Evolutionary time-scale of primate bocaviruses

Human bocavirus (HBoV) is associated with acute gastroenteritis in humans, occurring mostly in young children and elderly people. Four bocavirus genotypes (HBoV1–HBoV4) have been found so far. Since there were no data on the contribution of HBoV to gastroenteritis in Russia, 1781 fecal samples collected from infants hospitalized with acute gastroenteritis in Novosibirsk, Russia during one year were tested for the presence of nucleic acids from HBoV and three major gastrointestinal viruses (rotavirus A, norovirus II, and astrovirus). HBoV was detected only in 1.9% of the samples: HBoV1 was detected in 0.6% and HBoV2, in 1.3%. Complete genome sequencing of three Novosibirsk isolates was performed. An evolutionary analysis of these sequences and the available sequences of human and great apes bocaviruses demonstrated that the current HBoV genotypes diverged comparatively recently, about 60–300 years ago. The independent evolution of bocaviruses from chimpanzees and gorillas commenced at the same time period. This suggests that these isolates of great apes bocaviruses belong to separate genotypes within the species of human bocavirus, which is actually the primate bocavirus. The rate of mutation accumulation in the genome of primate bocaviruses has been estimated as approximately 9 × 10^–4 substitutions/site/year. It has been demonstrated that HBoV1 diverged from the ancestor common with chimpanzee bocavirus approximately 60–80 years ago, while HBoV4 separated from great apes bocaviruses about 200–300 years ago. The hypothesis postulating independent evolution of HBoV1 and HBoV4 genotypes from primate bocaviruses has been proposed.     

Human bocavirus in hospitalized children with acute gastroenteritis in Russia from 2010 to 2012

Human bocavirus (HBoV) can cause respiratory diseases and is detectable in the stool samples of patients with gastroenteritis. To assess the prevalence of HBoV in children hospitalized with acute gastroenteritis in Novosibirsk, Russia, as well as its genetic diversity and the potential role in the etiology of gastroenteritis in this region, a total of 5502 stool samples from children hospitalized with gastroenteritis from 2010 to 2012, n = 5250, and healthy children, n = 252, were assayed for the presence of HBoV DNA by semi-nested PCR. The HBoV DNA was found in 1.2% of stool samples from children, with gastroenteritis varying from 0.5% in 2012 to 1.7% in 2011. The prevalence of HBoV in healthy children was 0.3%. HBoV strains were detected throughout the year with an increase in the fall–winter season. In 87% of cases, HBoV was detected in children before 1 year of age. All known HBoV genetic variants have been detected in Novosibirsk, although with different prevalences: HBoV2 > HBoV1 > HBoV4 > HBoV3. At the beginning of 2011, HBoV2 replaced HBoV1 as the most prevalent variant. The median age of children with detected HBoV1 was 8.3 months, and that with HBoV2 was 8.0 months. All HBoV-positive samples were assayed for the presence of the rotaviruses A and C, norovirus GII, astrovirus, enterovirus, adenovirus F, Salmonella spp., Campylobacter spp., Shigella spp., and EIEC. HBoV1 and HBoV2 as single agents were found in 45.8% and 60% samples, respectively, although this difference was not statistically significant. In the case of co-infections, HBoV was most frequently recorded with rotavirus A and norovirus GII. This study demonstrated that the detection rate of HBoV in stool samples from children with gastroenteritis was low, although both HBoV1 and HBoV2 could be found as the sole agents in children with gastroenteritis in Novosibirsk.     

杭州地区急性呼吸道感染患儿人博卡病毒的检测及分析

目的了解杭州地区人博卡病毒(HBoV)的流行情况和基因变异情况。方法对杭州地区160例儿童急性呼吸道感染患者咽拭子样本进行HBoV检测,并对阳性样本进行常见呼吸道病毒的检测,最终选取HBoV单一阳性的HZ1株和HZ2株进行VP1基因扩增,将获得的序列上传Genbank并与其他国家和地区的8株HBoV的VP1基因进行序列比对和分析。结果杭州地区HBoV的检出率为6.9%(11/160),与其他常见呼吸道病毒的合并感染率达到45.5%(5/11),HZ1株和HZ2株与其他国家和地区的8株HBoV的VP1基因相似性达到99%以上。结论研究证明杭州地区急性呼吸道感染患儿中存在HBoV的感染,与其他呼吸道病毒有较高的合并感染,从分离到的HZ1株和HZ2株来看,杭州地区流行的是ST2基因型,其外壳蛋白VP1基因变异很小。     

Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4

Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1–4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011–11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2.     

