Human parvovirus 4 ‘PARV4’ remains elusive despite a decade of study

Population prevalence of infection in different geographic locations

The most substantial updates to the PARV4 literature are studies to determine the prevalence of infection in geographic areas that had not previously been represented. This includes new studies in Scandinavia^17,18, the Middle East¹⁹, and South America^20,21; we have summarised these data in Table 2. Below, we discuss these findings with further reference to their applicability to understanding risk factors for infection.

Risk factors for PARV4 infection

Based on the strong relationship between PARV4 and HIV, HBV, and HCV co-infection in North American and European populations, several studies have compared PARV4 exposure (screening for PARV4 IgG or DNA or both) in HIV-positive and HIV-negative groups in different settings. In Iran, HIV infection (with or without HCV co-infection) was reported to be associated with a high prevalence of PARV4 viraemia (35%)¹⁹. However, in this study, the background prevalence of viraemia in healthy blood donors was also high at 17%¹⁹. Higher seroprevalence was also reported in Scandinavian subjects in the setting of HIV infection; the PARV4 IgG detection frequency was 8% among HIV-positive children¹⁷, while the background population seropositivity rate was less than 1% in adults and absent in children^18,22. Stratification of risk factors in a French study reported the highest PARV4 prevalence in PWIDs, especially among those who were HIV-infected, but also among men who have sex with men (MSM), particularly those likely to have been exposed in the 1980s¹².

Our own study of a cohort of mothers and children in Kimberley, South Africa, was comparable to previous reports from sub-Saharan Africa in finding an overall PARV4 seroprevalence of 37% with no evidence that HIV infection was a risk factor²³. Although vertical transmission has previously been demonstrated in another setting²⁴, there was no concordance between maternal and child serostatus, suggesting that maternal transmission is probably not a major contributor to the overall burden of infection in South Africa²³. The clear trend towards increasing acquisition with age is consistent with a broader environmental source of PARV4 infection.

A potential association between PARV4 exposure and infection with another BBV, human T-cell lymphotrophic virus (HTLV), was described in a Brazilian study²¹. Among 113 subjects with HTLV-1 infection, seven (6%) were positive for PARV4 DNA, two of whom also had HIV or HCV infection or both. Based on a history of blood transfusion in one of these individuals, it was postulated that this represents the most likely route of acquisition of all three viruses. However, the remaining five cases had no other co-infecting BBVs and no history of injecting drug use or blood transfusion, leading to the conclusion that other transmission routes of PARV4 transmission may also be operating²¹. Another study published by the same group identified PARV4 viraemia in 2–5% of individuals either donating blood or receiving transfusions of blood products²⁰. Neither of these Brazilian studies screened for PARV4 IgG, so the prevalence of PARV4 exposure in this setting remains unknown^20,21.

Viral biology/immunology

PARV4 variants characterised to date fall into three distinct genetic groups (genotypes 1–3) but show relatively restricted sequence variability; in both the non-structural (NS) and capsid-encoding genome regions, there is less than 3% amino acid (8–9% nucleotide) sequence divergence between genotypes (representations of diversity can be found in Figure 2 and in Supplementary data 1). The even greater restriction of sequence diversity within genotypes supports the hypothesis for relatively recent transmission to human populations⁵. Relatively few new PARV4 sequences have been reported over the period that we have reviewed, and although incremental additions to the sequence database have been made over recent years (Figure 3), these have not substantially extended the known diversity of this virus.

Figure 2. Variability scan of human parvovirus 4 (PARV4) genomes, showing mean pairwise nucleotide distances of sequential 250 base fragments, incrementing by 25 bases between data points.

