Human Protoparvoviruses

Next-generation sequencing and metagenomics have revolutionized the discovery of novel viruses. In recent years, three novel protoparvoviruses have been discovered in fecal samples of humans: bufavirus (BuV) in 2012, tusavirus (TuV) in 2014, and cutavirus (CuV) in 2016. BuV has since been studied the most, disclosing three genotypes that also represent serotypes. Besides one nasal sample, BuV DNA has been found exclusively in diarrheal feces, but not in non-diarrheal feces, suggesting a causal relationship. According to both geno- and seroprevalences, BuV appears to be the most common of the three novel protoparvoviruses, whereas TuV DNA has been found in only a single fecal sample, with antibody detection being equally rare. Moreover, the TuV sequence is closer to those of non-human protoparvoviruses, and so the evidence of TuV being a human virus is thus far insufficient. Interestingly, besides in feces, CuV has also been detected in skin biopsies of patients with cutaneous T-cell lymphoma and a patient with melanoma, while all other skin samples have tested PCR negative. Even if preliminary disease associations exist, the full etiological roles of these viruses in human disease are yet to be resolved.Peer reviewe

ICTV Virus Taxonomy Profile : Parvoviridae

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.Non peer reviewe

Multiplex detection in tonsillar tissue of all known human polyomaviruses

Background: In the past few years, eleven new human viruses have joined the two previously known members JCPyV and BKPyV of the Polyomaviridae family, by virtue of molecular methods. Serology data suggest that infections with human polyomaviruses (HPyVs) occur since childhood and the viruses are widespread in the general population. However, the viral persistence sites and transmission routes are by and large unknown. Our previous studies demonstrated that the four new HPyVs KIPyV, WUPyV, MCPyV and TSPyV were present in the tonsils, and suggested lymphoid tissue as a persistent site of these emerging human viruses. We developed a Luminex-based multiplex assay for simultaneous detection of all 13 HPyVs known, and explored their occurrence in tonsillar tissues of children and adults mostly with tonsillitis or tonsillar hypertrophy. Methods: We set up and validated a new Luminex-based multiplex assay by using primer pairs and probes targeting the respective HPyV viral protein 1 (VP1) genes. With this assay we tested 78 tonsillar tissues for DNAs of 13 HPyVs. Results: The multiplex assay allowed for simultaneous detection of 13 HPyVs with high analytical sensitivity and specificity, with detection limits of 10(0)-10(2) copies per microliter, and identified correctly all 13 target sequences with no cross reactions. HPyV DNA altogether was found in 14 (17.9%) of 78 tonsils. The most prevalent HPyVs were HPyV6 (7.7%), TSPyV (3.8%) and WUPyV (3.8%). Mixed infection of two HPyVs occurred in one sample. Conclusions: The Luminex-based HPyV multiplex assay appears highly suitable for clinical diagnostic purposes and large-scale epidemiological studies. Additional evidence was acquired that the lymphoid system plays a role in HPyV infection and persistence. Thereby, shedding from this site during reactivation might take part in transmission of the newly found HPyVs.Peer reviewe

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors. IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.Peer reviewe

Bones hold the key to DNA virus history and epidemiology

DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970's, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20th century and provides the first evidence of the suitability of bone for exploration of DNA viruses.Peer reviewe

Microsphere-based antibody assays for human parvovirus B19V, CMV and T. gondii

Background: Human parvovirus B19 (B19V), cytomegalovirus (CMV) and Toxoplasma gondii (T. gondii) may cause intrauterine infections with potentially severe consequences to the fetus. Current serodiagnosis of these infections is based on detection of antibodies most often by EIA and individually for each pathogen. We developed singleplex and multiplex microsphere-based Suspension Immuno Assays (SIAs) for the simultaneous detection of IgG antibodies against B19V, CMV and T. gondii. Methods: We tested the performances of SIAs as compared to in-house and commercial reference assays using serum samples from well-characterized cohorts. Results: The IgG SIAs for CMV and T. gondii showed good concordance with the corresponding Vidas serodiagnostics. The B19V IgG SIA detected IgG in all samples collected >10 days after onset of symptoms and showed high concordance with EIAs (in-house and Biotrin). The serodiagnostics for these three pathogens performed well in multiplex format. Conclusions: We developed singleplex and multiplex IgG SIAs for the detection of anti-B19V,-CMV and -T. gondii antibodies. The SIAs were highly sensitive and specific, and had a wide dynamic range. These components thus should be suitable for construction of a multiplex test for antibody screening during pregnancy.Peer reviewe

Epidemiology of two human protoparvoviruses, bufavirus and tusavirus

Two human parvoviruses were recently discovered by metagenomics in Africa, bufavirus (BuV) in 2012 and tusavirus (TuV) in 2014. These viruses have been studied exclusively by PCR in stool and detected only in patients with diarrhoea, although at low prevalence. Three genotypes of BuV have been identified. We detected, by in-house EIA, BuV1-3 IgG antibodies in 7/228 children (3.1%) and 10/180 adults (5.6%), whereas TuV IgG was found in one child (0.4%). All children and 91% of the adults were Finnish, yet interestingly 3/6 adults of Indian origin were BuV-IgG positive. By competition EIA, no cross-reactivity between the BuVs was detected, indicating that the BuV genotypes represent distinct serotypes. Furthermore, we analysed by BuV qPCR stool and nasal swab samples from 955 children with gastroenteritis, respiratory illness, or both, and found BuV DNA in three stools (0.3%) and for the first time in a nasal swab (0.1%). This is the first study documenting the presence of BuV and TuV antibodies in humans. Although the seroprevalences of both viruses were low in Finland, our results indicate that BuV infections might be widespread in Asia. The BuV-specific humoral immune responses appeared to be strong and long-lasting, pointing to systemic infection in humans.Peer reviewe

Global Distribution of Human Protoparvoviruses

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.Peer reviewe

B-Cell Responses to Human Bocaviruses 1-4 : New Insights from a Childhood Follow-Up Study

Human bocaviruses (HBoVs) 1-4 are recently discovered, antigenically similar parvoviruses. We examined the hypothesis that the antigenic similarity of these viruses could give rise to clinically and diagnostically important immunological interactions. IgG and IgM EIAs as well as qPCR were used to study similar to 2000 sera collected from infancy to early adolescence at 3-6-month intervals from 109 children whose symptoms were recorded. We found that HBoV1-4-specific seroprevalences at age 6 years were 80%, 48%, 10%, and 0%, respectively. HBoV1 infections resulted in significantly weaker IgG responses among children who had pre-existing HBoV2 IgG, and vice versa. Furthermore, we documented a complete absence of virus type-specific immune responses in six viremic children who had pre-existing IgG for another bocavirus, indicating that not all HBoV infections can be diagnosed serologically. Our results strongly indicate that interactions between consecutive HBoV infections affect HBoV immunity via a phenomenon called "original antigenic sin", cross-protection, or both; however, without evident clinical consequences but with important ramifications for the serodiagnosis of HBoV infections. Serological data is likely to underestimate human exposure to these viruses.Peer reviewe

Epstein–Barr virus in bone marrow of rheumatoid arthritis patients predicts response to rituximab treatment

Objectives. Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients