Presence of herpesviruses, parvoviruses, and polyomaviruses in sinonasal lymphoma.

PURPOSE: Sinonasal lymphoma (SL) is a rare lymphatic neoplasm of the nasal cavities, paranasal sinuses and nasopharynx. Whereas some risk factors for SL subtypes have been identified, their aetiology is unknown. Along with other predisposing factors, the viral association of lymphomas, such as Epstein-Barr virus (EBV) and Burkitt and Hodgkin lymphomas, is well-established. Modern molecular biology techniques have enabled the discovery of novel human viruses, exemplified by the protoparvovirus cutavirus (CuV), associated with cutaneous T-cell lymphoma. These findings, and the anatomical location of the sinonasal tract with its rich microbiome and infectious agents, justify in-depth studies among SL. METHODS: We analysed the presence of 20 viruses of Orthoherpesviridae, Parvoviridae, and Polyomaviridae by qPCR in 24 SL tumours. We performed RNAscope in situ hybridisation (RISH) to localize the viruses. Parvovirus-specific IgG was analysed by enzyme immunoassay and targeted next-generation sequencing (NGS) was applied to detect CuV in plasma. RESULTS: We detected viral DNA in 15/24 (63%) tumours; nine of EBV, six of human herpesvirus (HHV) -7, four each of HHV-6B and parvovirus B19, two of cytomegalovirus, and one each of CuV and Merkel-cell polyomavirus. We found tumours with up to four viruses per tumour, and localized CuV and EBV DNAs by RISH. Two of the ten plasma samples exhibited CuV IgG, and one plasma sample demonstrated CuV viremia by NGS. CONCLUSION: Viruses were frequent findings in SL. The EBV detection rate was high in diffuse large B-cell lymphoma, and co-detections with other viruses were prevalent.

Viral DNA in submandibular gland tissue with an inflammatory disorder.

Background: The etiology behind different types of chronic sialadenitis (CS), some of which exhibit IgG4 overexpression, is unknown. Further, IgG4-related disease (IgG4-RD) commonly affects the submandibular gland, but its relationship to IgG4-overexpressing CS, and the antigen triggering IgG4 overexpression, remain unknown. Materials and Methods: By qPCR, we assessed the presence of 21 DNA-viruses causing IgG4 overexpression in submandibular gland tissue from patients with IgG4-positive and IgG4-negative CS. Healthy submandibular glands and glands with sialolithiasis without CS were used as controls. We examined the distribution of HHV-7, HHV-6B and B19V DNA, within virus PCR-positive tissues with RNAscope in-situ hybridization (RISH). Results: We detected DNA from seven viruses in 48/61 samples. EBV DNA was more prevalent within the IgG4-positive samples (6/29; 21%) than the IgG4-negative ones (1/19; 5.3%). B19V DNA was more prevalent within the IgG4-negative samples (5/19; 26%) than the IgG4-positive ones (4/29; 14%). The differences in virus prevalence were not statistically significant. Of the IgG4-RD samples (n = 3) one contained HHV-6B DNA. RISH only showed signals of HHV-7. Conclusions: None of the studied viruses are implicated as triggering IgG4-overexpression in CS. Although our results do not confirm viral etiology in the examined conditions, they provide valuable information on the prevalence of viruses in both diseased and healthy submandibular gland tissue.

WSV2023 - The second meeting of the world society for virology: One health - One world - One virology.

The Second International Conference of the World Society for Virology (WSV), hosted by Riga Stradins University, was held in Riga, Latvia, on June 15-17th, 2023. It prominently highlighted the recent advancements in different disciplines of virology. The conference had fourteen keynote speakers covering diverse topics, including emerging virus pseudotypes, Zika virus vaccine development, herpesvirus capsid mobility, parvovirus invasion strategies, influenza in animals and birds, West Nile virus and Marburg virus ecology, as well as the latest update in animal vaccines. Discussions further explored SARS-CoV-2 RNA replicons as vaccine candidates, SARS-CoV-2 in humans and animals, and the significance of plant viruses in the 'One Health' paradigm. The presence of the presidents from three virology societies, namely the American, Indian, and Korean Societies for Virology, highlighted the event's significance. Additionally, past president of the American Society for Virology (ASV), formally declared the partnership between ASV and WSV during the conference.

