n = 1619) and noninfected (n = 6940) bank voles (Myodes glareolus) from five vole cycles, normally representing increase, peak, and decline phase, to evaluate if infection status affected trapping success. If PUUV infection, as previously suggested, increases activity and/or mobility, we would expect a higher proportion of infected than noninfected specimens in the first trapping night. However, the proportion of PUUV-infected voles did not differ across the three trapping nights. We conclude that PUUV infection did not affect trapping success, confirming snap trapping as an appropriate trapping method for studies on PUUV prevalence and likely other orthohantaviruses.

Parkinson's disease (PD) is a neurodegenerative disease linked with the loss of dopaminergic neurons in the brain region called substantia nigra and caused by unknown pathogenic mechanisms. Two currently recognized prominent features of PD are an inflammatory response manifested by glial reaction and T-cell infiltration, as well as the presence of various toxic mediators derived from activated glial cells. PD or parkinsonism has been described after infection with several different viruses and it has therefore been hypothesized that a viral infection might play a role in the pathogenesis of the disease. We investigated formalin-fixed post-mortem brain tissue from 9 patients with Parkinson's disease and 11 controls for the presence of Ljungan virus (LV) antigen using a polyclonal antibody against the capsid protein of this recently identified picornavirus with neurotropic properties, suspected of being both a human and an animal pathogen. Evidence of viral antigen was found in 7 out of 9 Parkinson's disease cases and in only 1 out of 11 controls (p = 0.005). The picornavirus antigen was present in dopamine-containing neurons of the substantia nigra. We propose that LV or an LV-related virus initiates the pathological process underlying sporadic PD. LV-related picornavirus antigen has also been reported in patients with Alzheimer's disease. Potentially successful antiviral treatment in Alzheimer's disease suggests a similar treatment for Parkinson's disease. Amantadine, originally developed as an antiviral drug against influenza infection, has also been used for symptomatic treatment of patients with PD for more than 50 years and is still commonly used by neurologists today. The fact that amantadine also has an antiviral effect on picornaviruses opens the question of this drug being re-evaluated as potential PD therapy in combination with other antiviral compounds directed against picornaviruses.

We have earlier reported the finding of picornavirus antigen in amyloid plaques in the hippocampus of Alzheimer’s disease (AD) patients. As a result of this finding, three AD patients received antiviral therapy directed against picornavirus infection. Pleconaril, ribavirin and efavirenz were used in different combinations over the 4-8 year intervals of treatment. Patients were followed using the Mini Mental State Examination score and the Alzheimer’s Quick Test. Results including mostly unchanged cognitive function in two patients and a clear improvement in one patient are in contrast with the expected progression of AD.

We investigated formalin-fixed postmortem brain tissue from the hippocampus region of 18 AD cases and 11 age-matched controls using a polyclonal antibody against Ljungan virus (LV) capsid protein 1. Evidence of a LV antigen was found in all AD cases but in none of the control specimens (p < 0.0001). The antibodies reacted with neurons and astrocytes and also showed distinct positive reaction in the amyloid/neuritic plaques. The possible role of an incompletely characterized picornavirus as the etiologic agent in AD open up the possibility of treatment with antiviral therapy directed against picornaviruses. The positive result of such treatment in a small number of patients is presented separately back to back to this report.

We found an association between the abundance of rodents in the wild and onset of type 1 diabetes (T1D) in humans. A picornavirus named Ljungan virus (LV) was subsequently isolated from wild bank voles. Both picornavirus-like particles detected by electron microscopy and LV antigen visualized by immunohistochemistry was seen in islets of Langerhans in diabetic wild bank voles. LV antigen has also been found in islets of Langerhans in a patient with recent onset of T1D and in the commonly used Bio Breeding (BB) T1D rat model. We discuss the possibility of T1D and type 2 diabetes (T2D) as parts of a single disease entity. Antiviral compounds directed against picornavirus have been found to be an effective treatment of diabetes in BB rats. We propose using the same currently available antiviral compounds in clinical trials in humans. Antiviral treatment would have the potential to be both proof of concept for involvement of a picornavirus in diabetes pathogenesis and also present a first-generation therapy.

