Surveillance and molecular characterization of human influenza B viruses during 2006–2010 revealed co-circulation of Yamagata-like and Victoria-like strains in eastern India

Acute respiratory illness (ARI) is one of the major health problems in tropical countries of Asia, like India where approximately 0.5 million children in the age group of <5 years die annually. Previously we have reported the genetic characterization of influenza A (Inf-A) strains circulating in Kolkata, eastern India. This study was initiated to characterize the genetic diversity of the circulating influenza B (Inf-B) viruses. Of 3035 nasal/throat swabs, 494 (16.3%) samples were identified as influenza A/B positive by real time RT-PCR, of which 244 samples were confirmed having Inf-B infection. Comparison of nucleotide (nt) and amino acid (aa) sequences of HA and NA gene of Inf-B viruses revealed co-circulation of B/Yamagata and B/Victoria lineages. Of the 32 randomly selected Inf-B strains from Kolkata, seventeen strains possessed reassorted NA gene. There was a single Histidine to Asparagine substitution in the 131st position which is a part of 120 loop on HA1 region along with a deletion at position 178 in the Kolkata strains belonging to the Yamagata lineage. Amino acid substitution was observed at position 198 on NA gene in the strains B/Kol/542/2006, B/Kol/1373/2008, B/Kol/1880/2008, B/Kol/2044/2008 and in all the representative strains isolated during 2009 with respect to the circulating vaccine strains. This substitution is responsible for reduced sensitivity of neuraminidase inhibitors. The results highlight the importance of monitoring Inf-B viruses for development of antiviral resistance among circulating strains.     

Molecular detection and characterization of Influenza ‘C’ viruses from western India

Since 2003, India has had a well-established influenza surveillance network, though Influenza C virus was not the focus of study. We therefore retrospectively analyzed clinical samples from Pune, western India collected during January 2009 to August 2015, by real-time RT-PCR. Three of 2530 samples of patients with influenza-like illness (ILI) or severe acute respiratory illness (SARI) showed positivity for Influenza C virus infection, while 105 and 31 samples were positive for Influenza A and B viruses respectively. Influenza C viruses were successfully isolated using the embryonated egg system and whole genomes were sequenced and analyzed phylogenetically. HE gene-based phylogeny showed that two viruses C/India/P119564/2011 and C/India P121719/2012 clustered with the C/Sao Paulo/378/82 (SP82) lineage, whereas C/India/P135047/2013 clustered with the C/Kanagawa/1/76 (KA76) lineage. The internal gene of these viruses grouped in two lineages. The PB1, PB2, M and NS genes of the study viruses grouped with C/Yamagata/26/81 (YA81), while the P3 (PA) and NP genes grouped with C/Mississippi/80 (MS80). Bayesian clock studies conclude that the Indian strains may have emerged through multiple reassortment events.     

Molecular genetic analysis of the Influenza A(H1N1)pdm09 virus from lethal and recovered cases in Russia from 2009 to 2014: Deletions in the nucleoprotein

Influenza A(H1N1)pdm09 virus caused about 2000 laboratory confirmed lethal cases in Russia during 2009–2010 and 1302, 135 and 29 cases in the 2010–2011, 2012–2013 and 2013–2014 seasons respectively. The on average short duration (7.8 ± 5 days) of lethal cases of Influenza A(H1N1)pdm09 infections in Russia suggests primary viral rather than secondary bacterial pneumonia. Hemorrhagic syndrome was recorded in 36.6% of patients. An examination of 221 lung samples from lethal influenza cases for the presence of bacterial DNA that could cause pneumonia did not reveal bacterial superinfections in 86% of cases. Molecular-genetic analyses of Influenza A(H1N1)pdm09 viruses from lethal and recovered cases were performed. Amino acids G and N at position 222 of the influenza virus hemagglutinin, which increase the affinity for the lower respiratory tract receptors, were detected more often in the lungs of patients who died than in respiratory swabs collected from recovered patients (p < 0.0001 and p = 0.007). Viruses harboring various mutations (222D/G/N/S) was significantly associated with lung samples compared with respiratory swabs from recovered patients (p < 0.0001). Amino acid 222E, which increases the affinity for upper respiratory tract receptors, was found more frequently in recovered patients than in patients with lethal disease (27% versus 3%, p = 0.005). Phylogenetic analysis identified an isolated cluster of viruses in the 2009–2010 season that harbored amino acid 222E, which could explain the high transmissibility of the virus at the beginning of the pandemic. Bayesian skyline plot implied a decline in the effective population size of Influenza A(H1N1)pdm09 viruses in Russia from 2010–2011 to 2011–2012, followed by an increase in 2012–2013; this trend was accompanied by the increased genetic diversity of the hemagglutinin antigenic sites. Mutations of viral RNA leading to oseltamivir resistance were found in 2.8% of tested patients during only 2010–2011 season. Deletions in the nucleoprotein cDNA were found in influenza viruses from two patients.     

