Introduction
Human parvovirus B19V (B19V) is a DNA virus of the Parvoviridae family and genus Erythroparvovirus [
1]. B19V, an omnipresent pathogen, causing a broad spectrum of clinical manifestations such as childhood rash erythema infectiosum, arthralgias, and in pregnant women, it can cause fetal death (hydrops fetalis) [
2]. The infections of B19V are mild or asymptomatic and can lead to transient or persistent erythroid aplasia and aplastic crisis in people with underlying hematological disorders [
3]. Management of this viral infection is limited to symptomatic treatment sadly due to the lack of specific antiviral drugs/vaccines [
4,
5].
B19V virus has a single-stranded DNA genome of 5.6 kb, which codes for three proteins: a nonstructural protein (NS1) and two viral capsid proteins VP1 and VP2. VP2, a major capsid protein, has a pivotal role in the viral assembly of the B19 virus and is identified as an attractive molecular target for structure-based drug discovery [
6]. NS1 protein plays a significant role in viral replication, which is a pleiotropic nuclear phosphoprotein. It is a multi-functional protein that aids in cellular transcription, virus replication, cell death induction, and cellular promoters' transactivation [
7]. VP1, a minor capsid protein, has the same amino acid sequence as VP2, plus an additional 227 amino acids at the N-terminus called the VP1-unique region (VP1u). The VP1u exhibits relatively high sequence variability in persistently infected individuals and plays an essential role in eliciting specific immune responses [
8]. Substitution rates of B19V are unusually high, in the range of 1.0–4.0 × 10
−4 nucleotide substitutions per site per year, which is more similar to substitution rates of RNA viruses than either single-stranded or double-stranded DNA viruses [
9], which makes this virus unique.
The human B19V is classified into three distinct genotypes based on the NS1-VP1u region. Genotype 1 has a ∼10% sequence divergence from genotypes 2 and 3, and genotypes 2 and 3 have a ∼5% sequence divergence between each other [
10]. Genotypes 1 and 3 are further categorized into subtypes a and b with the divergence of about 5% [
11]. The distribution of the three genotypes is not spatially and temporally uniform: genotype 1 has a worldwide distribution [
12], Genotype 2 is found mainly in elderly adults in northern Europe [
13], and Genotype 3 is found in Sub-Saharan and West Africa, South America, and France [
14]. Many reports are available from distinct geographical areas, including Europe, Asia, South America, and Africa; reports on B19V genotypes from India is not yet available. Despite the recent reports of viral entry and genetic diversity, there is still a lack of sufficient data from distinct geographical locations to understand B19V evolution. Here, we performed assembly of B19V genome using metagenomics sequencing and studied sequence variation and the phylogenetic relationship of B19V isolated from South India to other global strains. Our study reports a distinct genotype for human B19V isolated from South India, with a 98.78% sequence similarity to currently circulating Genotype 2 strains. The study further helps to understand the nature of parvovirus evolution.
Discussion
Here we report a distinct genotype of the human B19 virus from South India through our screening, detection, sequencing, and phylogenetic analysis of the B19V genome from suspected clinical samples.
In our study, parvovirus B19 infection was shown among 33 of 456 patients when tested by nPCR; 30 among these were also positive by qPCR and were subsequently confirmed by sequencing 75% nPCR from patients with age. ≥ 50 years respectively (Additional file
1: Table S1). In terms of age, the prevalence of Parvovirus B19 infection is 15%, 50% and 85% in pre-school children, adults and elderlies, respectively [
32,
33].
Parvovirus B19 infection has spread across the globe and has been reported from all geographical regions. Different studies have emphasized the high prevalence of this disease than commonly estimated, and its seroprevalence can vary depending on age and geographical location [
34]. Another point to consider is that the earlier study was probably conducted in a period that did not include an epidemic year for B19V [
35,
36]. Therefore, the application of PCR has a higher diagnostic value and can help reach definite results. The present study's superiority is simultaneous conductance of PCR tests which can dramatically enhance the diagnostic power.
B19V genotypes and subtypes have a high rate of synonymous to non-synonymous nucleotide changes per site, suggesting that NS1 and VP1/2 regions are under strong purifying selection [
37]. Other specific mutations (V21T, Q220E, T227S, N323S, M389I, S32T) were seen in the South-Indian strain, which is also present in Genotype 1,2 and 3 strains. These above-mentioned mutations might significantly influence other vital residues of the protein; for example, the previously reported GH loop (residues 298–328) is essential for the immunodominance properties of parvovirus [
38]. In the residues of an above-stated loop that governs immunodominance in human B19V, residual mutation N323S was observed. This substitution of Asparagine (Asp) to Serine (Ser) at position 323 may be resulting in sustained angiogenesis and cell proliferation [
38].
Scientists have found a very high rate of nucleotide substitutions comparable to the substitution rates of ssRNA viruses and, on average, 1–2 × 10
−4 substitutions/site/year [
39]. This significantly high mutation rate might contribute to an efficient adaptive nature of the virus, and thus, for their wide variety of cellular tropism in humans. Therefore, this virus is thought to be evolved rapidly. But a recent publication has shown the existence of B19 in the human population present around 7000 years ago [
40]. Therefore, the study shows that the virus had a much lower mutation rate in its long-ago ancestors. Though the reason behind the acceleration in the mutation rate is not known, it is inferred that the virus is going through a rapid change in recent times. The small peptide of 60–100 amino acids from the VP-1 protein sequence has the maximum antibody-producing ability and can be used as a peptide vaccine [
41]. These higher substitution rates might affect the virus' tropism for different host cells and account for its persistence; thereby affecting the vaccination strategies against it.
The novel mutations we observed in our study may significantly affect selection pressure and exemplifies the crucial role of VP1 protein in the viral life cycle, especially the enzymatic function of B19V phospholipase A2 (vPLA2) with diversifying substitutions, critical for long-term persistent infection. One of the novel mutations, I283V in the South Indian B19 isolate, was seen closer to its known antigenic determinant sites. This might help the virus evade host neutralizing antibodies. However, in our study, there were positive and negative selected sites; this could be either the inherent nature of the strains or the result of a smaller number of samples sequenced. A further diverse set of samples needs to be analyzed, and in-depth studies requires to be performed on a more extensive collection of samples.
Acknowledgements
The authors would like to acknowledge the clinicians and the patients for their cooperation for support in every possible way. We also thank Dr. Lakshmi Soundararajan and Dr Malini, MedGenome Labs Ltd., Bangalore, India for helping viral whole genome sequencing.
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