A Picornavirus (Ljungan virus [LV]) originally found in bank voles has been associated with type 1 diabetes (T1D) in its wild rodent reservoir, but also associated with T1D in a laboratory rat model for the disease, the diabetes prone (DP) Bio Breeding (BB) rat. Successful treatment of diabetes in this rat model, using experimental antiviral compounds directed against picornavirus, has been reported. In the present study we show significant clinical response in DP-BB rats using antiviral compounds available for human use (Pleconaril, Efavirenz, and Ribavirin). Presence of LV picornavirus antigen has been detected in islets of Langerhans from both human and the T1D rat model with clear morphological similarity. Based on these data it would be of interest to test antiviral treatment in patients with newly diagnosed T1D. Successful outcome will offer both proof of concept regarding the role of virus involvement in the disease and possibly a first generation treatment interrupting a persistent infection and stopping β-cell destruction.

A picornavirus (Ljungan virus) has been associated with diabetes in its wild rodent reservoir and in diabetes-prone biobreeding (DP-BB) rats. We attempted to alter the development of diabetes in DP-BB rats using two anti-picornavirus compounds (pleconaril and APO-N039), singly or in combination. Antiviral therapy was initiated 2 weeks before expected onset of diabetes. Pleconaril or APO-N039 alone did not affect the debut of diabetes. However, animals receiving a combination of both compounds were protected for at least the entire period of treatment (4 weeks after expected time of diabetes onset). Immunohistochemistry demonstrated that the presence and distribution of virus antigen in the pancreatic islets coincided with the clinical status of the animal. Data indicate that a treatable picornavirus can be involved in the cellular assault resulting in diabetes and in these cases the disease mechanism appears to involve a virus present in the pancreatic beta cell mass itself.

Ljungan virus (LV) (family Picornaviridae, genus Parechovirus) is a suspected zoonotic pathogen with associations to human disease in Sweden. LV is a single-stranded RNA virus with a positive sense genome. There are five published Ljungan virus strains, three isolated from Sweden and two from America, and are classified into four genotypes. A further two strains described here were isolated from wild bank voles (Myodes glareolus) caught in Västmanlands county, Sweden in 1994. These strains were sequenced using next generation pyrosequencing technology on the GS454flx platform. Genetic and phylogenetic analysis of the obtained genomes confirms isolates LV340 and LV342 as two new putative members of genotype 2 along with LV145SL, with 92% and 99% nucleotide identities respectively. Only two codon sites throughout the entire genome were identified as undergoing positive selection, both situated within the VP3 structural region, in or near to major antigenic sites. Whilst these two strains do not constitute new genotypes they provide evidence, though weakly supported, which suggests the evolution of Ljungan viruses to be relatively slow, a characteristic unlike other picornaviruses. Additional genomic sequences are urgently required for Ljungan virus strains, particularly from different locations or hosts, to fully understand the evolutionary and epidemiological properties of this potentially zoonotic virus.

Zoonoses are major contributors to emerging infectious diseases globally. Hemorrhagic fever with renal syndrome (HFRS) is a zoonosis caused by rodent‐borne hantaviruses. In Europe, Puumala hantavirus (PUUV) carried and shed by the bank vole ( Myodes glareolus ), is the most common cause of HFRS. We explore the relationship of PUUV infection in bank voles, as measured by PUUV antibody detection, with habitat and landscape scale properties during two successive vole cycles in boreal Sweden. Our analysis revealed that PUUV infection in the population was not uniform between cycles and across different landscapes. The mean density index of PUUV antibody positive and negative bank voles were highest in old forest, second highest in cut‐over forest (approx. 0–30 years old) and lowest on mires. Most importantly, old forest was the core habitat, where PUUV antibody positive bank voles were found through the low density phase and the transition between successive vole cycles. In spring, occurrence of antibody positive voles was negatively related to the proportion of cut‐over forest in the surrounding landscape, suggesting that large scale human induced land‐use change altered the occurrence of PUUV infection in voles which has not been shown before. Dependence of PUUV infection on habitat and landscape structure, and the variation in infection load within and between cycles are of importance for human risk assessment.