天津地区儿童人博卡病毒感染及基因型分析

目的 了解天津地区急性下呼吸道感染儿童中人博卡病毒(HBoV)的感染情况及病毒基因特性.方法 采集2008年9月- 2009年5月急性下呼吸道感染住院儿童的鼻咽吸取物标本202份,进行PCR扩增来检测HBoVNP-1基因,对VP1及VP2基因进行扩增及序列测定,与GenBank中登录的序列进行比较,分析序列特征并绘制种系发生树,同时采用多重PCR方法检测其他12种呼吸道病毒及WU多瘤病毒.结果 202份标本中,6.9％( 14/202)为HBoV阳性,所有阳性患儿的年龄均≤6岁,6月～12月龄患儿感染率最高,为14.3％ (6/42);所有感染者均为肺炎和喘息性支气管炎患儿,各月份均有检出;HBoV与其他病毒的混合感染率为85.7％ (12/14),其中58.3％ (7/12)为3种以上呼吸道病毒混合感染;VP1/VP2基因种系发生树分析表明13株HBoV均属于Ib簇,同源性分析表明,天津地区HBoV VP1基因与从GenBank下载的Ⅰ型序列比较,核酸及氨基酸的同源性分别为99.0％～ 99.8％及99.0％ ～99.9％.结论 天津地区存在HBoV流行,感染者多为＜6岁儿童,HBoV与其他呼吸道病毒混合感染常见,HBoV病毒均属于Ib簇.     

Circulating of human bocavirus 1, 2, 3, and 4 in pediatric patients with acute gastroenteritis in Thailand

Human bocavirus (HBoV) is a newly identified human parvovirus that associated with respiratory and gastrointestinal diseases. Epidemiological surveillance of HBoV was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. Among a total of 222 fecal specimens tested, 17 (7.7%) were positive for HBoV by PCR. Of the 17 HBoV positive samples, double- or triple-infections together with other enteric viruses were found in 10 (58.8%) pediatric patients, while monoinfection with HBoV alone was detected in seven (41.2%) cases. Mixed infection among HBoV with norovirus GII was frequently observed in this population. The partial VP1 nucleotide sequences of all 17 HBoV strains demonstrated that all four species of HBoV were found in the specimens tested. Eleven strains were HBoV1. Other three strains showed the sequence identity with HBoV2, which were most closely related to the HBoV2A. In addition, other two HBoV strains showed the highest level of nucleotide sequence identity with the HBoV3. It was surprisingly to observe that one Thai HBoV strain showed a unique characteristic similar to the HBoV4, a rare species of HBoV found in acute gastroenteritis patients. In summary, this study presents the genetic background information of HBoV circulated in acute gastroenteritis children in Chiang Mai, Thailand and it was clearly demonstrated that HBoVs circulated in this area were genetically diverse as all four species of HBoVs (HBoV1-4) were detected in the fecal specimens collected from pediatric patients admitted to the hospitals in this area.     

Genetic analysis of human rhinovirus species A to C detected in patients with acute respiratory infection in Kumamoto prefecture,Japan 2011–2012

We performed detailed genetic analysis of the VP4/VP2 coding region in human rhinovirus species A to C (HRV-ABC) strains detected in patients with a variety of acute respiratory infections in Kumamoto, Japan in the period 2011–12. The phylogenetic tree and evolutionary timescale were obtained by the Bayesian Markov chain Monte Carlo method. Phylogenetic analyses showed that the present HRV-A, -B, and -C strains belonged to 25, 4, and 18 genotypes, respectively. Some new genotypes were confirmed as prevalent strains of HRV-C. An ancestor of the present HRV-ABCs could be dated back to about 20,000 years ago. The present HRV-A and -C strains have wide genetic divergence (pairwise distance >0.2) with rapid evolutionary rates (around 7 × 10⁻⁴ to 4 × 10⁻³ substitutions/site/year). Over 100 sites were found to be under negative selection, while no positively selected sites were found in the analyzed region. No evidence of recombination events was found in this region of the present strains. Our results indicate that the present HRV strains have rapidly evolved and subsequently diverged over a long period into multiple genotypes.     