Which influenza vaccine formulation should be used in Kenya? A comparison of influenza isolates from Kenya to vaccine strains, 2007–2013

IntroductionEvery year the World Health Organization (WHO) recommends which influenza virus strains should be included in a northern hemisphere (NH) and a southern hemisphere (SH) influenza vaccine. To determine the best vaccine formulation for Kenya, we compared influenza viruses collected in Kenya from April 2007 to May 2013 to WHO vaccine strains.MethodsWe collected nasopharyngeal and oropharyngeal (NP/OP) specimens from patients with respiratory illness, tested them for influenza, isolated influenza viruses from a proportion of positive specimens, tested the isolates for antigenic relatedness to vaccine strains, and determined the percentage match between circulating viruses and SH or NH influenza vaccine composition and schedule.ResultsDuring the six years, 7.336 of the 60,072 (12.2%) NP/OP specimens we collected were positive for influenza: 30,167 specimens were collected during the SH seasons and 3717 (12.3%) were positive for influenza; 2903 (78.1%) influenza A, 902 (24.2%) influenza B, and 88 (2.4%) influenza A and B positive specimens. We collected 30,131 specimens during the NH seasons and 3978 (13.2%) were positive for influenza; 3181 (80.0%) influenza A, 851 (21.4%) influenza B, and 54 (1.4%) influenza A and B positive specimens. Overall, 362/460 (78.7%) isolates from the SH seasons and 316/338 (93.5%) isolates from the NH seasons were matched to the SH and the NH vaccine strains, respectively (p < 0.001). Overall, 53.6% and 46.4% SH and NH vaccines, respectively, matched circulating strains in terms of vaccine strains and timing.ConclusionIn six years of surveillance in Kenya, influenza circulated at nearly equal levels during the SH and the NH influenza seasons. Circulating viruses were matched to vaccine strains. The vaccine match decreased when both vaccine strains and timing were taken into consideration. Either vaccine formulation could be suitable for use in Kenya but the optimal timing for influenza vaccination needs to be determined.     

Effect of seasonal vaccination on the selection of influenza A/H3N2 epidemic variants

The effect of vaccination on the dynamics of influenza virus variants remains largely unknown in humans, unlike in poultry. In this study, we compared influenza hemagglutinin (HA) gene sequences isolated from vaccinated and unvaccinated populations with the yearly vaccine strains. In total, 181 influenza A/H3N2 virus samples isolated from 82 vaccinated and 99 unvaccinated patients (2011–15, four Japanese influenza seasons) were genetically analyzed using a next-generation sequencer. Amino acid (AA) differences from corresponding vaccine strains were found in 74 of 329 HA1 sites. There was a maximum of four AA differences within the epitopes in the former three seasons (2011–14) and fifteen in the latter season (2014–15). Deviation to a greater number of AA differences was found more significantly in the isolates from vaccinated patients as compared to unvaccinated patients (P = 0.0005 in 2011–14; P = 0.0096 in 2014–15). AA difference rates within epitopes were also significantly higher in the isolates from vaccinated patients than from unvaccinated patients (2.64% vs. 2.14% for 2011–14, P = 0.033; 7.78% vs. 6.59% for 2014–15, P = 0.058). The AA differences at seven sites (48I-278K, 128A-142G, 145S, 158K, and 193S) became dominant in the following seasons. In all of these sites, the dominance was retained during the mismatch of isolates with the vaccine strains and was lost after vaccine match. Our data suggest that in humans, immune pressure induced by vaccination works to select influenza variants genetically distant from vaccine strains.     