Ljungan virus is a recently identified member of the family Picornaviridae that was isolated from bank voles in Sweden. It has been shown to cause type 1 diabetes-like symptoms and myocarditis in bank voles (Myodes glareolus), and it has been suggested that it has zoonotic potential. Here, we show for the first time that Ljungan virus is prevalent (20–27 % positive by PCR) in four species of UK rodent (Myodes glareolus [bank vole], Apodemus sylvaticus [wood mouse], Microtus agrestis [field vole] and Mus musculus [house mouse]). Sequence analysis showed that Ljungan virus of genotypes 1 and 2 were present, although genotype 1 was more prevalent and more frequently associated with brain tissue. This study highlights the prevalence of Ljungan virus in the UK and the need for confirmation of its zoonotic potential.

Tick-borne encephalitis (TBE) is a zoonotic disease, transmitted mainly by the bite of ticks. The TBE virus (TBEV) belongs to the family Flaviviridae, genus Flavivirus and is able to cause meningoencephalitis. For serological TBEV detection, the neutralization test (NT) is the most specific assay available. Different NT protocols are used in the laboratories, and until now the performance of these NTs has never been tested in an external quality assessment (EQA). In this EQA, we compared the results of eight European laboratories in detecting 17 samples (11 TBEV positive, five flavivirus cross reactive, and one negative sample) by NT. Furthermore, 14 of these EQA samples and 15 additional samples were tested in different commercial assays: 15 immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) and an immunofluorescence assay (IFA). Four laboratories showed a good NT EQA performance, whereas four laboratories had some sensitivity problems. Additionally, two of these laboratories showed a lack in specificity, misidentifying a dengue-positive sample as TBEV positive. The comparison of the commercial ELISAs revealed a high sensitivity in all assays, but as expected for IgG, the ELISAs showed a high degree of flavivirus cross reactivity. The assessment of Vienna Units in some of the ELISAs revealed deviations in the standards used by the different companies. Therefore, these standards should be revised. Generally, in this EQA, we found that reliable NT protocols are used in most of the laboratories, and the evaluation of the IgG ELISAs and the IFA showed a good agreement.

Thurner et al. (1) identifies spikes in diabetes incidence using year-of-birth data from Austria over the last century. They intriguingly associate these peaks with intervals of nationwide famine with the disease onset apparently occurring decades later. The information provided by Thurner et al. is a valuable supplement to previous studies on the Dutch WWII famine that also demonstrate a relationship to later diabetes onset (2). Based on our studies of a rodent-borne Picornavirus*, we suggest an alternative interpretation involving an infectious contribution to the diabetes.

Hantaviruses are endemic throughout the world and hosted by rodents and insectivores. Two human zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are caused by hantaviruses and case fatality rates have reached 12% for HFRS and 50% for HPS in some outbreaks. Symptomatic hantavirus infections in Europe are summarised as HFRS mainly due to Puumala, Dobrava-Belgrade and Saaremaa virus. While HFRS has an overall low incidence in Europe, the number of cases varies from 100 per year in all Eastern and Southern Europe up to 1,000 per year only in Finland. To assess the quality of hantavirus diagnostics, the European Network for the Diagnostics of “Imported” Viral Diseases (ENIVD) organised a first external quality assurance (EQA) in 2002. The purpose of this second EQA study is to collect updated information on the efficiency and accurateness of hantavirus serological methods applied by expert laboratories. A serum panel of 14 samples was sent to 28 participants in Europe of which 27 sent results. Performance in hantavirus diagnosis varied not only on the method used but also on the laboratories and the subclass of antibodies tested. Commercial and in-house assays performed almost equally. Enzyme immunoassays were mainly used but did not show the best performances while immunoblot assays were the less employed and showed overall better performances. IgM antibodies were not detected in 61% of the positive IgM samples and IgM detection was not performed by 7% of the laboratories indicating a risk of overlooking acute infections in patients. Uneven performances using the same method is indicating that there is still a need for improving testing conditions and standardizing protocols.