Prevalence, tropism, and activity of cutavirus in circulating blood lymphocytes, stool, and skin biopsy specimens of patients with cutaneous T-cell lymphoma and parapsoriasis en plaques.

A significant association has been established between a newly emerging human parvovirus, cutavirus (CuV), and cutaneous T-cell lymphoma/mycosis fungoides (CTCL/MF) and its precursor parapsoriasis en plaques (PP). CTCL is a heterogeneous group of skin malignancies of T cells, the cause of which remains unknown. This study aimed to determine the activity, spread, and cell tropism of the skin-persistent CuV. CuV DNA was detected in both skin biopsies (6/20, 30%) and peripheral blood mononuclear cells (PBMCs) (4/29, 13.8%) from 49 CTCL/MF or PP patients, while none from 33 patients with any other type of skin disease or healthy subjects harbored CuV DNA. CuV DNA persisted in the skin or PBMCs for up to 15 years, despite circulating CuV-specific IgG. Spliced CuV mRNA was expressed in skin, indicating viral activity. Also, both of two available stool samples contained encapsidated CuV genomes, suggesting that the patients excrete infectious virus into the environment. Finally, CuV was observed to target circulating and skin-resident CD4 + T cells and some skin keratinocytes and macrophages. This is especially intriguing as malignant T cells in CTCL develop from CD4 + T cells. Hence, CuV should be further investigated for the overall role it plays in the complex tumor microenvironment of CTCL/MF.

Human bocavirus 1 coinfection is associated with decreased cytokine expression in the rhinovirus-induced first wheezing episode in children.

BACKGROUND: Rhinovirus (RV)-induced first wheezing episodes in children are associated with a markedly increased risk of asthma. Previous studies have suggested that human bocavirus 1 (HBoV1) may modify RV-induced immune responses in young children. We investigated cytokine profiles of sole RV- and dual RV-HBoV1-induced first wheezing episodes, and their association with severity and prognosis. METHODS: Fifty-two children infected with only RV and nine children infected with dual RV-HBoV1, aged 3-23 months, with severe first wheezing episodes were recruited. At acute illness and 2 weeks later, peripheral blood mononuclear cells were isolated, and stimulated with anti-CD3/anti-CD28 in vitro. Multiplex ELISA was used to quantitatively identify 56 different cytokines at both study points. Patients were prospectively followed for 4 years. RESULTS: The mean age of the children was 14.3 months, and 30% were sensitized. During the acute illness, the adjusted analyses revealed a decrease in the expression of IL-1b, MIP-1b, Regulated upon activation, normal T cell expressed and presumably secreted (CCL5), TNF-a, TARC, and ENA-78 in the RV-HBoV1 group compared with the RV group. In the convalescence phase, the RV-HBoV1 group was characterized by decreased expression of Fractalkine, MCP-3, and IL-8 compared to the RV group. Furthermore, the hospitalization time was associated with the virus group and cytokine response (interaction p < 0.05), signifying that increased levels of epidermal growth factor and MIP-1b were related with a shorter duration of hospitalization in the RV-HBoV1 coinfection group but not in the RV group. CONCLUSIONS: Different cytokine response profiles were detected between the RV and the RV-HBoV1 groups. Our results show the idea that RV-induced immune responses may be suppressed by HBoV1.

Significant Association of Cutavirus With Parapsoriasis en Plaques: High Prevalence Both in Skin Swab and Biopsy Samples.

Cutavirus (CuV) is associated with cutaneous T-cell lymphoma (CTCL), of which parapsoriasis is a precursor. Our study reveals a significantly higher CuV-DNA prevalence in skin swabs of parapsoriasis patients (6/13; 46.2%) versus those of healthy adults (1/51; 1.96%). Eight patients (8/12; 66.7%) had CuV DNA in biopsied skin, and 4 developed CTCL.

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma.

Etiology of vestibular schwannoma (VS) is unknown. Viruses can infect and reside in neural tissues for decades, and new viruses with unknown tumorigenic potential have been discovered. The presence of herpesvirus, polyomavirus, parvovirus, and anellovirus DNA was analyzed by quantitative PCR in 46 formalin-fixed paraffin-embedded VS samples. Five samples were analyzed by targeted next-generation sequencing. Viral DNA was detected altogether in 24/46 (52%) tumor samples, mostly representing anelloviruses (46%). Our findings show frequent persistence of anelloviruses, considered normal virome, in VS. None of the other viruses showed an extensive presence, thereby suggesting insignificant role in VS.