Genotype circulation pattern of human respiratory syncytial virus in Iran

In order to have information on the molecular epidemiology and genetic circulation pattern of human respiratory syncytial virus (HRSV) in Iran, we studied the genetic variability of both group A and B HRSV strains during seven consecutive years by sequencing the hypervariable C-terminal domain of G protein. A total of 485 children <2 years of age who were negative for influenza viruses, screened for the presence of HRSV in this research. HRSV was detected in 94 (19.38%) of the samples using nested RT-PCR. Group A viruses were isolated during each year, while group B viruses were isolated during 2009 and 2013. Phylogenetic analysis showed that all HRSV group A viruses belonged to three genotypes: GA1, GA2, GA5 and the group B viruses were in BA genotype.     

Genetic variability of human respiratory syncytial virus in Pune,Western India

Human respiratory syncytial virus (RSV) is one of the most important respiratory viruses causing acute respiratory tract infections amongst children. Based on genotyping of the attachment glycoprotein (G) gene, it is divided into two groups, RSV-A and RSV-B. Infection with one group does not confer immunity against the other and children infected with one antigenic group are more likely to be reinfected with the heterologous group. We tested 854 samples of patients with influenza like illness (ILI)/severe respiratory illness (SARI) during the period 2009–2012 for RSV using a conventional multiplex RT-PCR and found 159 (18.61%) samples to be positive for RSV of which 130 (15.22%) were positive for RSV-B and 29 (3.39%) for RSV-A suggesting that RSV-B was the predominant group circulating in Western India during the study period. Seasonal RSV outbreaks were observed in the monsoon and winter months. RSV was more prevalent amongst children in the 0–24 month age group (21.53%) in comparison to children in the 24–60 month age group (13.01%). Phylogenetic analysis using the G gene of 27 representative RSV-A positive samples revealed that all sequences belonged to the NA1 genotype. Of these, 5 sequences exhibited the novel 72 nucleotide duplication in the C-terminal of the G gene first reported from Ontario, Canada and clustered in the newly designated ON1 genotype. Also, 32 of the 33 RSV-B sequences exhibited the 60 nucleotide duplication associated with genotype BA and phylogenetic analysis showed that these sequences belonged to the genotype BA9 and BA12. We also found one RSV-B sequence belonging to genotype GB2, which has not been previously reported in India.     

Genetic drift influenza A(H3N2) virus hemagglutinin (HA) variants originated during the last pandemic turn out to be predominant in the 2011–2012 season in Northern Italy

Influenza A(H3N2) virus is once again the predominant strain after the 2009 pandemic. Its molecular epidemiology and phylogeny were investigated during the 2011–2012 season in Northern Italy.The epidemiological and virological influenza surveillance was carried out within the framework of the Italian Influenza Surveillance Network. The hemagglutinin (HA) gene of the A(H3N2) viruses detected was analyzed by means of a time-scaled phylogenetic approach.In Northern Italy, the 2011–2012 epidemic wave was sustained almost exclusively by influenza A(H3N2) viruses (87.2% of total influenza virus detections). The consultation rates for influenza-like illness (ILI) in the age group ⩾65 years were 1.5 to 6-fold higher than those registered during the previous eight epidemics: A(H3N2) was the only virus identified in this group. The phylogenetic analysis of A(H3N2) viruses showed viruses belonging to the A/Victoria/208/2009 genetic clade, characterized by substitutions in HA antigenic sites with respect to the A/Perth/16/2009-like 2011–2012 vaccine strain. About one-third of analyzed sequences fell into group 6 and two thirds into group 3 (subdivided into 3A, 3B, and 3C). The time scale reconstruction of the phylogeny showed several independent introductions of A(H3N2) groups between summer and winter of 2011. However, the common origin of all the circulating A(H3N2) strains dated back to the 2009 pandemic period (November 2009).The time scale phylogenetic approach is of particular importance for the evaluation of the introduction and circulation of new variants in the area. Therefore, it should be implemented within the framework of influenza virological surveillance.     

