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.

Human bocavirus: Current knowledge and future challenges

Human bocavirus(HBoV) is a parvovirus isolated about a decade ago and found worldwide in both respiratory samples, mainly from early life and children of 6-24 mo of age with acute respiratory infection, and in stool samples, from patients with gastroenteritis. Since then, other viruses related to the first HBoV isolate(HBoV 1), namely HBoV 2, HBoV 3 and HBoV 4, have been detected principally in human faeces. HBo Vs are small nonenveloped single-stranded DNA viruses of about 5300 nucleotides, consisting of three open reading frames encoding the first two the non-structural protein 1(NS1) and nuclear phosphoprotein(NP1) and the third the viral capsid proteins 1 and 2(VP1 and VP2). HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models for the difficulties in replicating the virus in in vitro cell cultures, and the fact that HBo V infection is frequently accompanied by at least another viral and/or bacterial respiratory and/or gastroenteric pathogen infection. Current diagnostic methods to support HBoV detection include polymerase chain reaction, real-time PCR, enzymelinked immunosorbent assay and enzyme immunoassay using recombinant VP2 or virus-like particle capsid proteins, although sequence-independent amplification techniques combined with next-generation sequencing platforms promise rapid and simultaneous detection of the pathogens in the future. This review presents the current knowledge on HBoV genotypes with emphasis on taxonomy, phylogenetic relationship and genomic analysis, biology, epidemiology, pathogenesis and diagnostic methods. The emerging discussion on HBoV s as true pathogen or innocent bystander is also emphasized.

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.

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 with Acute Respiratory Infection in Nairobi, Kenya

Background: Acute respiratory infection (ARI) is a leading cause of morbidity and mortality in children under five years of age in developing countries with viruses contributing significantly to this problem. The recently identified parvovirus, Human Bocavirus (HBoV), has also been associated with ARI. Objective: To determine the frequency of HBoV in patients with ARI. Materials and Methods: Samples from 125 consenting patients with influenza like illness signs and symptoms were collected. DNA was extracted from these samples using the QIAamp DNA blood mini kit (Qiagen, Germany). Conventional PCR was carried out and the amplicons were examined in 2% agarose gels stained with ethidium bromide. This was followed by sequencing of the HBoV positive samples. Results: Twenty one (16.8%) patients were found to have HBoV infection. Males (n = 61.9%) were mainly infected with HBoV. Local HBoV strains had 98.9% - 100% similarities and were found to cluster together with other strains obtained elsewhere. Conclusion: These findings suggest that HBoV plays a role in respiratory tract infections in children in Kenya just like it has been found elsewhere. It also sheds light on multiple infections associated with HBoV infections in Kenya.

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.

Human bocavirus in children suffering from acute lower respiratory tract infection in Beijing Children＇s Hospital

Background Human bocavirus （HBoV） is a parvovirus recently found to possibly cause respiratory tract disease in children and adults. This study investigated HBoV infection and its clinical characteristics in children younger than five years of age suffering from acute lower respiratory tract infection in Beijing Children＇s Hospital. Methods Nasopharyngeal aspirates were collected from children suffering from acute lower respiratory tract infection during the winters of 2004 to 2006 （from November through the following February）. HBoV was detected by polymerase chain reaction amplification and virus isolation and the amplification products were sequenced for identification. Results HBoV infection was detected in 16 of 333 study subjects. Coinfections with respiratory syncytial virus were detected in 3 of 16 HBoV positive patients with acute lower respiratory tract infection. The median age for HBoV positive children was 8 months （mean age, 17 months; range, 3 to 57 months）. Among the HBoV positive children, 14 were younger than 3 years old, 9 were younger than 1 year old and 7 were younger than 6 months. These 16 positive HBoV children exhibited coughing and abnormal chest radiography findings and more than 60% of these children had wheezing and fever. Ten children were clinically diagnosed with pneumonia, 2 bronchiolitis, 2 acute bronchitis and 2 asthma. One child died. Conclusions HBoV was detected in about 5% of children with acute lower respiratory infection seen in Beijing Children＇s HosPital. Further investigations regarding clinical and epidemiologic characteristics of HBoV infection are needed.

Porcine Bocavirus: A 10-Year History since Its Discovery

Porcine bocavirus(PBoV)is a single-stranded DNA virus,belongs to the genus Bocaparvovirus of family Parvoviridae.It was discovered along with porcine circovirus 2(PCV 2)and torque tenovirus(TTV)in the lymph nodes of pigs suffering from postweaning multisystemic wasting syndrome(PMWS)in Sweden in 2009.PBoV has been reported throughout the world,mostly in weaning piglets,and has a broad range of tissue tropism.Since PBoV is prevalent in healthy as well as clinically infected pigs and is mostly associated with coinfection with other viruses,the pathogenic nature of PBoV is still unclear.Currently,there are no cell lines available for the study of PBoV,and animal model experiments have not been described.This review summarizes the current state of knowledge about PBoV,including the epidemiology,evolution analysis,detection methods,pathogenesis and public health concerns.

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.

Ungulate bocaparvovirus 4 and rodent bocavirus are different genotypes of the same species of virus

Bocaviruses are associated with many human infectious diseases, such as respiratory tract infections, gastroenteritis, and hepatitis. Rats are known to be reservoirs of bocaviruses, including rodent bocavirus and rat bocavirus.Recently, ungulate bocaparvovirus 4, a known porcine bocavirus, has also been found in rats. Thus, investigating bocaviruses in rats is important for determining the origin of the viruses and preventing and controlling their transmission. To the best of our knowledge, no study to date has investigated bocaviruses in the livers of rats. In this report, a total of 624 rats were trapped in southern China between 2014 and 2017. Liver and serum samples from rats were tested for the prevalence of bocaviruses using PCR. Sequences related to ungulate bocaparvovirus 4and rodent bocavirus were detected in both liver and serum samples. Interestingly, the prevalence of ungulate bocaparvovirus 4(reference strain: KJ622366.1) was higher than that of rodent bocavirus(reference strain:KY927868.1) in both liver(2.24% and 0.64%, respectively) and serum samples(2.19% and 0.44%, respectively).The NS1 regions of ungulate bocaparvovirus 4 and rodent bocavirus related sequences displayed over 84% and88% identity at the nucleic acid and amino acid levels, respectively. Furthermore, these sequences had similar genomic structure, genomic features, and codon usage bias, and shared a common ancestor. These viruses also displayed greater adaptability to rats than pigs. Our results suggested that ungulate bocaparvovirus 4 and rodent bocavirus may originate from rats and may be different genotypes of the same bocavirus species.

2014～2015年黑龙江牡丹江地区犬博卡病毒的分子流行病学调查

为了调查黑龙江牡丹江地区犬博卡病毒流行情况,在2014年5月至2015年4月,在黑龙江牡丹江地区采集40份腹泻犬的粪便拭子样品,利用PCR扩增方法进行鉴定和混合感染检测,进一步做进化树进行分析。检测结果显示,在40份样品中,8份样品为CBoV阳性（20%,8/40）;在8份CBoV阳性样品中,与CPV-2、CCoV、CaKV混合感染率分别为37.3%（3/8）、25%（2/8）、25%（2/8）。序列分析表明,8个CBoV毒株的NS1片段在核苷酸水平的同源性为94.2%～100%,在氨基酸水平的同源性为91.7%~100%。进化树分析显示,与韩国、美国、德国和中国香港的CBoV毒株相比,8个中国CBoV毒株表现出遗传多样性。研究揭示了黑龙江牡丹江地区流行的CBoV毒株显示出遗传多样性以及与其他肠道病毒的混合感染情况。