Capsid Structure of Aleutian Mink Disease Virus and Human Parvovirus 4: New Faces in the Parvovirus Family Portrait.

Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10-30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.

Prevalence, Cell Tropism, and Clinical Impact of Human Parvovirus Persistence in Adenomatous, Cancerous, Inflamed, and Healthy Intestinal Mucosa.

Parvoviruses are single-stranded DNA viruses, infecting many animals from insects to humans. Human parvovirus B19 (B19V) causes erythema infectiosum, arthropathy, anemia, and fetal death, and human bocavirus (HBoV) 1 causes respiratory tract infections, while HBoV2-4 are enteric. Parvoviral genomes can persist in diverse non-permissive tissues after acute infection, but the host-cell tropism and the impact of their tissue persistence are poorly studied. We searched for parvoviral DNA in a total of 427 intestinal biopsy specimens, as paired disease-affected and healthy mucosa, obtained from 130 patients with malignancy, ulcerative colitis (UC), or adenomas, and in similar intestinal segments from 55 healthy subjects. Only three (1.6%) individuals exhibited intestinal HBoV DNA (one each of HBoV1, 2, and 3). Conversely, B19V DNA persisted frequently in the intestine, with 50, 47, 31, and 27% detection rates in the patients with malignancy, UC, or adenomas, and in the healthy subjects, respectively. Intra-individually, B19V DNA persisted significantly more often in the healthy intestinal segments than in the inflamed colons of UC patients. The highest loads of B19V DNA were seen in the ileum and colon specimens of two healthy individuals. With dual-RNAscope in situ hybridization and immunohistochemistry assays, we located the B19V persistence sites of these intestines in mucosal B cells of lymphoid follicles and vascular endothelial cells. Viral messenger RNA transcription remained, however, undetected. RNA sequencing (RNA-seq) identified 272 differentially expressed cellular genes between B19V DNA-positive and -negative healthy ileum biopsy specimens. Pathway enrichment analysis revealed that B19V persistence activated the intestinal cell viability and inhibited apoptosis. Lifelong B19V DNA persistence thus modulates host gene expression, which may lead to clinical outcomes.

Torque Teno Virus Primary Infection Kinetics in Early Childhood.

Human torque teno viruses (TTVs) are a diverse group of small nonenveloped viruses with circular, single-stranded DNA genomes. These elusive anelloviruses are harbored in the blood stream of most humans and have thus been considered part of the normal flora. Whether the primary infection as a rule take(s) place before or after birth has been debated. The aim of our study was to determine the time of TTV primary infection and the viral load and strain variations during infancy and follow-up for up to 7 years. TTV DNAs were quantified in serial serum samples from 102 children by a pan-TTV quantitative PCR, and the amplicons from representative time points were cloned and sequenced to disclose the TTV strain diversity. We detected an unequivocal rise in TTV-DNA prevalence, from 39% at 4 months of age to 93% at 2 years; all children but one, 99%, became TTV-DNA positive before age 4 years. The TTV-DNA quantities ranged from 5 × 101 to 4 × 107 copies/mL, both within and between the children. In conclusion, TTV primary infections occur mainly after birth, and increase during the first two years with high intra- and interindividual variation in both DNA quantities and virus strains.

The long-term prognostic value of serum 25(OH)D, albumin, and LL-37 levels in acute respiratory diseases among older adults.