Avian influenza virus ecology in Iceland shorebirds: Intercontinental reassortment and movement

Shorebirds are a primary reservoir of avian influenza viruses (AIV). We conducted surveillance studies in Iceland shorebird populations for 3 years, documenting high serological evidence of AIV exposure in shorebirds, primarily in Ruddy Turnstones (Arenaria interpres; seroprevalence = 75%). However, little evidence of virus infection was found in these shorebird populations and only two turnstone AIVs (H2N7; H5N1) were able to be phylogenetically examined. These analyses showed that viruses from Iceland shorebirds were primarily derived from Eurasian lineage viruses, yet the H2 hemagglutinin gene segment was from a North American lineage previously detected in a gull from Iceland the previous year. The H5N1 virus was determined to be low pathogenic, however the PB2 gene was closely related to the PB2 from highly pathogenic H5N1 isolates from China. Multiple lines of evidence suggest that the turnstones were infected with at least one of these AIV while in Iceland and confirm Iceland as an important location where AIV from different continents interact and reassort, creating new virus genomes. Mounting data warrant continued surveillance for AIV in wild birds in the North Atlantic, including Canada, Greenland, and the northeast USA to determine the risks of new AI viruses and their intercontinental movement in this region.     

Particle quantification of influenza viruses by high performance liquid chromatography

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris–HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 10⁹ and 1 × 10¹¹ virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 10⁸ and 4.35 × 10⁹ whereas the limit of quantification was between 6.90 × 10⁸ and 1.45 × 10¹⁰ VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.     

Does seasonal vaccination affect the clinical presentation of influenza among the elderly? A cross-sectional analysis in the outpatient setting in France, 2003–2014

Vaccine-induced protection against influenza is not optimal, however it has been suggested that the vaccine may reduce the severity of symptoms among those who develop illness despite being vaccinated. We tested this hypothesis within a countrywide, sentinel general practitioners-based surveillance system in France. We included 2277 individuals aged 65 years or older (of whom 1293 had been vaccinated against influenza, 56.8%) who consulted a general practitioner because of an acute respiratory infection (ARI) during 2003–2014. All patients were taken a nasopharyngeal swab, and information was collected on demographic characteristics and symptoms at disease onset. All specimens were tested for respiratory viruses and, if positive for influenza, the virus type and subtype were determined. We compared the average maximum temperature and the frequency of each symptom, between non-vaccinated and vaccinated influenza patients. We then used logistic regression models to calculate the odds of presenting with each symptom between vaccinated vs. non-vaccinated patients, adjusting by age group, virus (sub)type and season. Overall, 675 ARI patients (29.6%) tested positive for influenza. The A(H3) virus caused the majority of cases (55.1%), followed by influenza B (22.9%), A not-subtyped (11.7%), and A(H1) (10.3%) viruses. Compared to non-vaccinated influenza patients, those who had been vaccinated had a slightly reduced maximum temperature and presented less frequently with myalgia, shivering and headache. In stratified analyses, the observed effect was limited to patients infected with A(H3) or type B viruses. After adjusting by age group, virus (sub)type and season, the difference remained statistically significant only for headache, which was less frequent among vaccinated individuals (odds ratio 0.69, 95% confidence intervals 0.48–0.98). In conclusion, the vaccine was found to be modestly associated with less severe clinical presentation of influenza among the elderly. Our findings reinforce the need for influenza vaccines providing better protection.     

