Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1

Human bocavirus(HBoV)1 is considered an important pathogen that mainly affects infants aged 6–24 months,but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging.Herein,we present a novel faster,lower cost,reliable method for the detection of HBoV1,which integrates a recombinase polymerase amplification(RPA)assay with the CRISPR/Cas12a system,designated the RPA-Cas12a-fluorescence assay.The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37℃without the need for sophisticated instruments.The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens.Furthermore,the method was appraised using 28 clinical samples,and displayed high accuracy with positive and negative predictive agreement of 90.9%and 100%,respectively.Therefore,our proposed rapid and sensitive HBoV1 detection method,the RPA-Cas12a-fluorescence assay,shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care.The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection.The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.

Detection of Human Bocavirus in Children with Acute Respiratory Tract Infections in Lanzhou and Nanjing,China

Objective The aim of this study was to explore the prevalent characteristics of HBoV1 and its co-infection.Methods PCR was used to detect HBoV1-DNA（HBoV1） and other viruses.A multivariate logistic regression model was used to explore possibility of co-detected for related viruses.Results The positivity rates in Nanjing and Lanzhou were 9.38%（74/789） and 11.62%（161/1386）,respectively（P〉0.05）.The HBoV1 positive group was younger than negative group（P〈0.05）.Seasonal differences were noted,with a higher frequency of infection in December and July.HBoV1-positive children [72.34%（169/235）] were co-infected with other respiratory viruses.Multifactorial analysis showed no correlations between HBoV1 and the clinical classification,region,gender,age,or treatment as an outpatient or in a hospital.Correlations were identified between HBoV1 infections with ADV（OR=1.53,95% CI 1.03-2.28）,RSV（OR=0.71,95% CI 0.52-0.98）,and IFVA（OR=1.77,95% CI 1.00-3.13）.Conclusions Presence of HBoV1 in nasopharyngeal aspirates did not correlate with region or gender,although the prevalence of HBoV1 was higher in younger children.There were no correlations between HBoV1 and other variables,except for the season and ADV,RSV,or IFVA infections.

Human bocavirus infection in children hospitalized with lower respiratory tract infections:Does viral load affect disease course?

Objective:To examine the effects of human bocavirus type 1(HBoV1)on the course of lower respiratory tract infections in cases of monoinfection and coinfection,and the effects of HBoV1 viral load on the disease in children under six years old hospitalized with a diagnosis of HBoV1-associated lower respiratory tract infections.Methods:Children under six years of age,who were hospitalized with the diagnosis of lower respiratory tract infection due to HBoV1 between 1 January 2021 and 1 January 2022 were included in the study.Laboratory confirmation of the respiratory pathogens was performed using polymerase chain reaction(PCR).Results:Fifty-four(16.4%)children with HBoV1 among 329 children whose PCR was positive with bacterial/viral agent in nasopharyngeal swab samples were included in the study.There were 28(51.9%)males and 26(48.1%)females with a median age 23.4 months[interquartile range(IQR):13.2,30.0 months](min-max:1 month-68 months).HBoV1 was detected as a monoinfecton in 26(48.1%)children,and as a coinfection with other respiratory agents in 28 children(51.9%).In multiple regression analysis,coinfection(P=0.032)was associated with the length of hospitalization(P<0.001;R^(2)=0.166).There was a negative correlation(r=−0.281,P=0.040)between cough and cycle threshold.Fever was found to be positively correlated with C-reactive protein(r=0.568,P<0.001)and procalcitonin(r=0.472;P=0.001).Conclusions:Although we found a higher HBoV1 viral load in children with more cough symptoms in our study,it had no effect on the severity of the disease,such as length of hospital stay and need for intensive care.Coinfection was found to affect the length of hospitalization.