BACKGROUND: Older adults are more susceptible to respiratory tract infection than healthy working age adults. The increased susceptibility of older adults is thought to be interlinked with vitamin D status, nourishment, and immunological state in general. Data are scarce whether these parameters could serve as prognostic markers. AIM: To study whether serum 25(OH)D, albumin, and LL-37 level could give prognostic value of long-term survival in the older adults with multimorbidity and acute respiratory infection. METHODS: Consecutive episodes of hospital care of patients 65 years and older with respiratory symptoms were prospectively studied as a cohort. Standard clinical questionnaire was filled by the study physician. Laboratory markers included serum levels of 25(OH)D, albumin and LL-37, C-reactive protein (CRP), white blood cell count (WBC) and polymerase chain reaction diagnostics for 14 respiratory viruses. Pneumonia was confirmed by chest radiographs. Respiratory illness severity, death at ward, length of hospital stays, and 5-year survival were used as outcomes. RESULTS: In total, 289 older adult patients with mean age of 83 years were included in the study. Serum 25(OH)D deficiency (< 50 nmol/liter) was present in 59% and hypoalbuminemia (< 3.5 g/dL) in 55% of the study patients. Low serum albumin level was associated to one, two- and five-year mortality after hospital stay (all P < .05). In addition, it was associated with pneumonia, dyspnea, over 13-night long stay at ward and death at ward (all P < .05). No associations were seen between serum 25(OH)D and LL-37 levels and disease severity, short-term clinical outcome, or long-term survival. Associations between serum 25(OH)D, albumin, and LL-37 levels and respiratory virus presence were not seen. CONCLUSIONS: Serum albumin level on admission seems to give valuable information about the patients' general health and recovery potential in treating older adults with respiratory symptoms. Serum 25(OH)D and LL-37 had no associations with disease severity or long- and short-term prognosis among older adults hospitalized with respiratory symptoms.

World Society for Virology first international conference: Tackling global virus epidemics.

This communication summarizes the presentations given at the 1st international conference of the World Society for Virology (WSV) held virtually during 16-18 June 2021, under the theme of tackling global viral epidemics. The purpose of this biennial meeting is to foster international collaborations and address important viral epidemics in different hosts. The first day included two sessions exclusively on SARS-CoV-2 and COVID-19. The other two days included one plenary and three parallel sessions each. Last not least, 16 sessions covered 140 on-demand submitted talks. In total, 270 scientists from 49 countries attended the meeting, including 40 invited keynote speakers.

Human bocavirus 1 respiratory tract reactivations or reinfections in two adults, contributing to neurological deficits and death.

Human bocavirus 1 (HBoV1) of the family Parvoviridae causes mild to life-threatening respiratory tract infections in young children, but, due to widespread immunity, it is uncommon in adults. HBoV1 reinfections or reactivations leading to casualties are rare, but might be underdiagnosed. We report two young adults, one previously healthy and one immunosuppressed, with rare diagnostic patterns of HBoV1 respiratory tract infection. Both patients exhibited very high loads of HBoV1 DNA in respiratory samples. The immunosuppressed patient was also HBoV1 DNA-positive in blood, stool and a colon biopsy, but exhibited prior HBoV1-specific high-avidity IgG and weak IgM positivity 9 months before the respiratory symptoms. Likewise, the previously healthy patient exhibited HBoV1 IgG of high avidity and very weak IgM in serum, pointing to prior immunity, but with a seroconversion in cerebrospinal fluid. This patient also showed strong HBoV2 cross-reactivity. The molecular and serological results, together with their ages, suggest that both patients exhibited unusual reinfection or reactivation of HBoV1, contributing to neurological deficits and death.

The presence of herpesviruses in malignant but not in benign or recurrent pleomorphic adenomas.

BACKGROUND: The etiology of salivary gland tumors is mainly unknown. The anatomical location of the salivary glands, with the mucosal pathway to the oral cavity and its rich microbiome, raises the question of potential viral background. OBJECTIVE: This study focuses on the potential presence of herpes-, polyoma- and parvoviruses in pleomorphic adenoma (PA), recurrent pleomorphic adenoma (RPA) and carcinoma ex pleomorphic adenoma (CaxPA). METHODS: Thirty different viruses were analyzed by PCR-based assays in 68 formalin-fixed paraffin-embedded salivary gland tumors (25 PA, 31 RPA and 12 CaxPA). RESULTS: Virus DNA was detected altogether in 19/68 (28%) tumor samples. Human herpesviruses 6B and 7 (HHV-6B and HHV-7) and Epstein-Barr virus (EBV) were frequently and almost exclusively found in CaxPA (5/12, 7/12, and 3/12, respectively). Within the 7 CaxPA that were virus-positive, 3 samples contained 3, and 1 sample even 4, different viruses. Infrequent viral positivity was shown for parvovirus B19 and cutavirus, as well as Merkel cell and Malawi polyomaviruses. CONCLUSIONS: Our unexpected finding of herpesvirus DNA almost exclusively in CaxPA tissues deserves further in-depth studies.

Persistent human bocavirus 1 infection and tonsillar immune responses.