Visualizing knowledge and attitude factors related to influenza vaccination of physicians

PurposeTo characterize groups of primary healthcare physicians according to sociodemographic data, years of professional experience and knowledge of and attitudes to influenza, and to evaluate differences between groups with respect to influenza vaccination in the 2011–2012 season.MethodsWe carried out an anonymous web survey of Spanish primary healthcare physicians in 2012. Information on vaccination, and knowledge of and attitudes to influenza was collected. Multiple correspondence analysis and cluster analysis were used to define groups of physicians.ResultsWe included 835 physicians and identified three types. Type B were physicians with low professional experience of influenza. Types A and C were physicians with high professional experience with influenza, type A also had a high awareness of influenza and seasonal vaccination. Types A and C were older and more often male than type B (p < 0.0001). Knowledge of influenza was greatest in type A and lowest in type B. Awareness of influenza was greatest in type A and lowest in type C. In type A, 71.0% of physicians were vaccinated in the 2011–2012 season, compared with 48.1% and 33.6% from types B and C, respectively (p < 0.001).ConclusionsAdditional efforts should be made to increase interest and concerns about preventing the transmission of influenza in physicians who do not believe influenza is a severe disease and are not concerned about its transmission.     

Molecular detection and characterization of respiratory syncytial virus B genotypes circulating in Pakistani children

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infections in young children, but very little is known about its epidemiology and circulating genotypes in Pakistan. This study analyzed the epidemiological and molecular characteristics of RSV B genotypes in Pakistani children below 5 years with acute respiratory tract infections (ARIs) during three consecutive winter seasons from 2010 to 2013. A total of 1941 samples were analyzed for RSV infection by real time PCR and 24% (472/1941) samples were found positive out of which 22.3% (105/472) were sub-typed as RSV-B. The frequency of outpatient cases was higher (62.5%; 295/472) as compared to hospitalized patients (37.5%; 177/472). Patient ages ranged from 2 month to 5 years with a mean age of 1.48 ± 1.2 (years) and a median age of 1 year. Children below one year made up the highest percentage of enrolled subjects and male to female ratio of RSVB positive cases was nearly equivalent (1:1.1). The most common clinical symptoms were cough (96%), fever (80%) and sore throat (50%). All Pak RSVB strains ascribed to the BA genotype showing 91.9–97.1% and 86.2–95.3% homology at the nucleotide and amino acid levels respectively in comparison to BA prototype strain. On phylogenetic analysis, three genotypes of Pakistan RSV B viruses were observed; BA-9 and BA-10 which have been reported previously from other regions, and a third novel genotype assigned as BA-13 which formed a distinct cluster with protein length of 319 AA and showed 9–11 unique AA substitutions. All the RSV B isolates had two potential N-glycosylation sites in HVR2 of G protein and with heavy O-glycosylation of serine and threonine residues (G scores of 0.5–0.7). This study highlights the diversity of RSVB viruses and the significance of RSV as a dominant viral etiologic agent of pediatric ARI. It also emphasizes the need for continued molecular surveillance for early detection of prevalent and newly emerging genotypes to understand epidemiology of RSV infections in various regions of Pakistan.     

Egg-adaptive mutations in H3N2v vaccine virus enhance egg-based production without loss of antigenicity or immunogenicity

The recently detected zoonotic H3N2 variant influenza A (H3N2v) viruses have caused 343 documented cases of human infection linked to contact with swine. An effective vaccine is needed for these viruses, which may acquire transmissibility among humans. However, viruses isolated from human cases do not replicate well in embryonated chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we sought to identify egg-adaptive mutations in surface proteins that increase the yield of candidate vaccine viruses (CVVs) in eggs while preserving their immunizing effectiveness. After serial passage of a representative H3N2v isolate (A/Indiana/08/2011), we identified several egg-adaptive combinations of HA mutations and assessed the egg-based replication, antigenicity, and immunogenicity of A/Puerto Rico/8/34 (H1N1, PR8)-based 6 + 2 reverse genetics CVVs carrying these mutations. Here we demonstrate that the respective combined HA substitutions G186₁V + N246₁K, N165₁K + G186₁V, T128₁N + N165₁K + R76₂G, and T128₁N + N165₁K + I10₂M, all identified after egg passage, enhanced the replication of the CVVs in eggs without substantially affecting their antigenicity or immunogenicity. The mutations were stable, and the mutant viruses acquired no additional substitutions during six subsequent egg passages. We found two crucial mutations, G186V, which was previously defined, and N246K, which in combination improved virus yield in eggs without significantly impacting antigenicity or immunogenicity. This combination of egg-adaptive mutations appears to most effectively generate high egg-based yields of influenza A/Indiana/08/2011-like CVVs.     

