Detection of Head-to-Tail DNA Sequences of Human Bocavirus in Clinical Samples

Parvoviruses are single stranded DNA viruses that replicate in a so called “rolling-hairpin” mechanism, a variant of the rolling circle replication known for bacteriophages like ϕX174. The replication intermediates of parvoviruses thus are concatemers of head-to-head or tail-to-tail structure. Surprisingly, in case of the novel human bocavirus, neither head-to-head nor tail-to-tail DNA sequences were detected in clinical isolates; in contrast head-to-tail DNA sequences were identified by PCR and sequencing. Thereby, the head-to-tail sequences were linked by a novel sequence of 54 bp of which 20 bp also occur as conserved structures of the palindromic ends of parvovirus MVC which in turn is a close relative to human bocavirus

Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65.

Human bocavirus (HBoV), a parvovirus, is a single-stranded DNA etiologic agent causing lower respiratory tract infections in young children worldwide. Nuclear factor kappa B (NF-κB) transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes. Previous investigation showed that HBoV infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator. Here we investigated whether HBoV proteins modulate TNF-α-mediated activation of the NF-κB signaling pathway. We showed that HBoV NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α. Overexpression of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase alpha (IKKα)-, IκB kinase beta (IKKβ)-, constitutively active mutant of IKKβ (IKKβ SS/EE)-, or p65-induced NF-κB activation was inhibited by NS1 and NS1-70. Furthermore, NS1 and NS1-70 didn't interfere with TNF-α-mediated IκBα phosphorylation and degradation, nor p65 nuclear translocation. Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65. Of note, NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536. Our findings together indicate that HBoV NS1 and NS1-70 inhibit NF-κB activation. This is the first time that HBoV has been shown to inhibit NF-κB activation, revealing a potential immune-evasion mechanism that is likely important for HBoV pathogenesis

Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells

Productive Parvovirus B19 Infection of Primary Human Erythroid Progenitor Cells at Hypoxia Is Regulated by STAT5A and MEK Signaling but not HIFα

Human parvovirus B19 (B19V) causes a variety of human diseases. Disease outcomes of bone marrow failure in patients with high turnover of red blood cells and immunocompromised conditions, and fetal hydrops in pregnant women are resulted from the targeting and destruction of specifically erythroid progenitors of the human bone marrow by B19V. Although the ex vivo expanded erythroid progenitor cells recently used for studies of B19V infection are highly permissive, they produce progeny viruses inefficiently. In the current study, we aimed to identify the mechanism that underlies productive B19V infection of erythroid progenitor cells cultured in a physiologically relevant environment. Here, we demonstrate an effective reverse genetic system of B19V, and that B19V infection of ex vivo expanded erythroid progenitor cells at 1% O2 (hypoxia) produces progeny viruses continuously and efficiently at a level of approximately 10 times higher than that seen in the context of normoxia. With regard to mechanism, we show that hypoxia promotes replication of the B19V genome within the nucleus, and that this is independent of the canonical PHD/HIFα pathway, but dependent on STAT5A and MEK/ERK signaling. We further show that simultaneous upregulation of STAT5A signaling and down-regulation of MEK/ERK signaling boosts the level of B19V infection in erythroid progenitor cells under normoxia to that in cells under hypoxia. We conclude that B19V infection of ex vivo expanded erythroid progenitor cells at hypoxia closely mimics native infection of erythroid progenitors in human bone marrow, maintains erythroid progenitors at a stage conducive to efficient production of progeny viruses, and is regulated by the STAT5A and MEK/ERK pathways

Chloroquine and Its Derivatives Exacerbate B19V-Associated Anemia by Promoting Viral Replication

Human parvovirus B19 (B19V) is typically associated with a childhood febrile illness known as erythema infectiosum. The infection usually resolves without consequence in healthy individuals. However, in patients with immunologic and/or hematologic disorders, B19V can cause a significant pathology. The virus infects and kills red cell precursors but anemia rarely supervenes unless there is pre-existing anemia such as in children living in malaria-endemic regions. The link between B19V infection and severe anemia has, however, only been confirmed in certain malaria-endemic countries in parallel with chloroquine (CQ) usage. This raises the possibility that CQ may increase the risk of severe anemia by promoting B19V infection. To test this hypothesis, we examined the direct effect of CQ and other commonly used antimalarial drugs on B19V infection in cultured cell lines. Additionally, we examined the correlation between B19V infection, hemoglobin levels and use of CQ in children from Papua New Guinea hospitalized with severe anemia. The results suggest strongly that CQ and its derivatives aggravate B19V-associated anemia by promoting B19V replication. Hence, careful consideration should be given in choosing the drug partnering artemisinin compounds in combination antimalarial therapy in order to minimize contribution of B19V to severe anemia

Investigating porcine parvoviruses genogroup 2 infection using in situ polymerase chain reaction

Abstract Background Porcine parvovirus 2 (PPV2) was detected in swine serum without showing any relationship with disease. The emergence of the virus seemed to be a unique event until other genetically highly similar parvoviruses were identified in China and, later in 2012, the presence of the virus was also described in Europe. PPV2 is widely distributed in pig populations where it is suspected to be involved in respiratory conditions, based on its frequent detection in lung samples. In order to investigate the potential pathogenic involvement of PPV2, 60 dead pigs were examined from two farms. They were necropsied and tested for PPV2 and PCV2 (Porcine circovirus type 2) by PCR; by Brown and Brenn (B&B) staining for bacteria; by immunohistochemistry (IHC) to detect CD3, Swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza (SIV), Mycoplasma hyopneumoniae (Mhyo); and by in situ hybridization (ISH) to detect ssDNA and dsDNA of PCV2. PPV2 positive samples were subjected to in situ polymerase chain reaction (IS-PCR) including double staining method to detect PPV2 and host cell markers. To calculate statistical difference we used GENMOD or LOGISTIC procedures in Statistical Analysis System (SAS®). Results We found that the PPV2 was localized mostly in lymphocytes in lungs, lymph nodes and liver. Neither CD3 antigen nor lysozyme was expressed by these infected cells. In contrast, low levels of SLAIIDQ were expressed by infected cells, suggesting that PPV2 may have a specific tropism for immature B lymphocytes and/or NK lymphocytes though possibly not T lymphocytes. Conclusion The overall conclusion of this study indicates that PPV2 may contribute to the pathogenesis of pneumonia

Substitution of adeno-associated virus Rep protein binding and nicking sites with human Chromosome 19 sequences

<p>Abstract</p> <p>Background</p> <p>Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated <it>AAVS1 </it>(also known as <it>MBS85</it>). Integration at <it>AAVS1 </it>requires the AAV2 replication (Rep) proteins and a DNA sequence within <it>AAVS1 </it>containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or <it>trs</it>). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced <it>trs</it>, but the sequences differ from those in <it>AAVS1</it>. These ITR sequences are required for replication and packaging.</p> <p>Results</p> <p>In this study we demonstrate that the <it>AAVS1 </it>RRS and <it>trs </it>can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of <it>AAVS1 </it>DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at <it>AAVS1</it>, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to <it>AAVS1 </it>sequences.</p> <p>Conclusions</p> <p>The ability of these <it>AAVS1 </it>sequences to substitute for the AAV2 RRS and <it>trs </it>provides indirect evidence that the stable secondary structure encompassing the <it>trs </it>is part of the AAV2 packaging signal.</p