BACKGROUND: Persistent human bocavirus 1 (HBoV1) infection is a common finding in patients suffering from chronic tonsillar disease. However, the associations between HBoV1 infection and specific immune reactions are not completely known. We aimed to compare in vivo expression of T-cell cytokines, transcription factors, and type I/III interferons in human tonsils between HBoV1-positive and -negative tonsillectomy patients. METHODS: Tonsil tissue samples, nasopharyngeal aspirate (NPA), and serum samples were obtained from 143 immunocompetent adult and child tonsillectomy patients. HBoV1 and 14 other respiratory viruses were detected in NPAs and tonsil tissues by polymerase chain reaction (PCR). Serology and semi-quantitative PCR were used for diagnosing HBoV1 infections. Expression of 14 cytokines and transcription factors (IFN-α, IFN-ß, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-ß, FOXP3, GATA3, RORC2, Tbet) was analyzed by quantitative reverse-transcription (RT)-PCR in tonsil tissues. RESULTS: HBoV1 was detected by PCR in NPA and tonsils from 25 (17%) study patients. Serology results indicated prior nonacute infections in 81% of cases. Tonsillar cytokine responses were affected by HBoV1 infection. The suppression of two transcription factors, RORC2 and FOXP3, was associated with HBoV1 infection (p < 0.05). Furthermore, intratonsillar HBoV1-DNA loads correlated negatively with IFN-λ family cytokines and IL-13. CONCLUSIONS: Our study shows distinctively decreased T-helper17 and T-regulatory type immune responses in local lymphoid tissue in HBoV1-positive tonsillectomy patients. HBoV1 may act as a suppressive immune modulator.

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections.

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0-6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.

Characterization of the GBoV1 Capsid and Its Antibody Interactions.

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.

Persistence of Human Bocavirus 1 in Tonsillar Germinal Centers and Antibody-Dependent Enhancement of Infection.

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies.IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding.

pH-Induced Conformational Changes of Human Bocavirus Capsids.

Human bocavirus 1 (HBoV1) and HBoV2-4 infect children and immunocompromised individuals, resulting in respiratory and gastrointestinal infections, respectively. Using cryo-electron microscopy and image reconstruction, the HBoV2 capsid structure was determined to 2.7 Å resolution at pH 7.4 and compared to the previously determined HBoV1, HBoV3, and HBoV4 structures. Consistent with previous findings, surface variable region (VR) III of the capsid protein VP3, proposed as a host tissue-tropism determinant, was structurally similar among the gastrointestinal strains HBoV2-4, but differed from HBoV1 with its tropism for the respiratory tract. Towards understanding the entry and trafficking properties of these viruses, HBoV1 and HBoV2 were further analyzed as species representatives of the two HBoV tropisms. Their cell surface glycan-binding characteristics were analyzed, and capsid structures determined to 2.5-2.7 Å resolution at pH 5.5 and 2.6, conditions normally encountered during infection. The data showed that glycans with terminal sialic acid, galactose, GlcNAc or heparan sulfate moieties do not facilitate HBoV1 or HBoV2 cellular attachment. With respect to trafficking, conformational changes common to both viruses were observed at low pH conditions localized to the VP N-terminus under the 5-fold channel, in the surface loops VR-I and VR-V and specific side-chain residues such as cysteines and histidines. The 5-fold conformational movements provide insight into the potential mechanism of VP N-terminal dynamics during HBoV infection and side-chain modifications highlight pH-sensitive regions of the capsid.IMPORTANCE Human bocaviruses (HBoVs) are associated with disease in humans. However, the lack of an animal model and a versatile cell culture system to study their life cycle limits the ability to develop specific treatments or vaccines. This study presents the structure of HBoV2, at 2.7 Å resolution, determined for comparison to the existing HBoV1, HBoV3, and HBoV4 structures, to enable the molecular characterization of strain and genus-specific capsid features contributing to tissue tropism and antigenicity. Furthermore, HBoV1 and HBoV2 structures determined under acidic conditions provide insight into capsid changes associated with endosomal and gastrointestinal acidification. Structural rearrangements of the capsid VP N-terminus, at the base of the 5-fold channel, demonstrate a disordering of a "basket" motif as pH decreases. These observations begin to unravel the molecular mechanism of HBoV infection and provide information for control strategies.