Impact of rotavirus vaccine on all-cause diarrhea and rotavirus hospitalizations in Madagascar

BackgroundRotavirus vaccine was introduced into the Extended Program on Immunization in Madagascar in May 2014. We analyzed trends in prevalence of all cause diarrhea and rotavirus hospitalization in children <5 years of age before and after vaccine introduction and assessed trend of circulating rotavirus genotypes at Centre Hospitalier Universitaire Mère Enfant Tsaralalàna (CHU MET).MethodsFrom January 2010 to December 2016, we reviewed the admission logbook to observe the rate of hospitalization caused by gastroenteritis among 19619 children <5 years of age admitted at the hospital. In June 2013–December 2016, active rotavirus surveillance was also conducted at CHUMET with support from WHO. Rotavirus antigen was detected by EIA from stool specimen of children who are eligible for rotavirus gastroenteritis surveillance at sentinel site laboratory and rotavirus positive specimens were further genotyped at Regional Reference Laboratory by RT-PCR.ResultsDiarrhea hospitalizations decreased after rotavirus vaccine introduction. The median proportion of annual hospitalizations due to diarrhea was 26% (range: 31–22%) before vaccine introduction; the proportion was 25% the year of vaccine introduction, 17% in 2015 and 16% in 2016. Rotavirus positivity paralleled patterns observed in diarrhea. Before vaccine introduction, 56% of stool specimens tested positive for rotavirus; the percent positive was 13% in 2015, 12% in 2016. Diverse genotypes were detected in the pre-vaccine period; the most common were G3P[8] (n = 53; 66%), G2P[4] (n = 12; 15%), and G1P[8] (n = 11; 14%). 6 distinct genotypes were found in 2015; the most common genotype was G2P[4] (n = 10; 67%), the remaining, 5, G12[P8], G3[P8], G1G3[P4], G3G12[P4][P8] and G1G3[NT] had one positive specimen each.ConclusionsFollowing rotavirus vaccine introduction all-cause diarrhea and rotavirus-specific hospitalizations declined dramatically. The most common genotypes detected in the pre-vaccine period were G3P[8] and G2P[4] in 2015, the post vaccine period.     

Tracking the molecular epidemiology of Brazilian Infectious bursal disease virus (IBDV) isolates

Infectious bursal disease is a highly contagious disease of young chickens caused by Infectious bursal disease virus (IBDV). Genome segment A encodes the capsid protein (VP2), while segment B encodes the RNA-dependent RNA polymerase (VP1). In the present study, we trace the molecular epidemiology of IBDV in Brazil by analyzing 29 isolates collected in the major regions of poultry production. To genetically characterize the isolates, phylogenetic and population dynamic analyses were conducted using 68 VP1 (2634 nt) and 102 VP2 (1356 nt) coding sequences from IBDV isolates from different regions of the world. Furthermore, the evolution of IBDV was analyzed by characterizing the selective forces that operated during the diversification of viral isolates. We show that IBDV isolates were introduced into Brazil mainly from the Netherlands and the USA. These introductions were associated with all Brazilian poultry production regions analyzed in this work. In addition, we show that the evolution of IBDV has been shaped by a combination of very low recombination rates and relatively high rates of nucleotide substitution (2.988 × 10⁻⁴ for VP1 and 3.2937 × 10⁻⁴ for VP2), which themselves are a function of purifying selection operating on VP1 and VP2. Furthermore, our extended Bayesian skyline plot suggests that the increase in the effective population size of isolates of IBDV is consistent with its epidemiological history, with a large increase during the emergence of acute outbreaks of IBD in the 1980s.     

Molecular epidemiology of noroviruses detected in Nepalese children with acute diarrhea between 2005 and 2011: Increase and predominance of minor genotype GII.13