Sequence comparisons were between 10 genotype-1 (DQ873386, DQ873387, DQ873388, DQ873389, EU175856, EU200667, EU546204, EU546210, EU546211, and NC007018), 13 genotype-2 (DQ873390, DQ873391, EU175855, EU546205, EU546206, EU546207, HQ593530, HQ593532, KJ541119, KJ541120, KJ541121, KM390024, and KM390025), and seven genotype-3 (EU874248, JN798193, JN798194, JN798195, JN798196, KU871314, and KU871315) complete or near complete genome sequences. A genome diagram drawn to scale is included showing the main non-structural (NS, ORF1) and structural (VP, ORF2) gene coding regions as well as the positions of the additional reading frames (ARF1 and ARF2) embedded in ORF2. All nucleotide positions are numbered based on the reference sequence NC007018. It is striking that the region containing the two small ORFs is the most conserved of the whole genome; this may be in part a general feature of ORFs (where less flexibility is likely to be tolerated) but could also point to an important structural or functional role of this region. ORF, open reading frame.

Figure 3. Phylogeny of published complete or near complete human parvovirus 4 (PARV4) genome sequences as inferred from complete NS (ORF1) nucleotide sequences (equivalent to nucleotides 283–2271 of the reference sequence NC007018).

Sequences published since 2014 are highlighted with filled circles. The evolutionary history was inferred by using the maximum likelihood model. The optimum maximum likelihood model (lowest Bayesian information criterion score and typically greatest maximum likelihood value for the nucleotide sequence alignment) was first determined and used for phylogenetic reconstruction. This was the Tamura 3-parameter model with a gamma distribution. Bootstrap support of branches (500 replications) is indicated.

The distribution of the three genotypes is summarised as follows:

Various reports provide preliminary evidence that PARV4 may not be completely cleared after acute infection, suggesting that it has the potential for latency and reactivation. Two studies in Taiwan have sought to describe serological responses to PARV4 infection by undertaking longitudinal follow-up of their cohorts^31,33. Among a small cohort of health-care workers, IgG seropositivity was strikingly high at 60%; half of these had a positive IgM that was sustained over the study period, and viraemia was detected at one of several sampling time-points³³. In the second study, this time focusing on a high-risk PWID cohort, IgM was reported as remaining positive for up to 21 months³¹. Meanwhile, in vitro experiments have demonstrated a sustained high-magnitude ex vivo CD8⁺ T-cell response to PARV4 NS peptide³⁴. Prolonged IgM-positivity and sustained high-magnitude T-cell responses together suggest a sustained or relapsing/remitting exposure to viral antigen. Tissue homing and sites of latency are unknown, but existing data suggest that bone marrow, the respiratory tract, liver and gut might represent potential sites of viral replication^{5,15,26,27,35,36}. Skin has also been highlighted as a potential reservoir site for B19V³⁷, although—to the best of our knowledge—no study has investigated this as a site of PARV4 replication. The role of capsid proteins in parvovirus infections has been recently reviewed³⁸, although there are no specific functional or structural data for PARV4.

Clinical associations of PARV4

To date, determining the clinical impact (if any) of PARV4 infection remains perhaps the most uncertain area of research into this virus, despite the medical importance of establishing such disease associations. A wide range of potential infection outcomes has been proposed (summarised in Table 1), but the evidence for a pathogenic role of PARV4 in any of these has not been significantly expanded in recent years.

The most significant addition to the literature builds on an earlier report linking PARV4 with an encephalitis syndrome in Bellary, South India³². Further efforts have been made to substantiate this important potential disease association by screening ten patients in Northern India with an acute encephalitis syndrome in whom other viral causes had been ruled out. The investigators identified PARV4 DNA in the cerebrospinal fluid (CSF) and blood of two³⁹. However, it remains uncertain whether PARV was actually the agent of the observed disease, or a bystander that was coincidentally detected in a highly exposed population. Neither this nor the previous study³² included a control group in which the background incidence of PARV4 infections could have been evaluated. It is additionally possible that detection of PARV4 DNA in CSF in these two studies simply represents reactivation of the virus in the central nervous system in the context of other pathology.

