key: cord-0824246-e0gkapj9 authors: Bagasi, Arwa A.; Howson-Wells, Hannah C.; Clark, Gemma; Tarr, Alexander W.; Soo, Shiu; Irving, William L.; McClure, C. Patrick title: Human Bocavirus infection and respiratory tract disease identified in a UK patient cohort date: 2020-05-21 journal: J Clin Virol DOI: 10.1016/j.jcv.2020.104453 sha: 787c54fa5d1d09f0bf455ce98aa98e55a6b2d6b5 doc_id: 824246 cord_uid: e0gkapj9 BACKGROUND: Since its first isolation in 2005, Human Bocavirus (HBoV) has been repeatedly associated with acute respiratory tract infections, although its role in pathogenicity remains unclear due to high co-infection rates. OBJECTIVES: To assess HBoV prevalence and associated disease in a cohort of respiratory patients in the East Midlands, UK between 2015 and 2019. STUDY DESIGN: We initially investigated the undiagnosed burden of HBoV in a retrospective paediatric cohort sampled between 2015 and 2017 using an in-house PCR assay. HBoV was subsequently incorporated into the standard respiratory diagnostic pathway and we audited a calendar year of HBoV positive results between 2018 and 2019. RESULTS: Our retrospective PCR screening of previously routine diagnostic-negative samples from juvenile patients identified a 9% (n = 30) prevalence of HBoV type 1. These apparentHBoV1 mono-infections were frequently associated with respiratory tract symptoms, often severe including ventilation, oxygen and steroid intervention with 31% (n = 9) of individuals requiring intensive care. When HBoV screening was subsequently adopted into the routine respiratory diagnostic pathway, year-round infections were observed in both children and adults peaking in February. 185 of 9098 (2.03%) individuals were found to be HBoV positive with children aged 12-24 months the principally infected group. However, HBoV infection was also observed in patients aged over 60, predominantly as a mono-infection. 23% of the 185 unique patients were HBoV monoinfectedand persistent low-level DNA positivity was observed in 15 individuals up to 6-months after initial presentation. CONCLUSION: HBoV1 is a prevalent respiratory infection in the UK capable of causing serious monoinfections. . Despite HBoV1's frequent detection in respiratory samples from subjects with acute respiratory tract infections (ARTIs), its role as a respiratory pathogen is not fully understood. High rates of co-infection (up to 70-80% in some studies) with predominantly Respiratory Syncytial Virus, Rhinovirus, Parainfluenza, Adenovirus [5, 6] and also frequent detection in asymptomatic subjects [7] , have promoted a hypothesis of HBoV1 as a "passenger" virus [5, 7, 8] . In vitro culture models of the virus have been described [9, 10] which could further elucidate pathogenicity. Clinically, HBoV1 infections are typically characterized by mild self-limiting acute respiratory symptoms including cough, rhinitis, acute otitis media, and pharyngitis [7, 11] . However, possibly due to the unknown pathogenicity and frequently asymptomatic or selflimiting nature of bocavirus infections, HBoV is often omitted from diagnostic investigation. Nevertheless, evidence to support HBoV1 as the aetiological agent in presentations of ARTI is growing, for instance by use of serological diagnosis using acute convalescent sera [12] and through correlation of HBoV1 viral loads with symptom severity in mono-infections [5, 11, 13] . Furthermore, the virus has been associated with respiratory symptoms in the absence of other viral, fungal, or bacterial agents, with a significant detection rate difference between cases and controls [5]. HBoV1 has been associated not only with self-limiting upper respiratory tract infections, but also lower respiratory tract symptoms including wheezing, bronchiolitis, respiratory distress, and pneumonia [5, 8] . Indeed, there are several documented cases of bocavirus infection associated with severe lower respiratory illness requiring hospitalization, oxygen therapy, and even intensive care [14] [15] [16] [17] . Taken together, these findings suggest HBoV1 can function in isolation as a respiratory pathogen. In order to better understand the epidemiology and disease burden (if any) imposed by HBoV infection, we retrospectively tested residual total nucleic acid (TNA) from 347 nasopharyngeal aspirates (NPAs) by pan-bocavirus degenerate PCR [18] . Samples were collected from January 2015 to April 2017 from individuals between 6 months and 5 years of J o u r n a l P r e -p r o o f age that were known to be negative for a panel of more established respiratory viruses. Having identified a significant number of bocavirus mono-infections associated with clinically severe disease, HBoV1 was included as a target in routine respiratory viral panel screening. HBoV1 detection was also reported throughout a calendar year (September 2018 to August 2019) in upper and lower respiratory tract samples, again identifying significant numbers of patients infected with HBoV1, mainly children but also adults, encompassing both mono-and co-infections. Overall, our findings support the hypothesis that HBoV1 can be a significant human pathogen responsible for clinically relevant respiratory disease. Nasopharyngeal aspirates (NPA) from children between 6 months and 5 years old were processed for routine diagnostic investigation at Nottingham University Hospitals Trust (NUHT) between January 2015 and April 2017 as previously described [19] . [20] to target the NS1 gene in 15 μl reactions comprising: 1.5 μl of QIAGEN 10x PCR buffer, 3 pmol of each primer, 6 nmol of