Genetic characterization of enterovirus strains identified in Hand,Foot and Mouth Disease (HFMD): Emergence of B1c,C1 subgenotypes,E2 sublineage of CVA16, EV71 and CVA6 strains in India

Hand, Foot and Mouth disease (HFMD) is a common childhood disease and caused due to Enterovirus-A (EV-A), EV-B and EV-C species worldwide. Cases of HFMD were reported from, Ahmedabad (Gujarat, 2012) and Pune (Maharashtra, 2013–2014) in India. The present study highlights the identification of EV strains (CVA16, CVA6, CVA4 and Echo12), characterization of subgenotypes of CVA16, CVA6 strains during 2012–14 and CVA16, CVA6, EV71 strains reported from the earlier study (2009–10) in HFMD cases from India. A total 158 clinical specimens collected from 64 HFMD cases (2012–2014) were included in the study. EV detection was carried out by 5′NCR based RT-PCR, molecular typing and subgenotyping was by VP1/2A junction or VP1, full VP1 gene amplification respectively followed by phylogenetic analysis. The present study reports 63.92% (101/158) EV positivity by RT-PCR. Ninety four of the 101 (93.06%) EV positive strains were amplified by VP1/2A junction or VP1 regions. Sequence analysis revealed the presence of CVA16 (61.7%), CVA6 (34.04%), CVA4 and Echo12 (4.3%). A total of 114 EV positive strains were genotyped using full and partial VP1 region. All CVA16 Indian strains (n = 70) clustered with rarely reported B1c subgenotype, CVA6 (n = 43) and EV71 (n = 1) strains clustered with sub-lineage E2 and C1 subgenotypes respectively. In summary, the study reports genetic characterization of CVA16, CVA6, CVA4 and Echo12 strains in HFMD cases from India. Circulation of B1c subgenotype of CVA16, E2 sub-lineage of CVA6 and C1 subgenotype of EV 71 strains in HFMD cases were reported for the first time from India. This study helps to understand the genotype distribution, genetic diversity of EV strains associated with HFMD from Eastern, Western and Southern regions in India.     

Detection of novel porcine bocaviruses in fecal samples of asymptomatic pigs in Cameroon

Improvements and widespread use of nucleic acid amplification and sequencing methods have led to the recognition of new virus diversity in various domestic animals, including pigs. In this study we utilized either virus species specific or broadly reactive PCR assays to describe the occurrence and genetic diversity of selected DNA viruses belonging to families Adenoviridae, Circoviridae, Anelloviridae and Parvoviridae in Cameroonian pigs. Fecal specimens were collected during spring of 2011. No adenoviruses, circoviruses and anelloviruses were detected, however, high prevalence and remarkable genetic diversity within the identified parvoviruses and, particularly, within bocaviruses was observed. PPV4 was the most prevalent virus (20%), followed by PBoV3 (18%), PBoV4 (18%), PBoV5 plus 6V/7V (16%), and PBoV1 plus PBoV2 (6%). The frequency of mixed infections with various combinations of these virus species reached 20%. Genetic analysis of the identified viruses showed that the capsid gene of PBoV1 and PBoV2 strains shared up to 91% and 94% nt sequence similarities to reference PBoV1 and PBoV2 strains, respectively. The identified PBoV3 and PBoV4 strains shared ⩽95% and ⩽98% nt identities with reference PBoV3 and PBoV4 strains, respectively, along the NS gene, whereas the PBoV5 strains shared 86% nt identities with Hungarian and 87% nt identities with Chinese PBoV5 strains along the capsid gene. In addition, a single PBoV5-like strain shared ⩽71% nt sequence identity with other PBoV5 strains. This is the first study to report evidence of the circulation of bocaviruses in Africa and contributes to our understanding of the impact of globalization on the dispersal of new and emerging viruses.