Distribution and clinical associations of Ljungan virus (Parechovirus B)

Worldwide, infectious diseases are responsible for death of 15 million people annually with a significant impact on public health and economic growth. Many of these diseases are zoonotic, that is, transmitted from wild or domestic animals to humans. The incidence of zoonotic diseases is increasing mainly as a result of more intense and frequent contacts between humans and between humans and other animals. Zoonotic pathogens include viruses, bacteria, fungi, rickettsia, protists, prions and protozoa. Viruses, in particular, are capable of rapidly adapting to new hosts, with rodents as the primary reservoir. “Rodentborne” viral infection in humans occurs by direct contact with feces, saliva, and urine of infected rodents, or by inhalation of viral particles from aerosolized rodent excrement. Among rodent-borne viruses, those belonging to the genera Mammarenavirus, Orthohantavirus and Orthopoxvirus are a particular focus of study both in humans and animals, since they represent some of the most widespread rodent-borne disease-causing pathogens. More recently, the interest in parechoviruses has been increasing because some are known to cause diseases in humans, while others are carried by rodents, although the zoonotic potential of rodent-borne parechoviruses has not been established. Ljungan virus (LV), which belongs to the species Parechovirus B, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998. It belongs to the Picornaviridae family, which includes many viruses that infect humans and other animals. Currently, there is little information about LV host range and epidemiology, but a few reports suggest an association between LV and human disease. The main aims of this doctoral thesis were 1) to establish the symptoms associated with LV in humans, 2) to investigate the association of LV with human central nervous system (CNS) disease, and 3) to determine the prevalence and distribution of LV in human and other animal populations in Europe. LV-associated symptoms were investigated in two human cohorts. Serum samples from Finnish patients hospitalized for suspected nephropathia epidemica (NE) caused by the Orthohantavirus Puumala virus (PUUV) were screened for the presence of lymphocytic choriomeningitis virus (LCMV, Arenavirus), cowpox virus (CPXV, Orthopoxvirus) and LV, in order to compare the disease outcomes in these patients and to establish if the co-existence of viruses could lead to an increase in the severity of symptoms. However, no unusual or additional manifestations between PUUV cases and PUUV-LV/LCMV/CPXV cases were detected (I). To determine if LV (together with the rodent-borne virus LCMV) could be one of the causes of neurological symptoms in Finnish patients with suspected CNS disease, anti-LV and LCMV antibodies were analyzed from serum and cerebrospinal fluid samples. LV- and LCMV-specific nucleic acids were also analyzed from the patient samples. However, no association between LV or LCMV antibodies or nucleic acids and the neurological manifestations in the patient cohort was detected (III). In order to improve the knowledge of the host and geographical distribution of LV, tissues from multiple rodent and insectivore species from ten European countries were screened for LV nucleic acids (II; IV). We confirmed that LV is widespread geographically, having been detected in at least one host species in nine out of ten countries involved in the study. Seventeen out of 21 species screened were LV PCR-positive, and the virus was detected for the first time in the northern red-backed vole (Myodes rutilus) and the tundra vole (Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for LV (overall PCR-prevalence: 15.2%). Male and subadult bank voles are significantly more likely to be LV-positive, and the prevalence has a temporal pattern (higher in autumn compared to spring and summer), possibly due to adult bank voles clearing the infection. Interestingly, higher levels of precipitation (rain and snow) at any given time, are associated with a lower LV prevalence six months later. In conclusion, LV is widespread geographically and found in many hosts that are reservoirs for rodent-borne zoonotic pathogens. However, the seroprevalence of LV or an LV-like virus in humans is above 40% and higher in younger patients (confirmed in this study and by others) suggesting that LV- or an LV-like virus might be transmitted by an alternative route. Thus far, LV has not been isolated from humans and has not definitively been confirmed as an infectious agent in humans. Despite high seroprevalence found in patient cohorts, LV was not detected in association with human CNS disease, and did not seem to cause disease symptoms. Therefore, this study adds to the growing body of evidence that LV is unlikely to cause zoonotic or non-zoonotic disease. However, since LV has been associated with other non-CNS symptoms in rodents, whether LV or LV-like viruses are potential human pathogens deserve further investigation.