Browsing by Author "Jokinen, Maija"

Parvoviruses are among the smallest known viruses. The parvovirus genome is a single stranded DNA, approximately 5 kb in size. The virion has a small (20 to 30 nm), rugged, non-enveloped icosahedral capsid. Parvoviruses can cause a number of diseases. Possibly the most recognized human parvovirus is parvovirus B19 (B19V), which can cause the so-called fifth disease, anemias and fetal death. Another relatively well characterised parvovirus is human bocavirus 1 (HBoV1), which causes respiratory tract infections in young children. Bufavirus (BuV) tusavirus (TuV) and cutavirus (CuV) are emerging parvoviruses, discovered during the years 2012-2016 using next generation sequencing methods. All three viruses were originally discovered in feces of patients suffering from diarrhea. BuV was originally found in Burkina Faso and has since been detected in fecal samples with polymerase chain reaction (PCR)-based methods from Europe, Asia and Africa. The seroprevalence of BuV differs between countries. TuV was found in a single stool sample from Tunisia, but no further reports of it have since emerged. CuV was found in 2016 and it has been linked to cutaneous T-cell lymphoma, but it is not known if the virus is the cause of the cancer or if the virus simply prefers quickly dividing cancer cells for its replication. BuV, TuV and CuV belong to the Protoparvovirus genus, but it is still unclear whether TuV is a human pathogen. More research is needed to study the epidemiology of these viruses and their role in illnesses. There were two main aims in this thesis: to set up an IgM µ-capture enzyme immunoassay (EIA) for human protoparvoviruses using BuV1 as an example and to screen three stool sample cohorts for BuV, TuV and CuV using an in-house multiplex quantitative PCR (qPCR). The IgM EIAs developed for B19V and HBoV1 was used as the base for developing human protoparvovirus IgM EIA, using Virus-like particles (VLP) as antigens. Setting up the EIA required a great amount of optimization and finally troubleshooting, since the assay did not work as expected. The troubleshooting revealed that the ambiguous results in the IgM µ-capture EIA were possibly due to degraded VLPs or that the sensitive µ-capture format requires extremely carefully purified VLPs. More optimizing is needed for this assay, however, the work done in this thesis offers a good base for further development of protoparvovirus IgM EIA. All three viruses were found in the stool samples during multiplex qPCR screening. Based on the qPCR and sequencing results one sample was positive for BuV DNA, one sample for TuV DNA and a total of 12 samples for CuV DNA. This is the first time TuV DNA has been found since its discovery. In addition to that, CuV DNA was identified in fecal samples for the first time since the discovery, previously CuV DNA had been found mostly in skin biopsies. As for TuV, based on the parvovirus phylogenetic analyses, its sequence is more closely related to rodent parvoviruses than CuV or BuV. More research is needed, possibly with animal and human samples, to establish the role of TuV as a human virus.