dNTPs, 0.375U QIAGEN HotStarTaq DNA polymerase, 1 μl of TNA template and nuclease-free water. PCR was thermocycled as follows: 95°C for 15 minutes, then 55 cycles of 95°C for 20 seconds, 55°C for 20 seconds, and 72°C for 45 seconds. Amplification of a 1798bp NS1 fragment was also performed as above, except with 45 cycles and 72°C for 120 seconds, using novel primers hBocaNS1f (TCTCAACCTGCTTTYACYTATGT) and hBocaNS1r (AGAATTGTCAGCRSTATGAGSAA). All positive pools and individual samples were confirmed by agarose gel electrophoresis and sequencing as previously described [19] . Phylogenetic analysis of sequences was performed using MEGA7 software (version 7.0.25) and all human reference sequences were downloaded from GenBank circa May 2017. were admitted to intensive care, with one patient dying of multi-organ failure and viral pneumonitis. Age of HBoV-infected individuals was significantly skewed toward the young (p=<0.0001, Chi-square test for trend), with 87% of unique HBoV-positive patients being aged 5 or under. A peak in positivity was seen in the 1-2-year-old age group, with 9% of these individuals Interestingly, of the 11 HBoV positive patients aged ≥60, seven were mono-infected and where co-infected, higher viral loads for HBoV were observed than for the co-infecting virus (data not shown). 11 individuals were sampled on more than one occasion and >1 day apart, ranging from a 13 to a 157 day period (Figure 3) prescriptions are erroneously prescribed for respiratory illnesses caused mainly by viruses [21] . Such overuse not only contributes to bacterial resistance, but can also affect commensal gastrointestinal microbiota required for healthy gut function and potentially cause unnecessary adverse effects in patients [21, 22] . Expanding routine tested-for viral respiratory panels has the potential to reduce this public health burden. We tested this hypothesis by archiving and re-screening apparently viral-negative respiratory TNA extracts for HBoV, a relatively recently discovered, but widely reported viral pathogen, identifying 9% HBoV1 positivity of NPAs in patients aged 6 months to 5 years. Importantly, 10 (34%) patients with undiagnosed HBoV1 positive patients were prescribed empirical antibiotic therapy; eight in the absence of diagnosed bacterial co-infection whilst two were not investigated. HBoV1 pathogenicity is not fully understood with HBoV frequently observed as a coinfection. Our initial in-house study supported a causal role of HBoV1 as a respiratory pathogen, with severe symptoms observed and intensive care required in the absence of coinfection with other typical viral or bacterial respiratory pathogens. Our subsequent one-year review of HBoV screening in routine diagnostic service confirmed previously observed high rates of co-infection with other viral pathogens. High rates of coinfections and the presence of HBoV1 in asymptomatic individuals could be explained by long persistence periods and high prevalence of the virus. HBoV1 was found to persist in mucosa for more than four months following primary infections, which increases the chances of co-existence with other viral or bacterial pathogens [23, 24] and the virus has been suggested to reactivate following a superinfection with another virus [4].We similarly observed persistence of HBoV in our cohort for up to 6 months. HBoV1's high prevalence could also explain the high rate of co-infections. One study estimated HBoV1 infection rate to be as high as 59% in a cohort of children with respiratory illness and another reported that ~90% of adults have HBoV-specific antibodies [23, 25] . It is clear that detection of HBoV DNA can persist for many months, and therefore the presence of such DNA does not necessarily indicate a recent infection. Our findings of frequent administration of oxygen, steroids and salbutamol in addition to ventilation and intensive care in HBoV1 mono-infection, supports previous reports of HBoV1 as a cause of serious lower respiratory tract infections and pneumonia [5, [13] [14] [15] . HBoV1 has been reported to have very low genetic diversity worldwide [18] . Our sequencing and phylogenetic analysis of a region of NS1 (circa 22% of the genome) indeed confirmed these findings and would indicate whole genome sequencing is required for more robust investigation and interpretation of epidemiology. Sequencing of this NS1 region did strongly suggest all detected samples were of HBoV type 1, but the possibility of recombination cannot be ruled out without whole genome sequencing [18] . With respect to HBoV1 seasonality, our study showed higher prevalence during the late autumn and winter periods, consistent with prevalence reported worldwide [1, [26] [27] [28] . However, few studies reported year-round detection and two studies reported peak detection during summer [29] [30] [31] [32] . The contradictory findings of these reports with many studies reporting winter and spring surges in HBoV1 infection rates could be explained by differences between strains isolated in different locations, but it could be also attributed to social or behavioural factors [33] . In summary, our study implicates HBoV1 as a currently prevalent respiratory pathogen in the UK capable of causing serious mono-infections. Increasing adoption of this relatively Supervision Gemma Clark: Resources, Conceptualization Alexander W. Tarr: Supervision, Formal analysis Shiu Soo: Resources, Conceptualization William L. Irving: Resources, Conceptualization, Supervision C. 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