Ljungan virus (LV) is a rodent pathogen that causes diabetes and myocarditis in its natural host. In addition, LV has been associated with human disease during pregnancy and of neonates, respectively. A panel of 22 monoclonal antibodies (mAbs) against first and second LV genotypes were produced by immunization of BALB/c mice with whole virus. Thirteen mAbs were class IgG antibodies and nine were class IgM antibodies; all of them contained kappa light chains. All mAbs were reactive with LV by capture enzyme-linked immunosorbent assay and indirect immunofluorescence assay. In addition, five mAbs showed a positive staining in immunohistochemistry. No mAb bound to denatured capsid proteins detected by western immunoblotting. In contrast, the target capsid protein(s) of 20 mAbs were identified by immune precipitation, revealing the conformational nature of epitopes required for mAb binding. None of the mAbs reacted with third and fourth LV genotypes. mAbs characterized should provide useful tools for the development of diagnostic assays and the investigation of LV first and second genotype properties and its pathogenesis.

AbstractThe Ljungan virus (LV), a member of the Parechovirus genus and Picornaviridae family was isolated originally from its wild reservoir, the bank vole (Myodes glareolus) at the Ljungan River in central Sweden [Niklasson et al., 1999]. LV is associated with diseases such as myocarditis, encephalitis, pregnancy related diseases, and diabetes in several species of wild rodent [Niklasson et al., 2006a]. The same outcomes can be induced in mice under controlled laboratory conditions [Niklasson et al., 2006b]. Several mouse and rat animal models used to study diabetes have been found to carry LV naturally [Niklasson et al., 2007]. The role of the viruses in diabetes pathogenesis has been investigated by treating these animals with antiviral compounds [Holmberg et al., 2009].Variation in the incidence of type 1 diabetes has been found to track the fluctuations in native rodent populations in central Sweden. Increased prevalence of antibodies to LV has been detected in Swedish type 1 diabetes cases. However, LV virus has not been isolated and its viral RNA has not been detected in type 1 diabetes. J. Med. Virol. 83:1673–1673, 2011. © 2011 Wiley‐Liss, Inc.

Bank voles develop glucose intolerance/diabetes mellitus when kept in captivity. We have characterized beta-cell function of glucose intolerant/diabetic animals, and found that this animal model has features of both human type 1 and type 2 diabetes. The aim of this study was to study the functional alterations of islets isolated from glucose tolerant bank voles after a prolonged exposure to various glucose concentrations in vitro. For this purpose, pancreatic islets from normal (glucose tolerant) male and female bank voles were cultured at different glucose concentrations (5.6, 11.1 (control), or 28 mM) whereupon islet functions were examined. Overall, islet insulin output was lowered at 5.6 mM glucose, and similar to control, or enhanced after culture in 28 mM glucose. High glucose culture led to decreased insulin contents, but there was no change in islet DNA content and in morphological assessments of cell death, with the latter findings suggesting that the so-called glucotoxicity had not evolved. A slight gender difference was observed in that islets isolated from females exhibited a glucose-regulated (pro)insulin biosynthesis rate and insulin gene expression. In conclusion, we have found that islets isolated from female and male bank voles are affected by glucose concentrations in vitro in that some signs of dysfunction were observed upon high glucose exposure. A minor gender difference was observed suggesting that the islets of the females may more readily adapt to the elevated glucose concentration than islets of the male bank voles. It could be that these in vitro gender differences observed may represent a mechanism underlying the gender difference in diabetes development observed among bank voles.