Human bocavirus 1 and 2 genotype-specific antibodies for rapid antigen testing in pediatric patients with acute respiratory infections

Background Previous serological studies of human bocavirus(HBoV)1 could not exclude cross-reactivity with the other three HBoVs,particularly HBoV2.Methods To search for genotype-specific antibodies against HBoV1 and HBoV2,the divergent regions(DRs)located on the major capsid protein VP3 were defined through viral amino acid alignment and structure prediction.DR-deduced peptides were used as antigens to harvest corresponding anti-DR rabbit sera.To determine their genotype specificities for HBoV1 and HBoV2,these sera samples were used as antibodies against the antigens VP3 of HBoV1 and HBoV2(expressed in Escherichia coli)in western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and bio-layer interferometry(BLI)assays.Subsequently,the antibodies were evaluated with clinical specimens from pediatric patients with acute respiratory tract infection by indirect immunofluorescence assay(IFA).Results There were four DRs(DR1–4)located on VP3 with different secondary and tertiary structures between HBoV1 and HBoV2.Regarding the reactivity with VP3 of HBoV1 or HBoV2 in WB and ELISA,high intra-genotype cross-reactivity of anti-HBoV1 or HBoV2 DR1,DR3,and DR4,but not anti-DR2,was observed.Genotype-specific binding capacity of anti-DR2 sera was confirmed by BLI and IFA,in which only anti-HBoV1 DR2 antibody reacted with HBoV1-positive respiratory specimens.Conclusion Antibodies against DR2,located on VP3 of HBoV1 or HBoV2,were genotype specific for HBoV1 and HBoV2,respectively.

Human bocavirus in children hospitalized for acute respiratory tract infection in Rome

Background The role of human bocavirus (HBoV) as a respiratory pathogen has not been fulfilled yet.We aimed to describe clinical and serological characteristics of children with HBoV hospitalized for acute respiratory tract infection and to evaluate whether differences occur between HBoV alone and in co-infection.Methods We retrospectively reviewed data from 60 children (median age of 6.2 months,range 0.6-70.9) hospitalized for acute respiratory symptoms,with HBoV detected from a respiratory sample,using a reverse transcriptase-PCR for 14 respiratory viruses (including respiratory syncytial virus (RSV),influenza virus A and B,human coronavirus OC43,229E,NL-63 and HUK1,adenovirus,rhinovirus,parainfluenza virus 1-3,and human metapneumovirus).Results HBoV was detected alone in 29 (48.3%) patients,while in co-infection with other viruses in 31 patients (51.7%),with a peak between December and January.Among the 60 patients,34 were bronchiolitis,19 wheezing,3 pneumonia,2 upper respiratory tract infection,and 2 whooping cough.Seven children (11.6%) required admission to the paediatric intensive care unit (PICU) for respiratory failure.No differences was observed in age,family history for atopy and/or asthma,clinical presentations,chest X-ray,or laboratory findings in children with HBoV alone vs.multiple viral detection.RSV was the most frequently co-detected virus (61.3%).When compared with HBoV detection alone,the co-detection of RSV and HBoV was associated with male sex (P=0.013),younger age (P=0.01),and lower blood neutrophil count (P=0.032).Conclusions HBoV can be detected alone and in co-infection respiratory samples of children with an acute respiratory tract infection.A cause-effect relationship between HBoV and respiratory infection is not clear,so further studies are needed to clarify this point.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Human bocavirus infections are common in Beijing population indicated by sero-antibody prevalence analysis

Background Human bocavirus （HBoV） is a newly identified human parvovirus that was originally detected in the respiratory secretions of children with respiratory infections. This study aimed to learn about the importance of HBoV infections by revealing the prevalence of serum antibodies against HBoV in Beijing population. Methods Two batches of serum specimens collected in different periods were tested by Western blotting for specific IgG against HBoV using recombinant VP2 as antigen. Results Out of 677 serum specimens collected during April 1996 to March 1997, 400 （59.1%） were positive and antibody positive rate for another batch of 141 serum specimens collected in August, 2005 from adults aged from 20 years to over 60 years was 78.7% （111/141）. Comparison of the sero-prevalence profiles for serum specimens collected during 1996-1997 to those collected in 2005 indicated that the antibody positive rate for specimens collected in 2005 was higher than that of the corresponding age groups collected during 1996-1997. Conclusions The data suggest that HBoV has been circulating in Beijing population for at least over 10 years, and most of children had been exposed to HBoV by age of 7 years. Higher HBoV antibody positive rate shown in the serum specimens collected in 2005 suggested that infections by HBoV have been increased in Beijing population in recent years.