Developing and refining methods for the study of PARV4

One approach to the determination of PARV4 serostatus is to use an enzyme-linked immunosorbent assay (ELISA)-based approach with an optical density read-out that is calibrated as positive or negative. However, the choice of a fixed cut-off above which threshold samples are termed ‘positive’ can be problematic and evidently has bearing on the final estimation of prevalence; the choice of this threshold evidently affects sensitivity and specificity of the test, and may need to be altered in light of the consideration of the primary reason for screening (diagnosis versus seroepidemiology). This effect is exemplified by recently published simulations, alongside discussion of alternative approaches that set out to quantify population prevalence⁴⁰. One of these is to use ‘mixture models’, which estimate population seroprevalence from the complete screened dataset rather than classifying each individual sample as positive or negative⁴⁰. Application of these new analytical methods to existing PARV4 serosurvey datasets has yet to be performed but may refine prevalence estimates and allow for better comparison between studies.

A novel polymerase chain reaction (PCR) assay for genotyping and quantifying PARV4 was published in 2014⁴¹. The authors report that their method reliably discriminates between PARV4 genotypes, and use quantitative PCR to measure viraemia⁴¹.

Practical implications: human tissue for transfusion and transplantation

Consistently high detection frequencies of anti-PARV4 antibodies in sera of haemophiliac individuals provide strong evidence for some transmission via the use of clotting factor concentrates^11,42. Although modern procedures for viral inactivation may effectively remove parvovirus infectivity, PARV4 is more robust than B19V to heat and other virus inactivation methods used in blood product manufacture, and there is ongoing discussion about the need to implement donation screening for PARV4 DNA and/or to enhance measures to ensure that the virus is eradicated from blood products for transfusion^10,43–45.

Blood donors have been screened as a component of several studies (see representative data in Table 1 for recent examples). PARV4 IgG screening of UK blood donors identified a seropositivity rate of 4.8%, but no donations were PCR-positive¹⁰. However, the results of such studies are disparate, as a previous study from the same group did find PARV4-positive pools⁴⁶. Studies outside the UK that have screened blood donors by PCR have found frequencies of 0% in Italy³⁵; 3–4% in Brazil²⁰, Thailand⁹, and North America²⁵; and as high as 17% in Iran¹⁹.

Solid organ transplant recipients are a more difficult population to assess and are unsurprisingly less well represented in the literature; no new articles on this topic have been published in the past two years. However, since 2008, an Italian study has reported finding PARV4 in a small number of renal transplant recipients³⁵, and a French cohort of lung transplant patients reports that 14% were viraemic⁴⁷. The clinical significance of these findings and whether these cases represent reactivation of autologous virus in the context of immunosuppression or transmission of donor virus at the time of organ transplantation have yet to be explored.

Insights from new animal data

There is increasing evidence that infections with porcine and bovine hokoviruses are endemic in both wild and domestic animals; most of these recent animal data are simply detection studies and do not provide any new data about pathogenicity. These include the frequent detection of porcine hokovirus in hunted wild boar in Portugal⁴⁸ and a bovine hokovirus in domestic yaks⁴⁹. In South America, another hokovirus was found to be endemic in a survey of Brazilian pig herds, in which post-weaning multi-systemic wasting syndrome was common⁵⁰, and a novel porcine hokovirus (referred to as porcine parvovirus 6, or PPV6) has been associated with foetal loss in pigs in China^51,52.

As livestock pathogens, these organisms are potentially implicated as a cause of significant economic losses in agriculture^52,53. However, since most of the suggested pathogenicity has been attributed to protoparvoviruses or copiparvoviruses, we cannot necessarily infer any significance to tetraparvoviruses. Nevertheless, the widespread isolation of parvoviruses, particularly from domestic animals, should raise concerns about the potential risk of inter-species transmission events leading to the possibility of more pathogenic viruses crossing into human populations.

A recent study of microRNA expression in porcine parvovirus infection describes upregulation of microRNAs that are known to be immune system regulators⁵². In the longer term, this kind of study may provide further insights into the nature and mechanism of host-virus interactions.