Noroviruses, an important cause of acute gastroenteritis, possess a highly divergent genome which was classified into five genogroups and dozens of genotypes. However, changes in genotype distribution over time were poorly understood, particularly in developing countries. We therefore conducted a molecular epidemiological study which characterized the norovirus strains detected in 4437 Nepalese children with acute diarrhea between November 2005 and January 2011 to gain insight into how their genotypes changed over time. Of the 356 samples positive for noroviruses, 277 (78%) were successfully genotyped into 22 capsid genotypes; GII.4 (n = 113), GII.3 (n = 38) and GII.13 (n = 37) were the majority. Interestingly, GII.13 accounted for only 1.7% (4/230) between 2005 and 2008 (period 1) but increased substantially to 26.2% (33/126) between 2009 and 2011 (period 2). Phylogenetic analysis of the VP1 nucleotide sequences of 35 GII.13 strains indicated that they clustered into two lineages named NPL2008 and NPL2009 to which two period 1 strains and 33 period 2 strains belonged, respectively. Lineage NPL2009 contained GII.13 strains that were detected in a large-scale gastroenteritis outbreak in Germany in 2012. Both Nepalese and German VP1 sequences carried two substitutions, H378N and V394Q, in the putative histo-blood group antigen (HBGA)-binding sites. As to the polymerase genotypes of Nepalese strains, the period 1 strains possessed GII.Pm, but the period 2 strains possessed GII.P13, GII.P16, and GII.P21. Together with recent reports on the predominance of GII.P13/GII.13 and GII.P16/GII.13 in India and GII.P16/GII.13 in European countries, this study predicts that genotype GII.13 which was previously regarded as a minor genotype has a potential to become an epidemiologically important genotype.     

Genomic constellation and evolution of Ghanaian G2P[4] rotavirus strains from a global perspective

Understanding of the genetic diversity and evolution of Rotavirus A (RVA) strains, a common cause of severe diarrhoea in children, needs to be based on the analysis at the whole genome level in the vaccine era. This study sequenced the whole genomes of six representative G2P[4] strains detected in Ghana from 2008 to 2013, and analysed them phylogenetically with a global collection of G2P[4] strains and African non-G2P[4] DS-1-like strains. The genotype constellation of the study strains was G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Strains from the same season were highly identical across the whole genome while strains from different seasons were more divergent from each other. The VP7, VP4, VP2, NSP1, and NSP5 genes belonged to lineage IVa; the VP6, VP1, NSP2, and NSP3 genes belonged to lineage V, and all these genes evolved in the same fashion as the global strains. In the NSP4 gene, lineages V (2008) and X (2009) were replaced by VI (2012/2013) whereas in the VP3 gene, lineage V (2008/2009) was replaced by VII (2012/2013) and these replacements coincided with the vaccine introduction period (2012). The evolutionary rate of the NSP4 gene was 1.2 × 10^− 3 substitutions/site/year and was rather comparable to that of the remaining 10 genes. The multiple NSP4 lineages were explained by intra-genotype reassortment with co-circulating African human DS-1-like strains bearing G2[6], G3P[6], G6[6] and G8. There was no explicit evidence of the contribution of animal RVA strains to the genome of the Ghanaian G2P[4] strains. In summary, this study revealed the dynamic evolution of the G2P[4] strains through intra-genotype reassortment events leading to African specific lineages such IX and X in the NSP4 gene. So far, there was no evidence of a recent direct involvement of animal RVA genes in the genome diversity of African G2P[4] strains.     

Recombination analysis based on the HAstV-2 and HAstV-4 complete genomes

Complete genome sequences of previously unstudied human astrovirus subgenotypes – HAstV-2a and HAstV-2c – and two isolates of a rare genotype HAstV-4 have been determined. These isolates were recovered from fecal samples of young children hospitalized with acute intestinal infections in Novosibirsk (Russia). Three of the four sequenced isolates (HAstV-2a, HAstV-2c, and HAstV-4) are recombinants. It has been shown that all known HAstV-2 genomes have emerged via recombination; the HAstV-1 and HAstV-4 genotypes contain both recombinant and non-recombinant isolates; and all HAstV-3, HAstV-5, and HAstV-6 whole-genome sequences display no reliable signs of recombination. The average mutation accumulation rate has been determined based on an extended ORF2 fragment and amounts to 1.0 × 10⁻³ substitutions per site per year. The evolutionary chronology of current HAstV genotypes has been reconstructed.     

Recombination in the evolution of human bocavirus

Whole genome sequencing of Novosibirsk human bocavirus (HBoV) isolates has detected an isolate that emerged via recombination between HBoV3 and HBoV4 genotypes. The recombination site is located between regions with abnormally low and abnormally high GC contents in the genome. This site is a bocavirus recombination hotspot and coincides with one of two parvovirus recombination hotspots. The Novosibirsk recombinant isolate, which is similar to a previously studied isolate from Thailand, utilizes the strategy of borrowing ORF3, which encodes structural proteins, of a rare genotype HBoV4. The role of recombination in HBoV evolution is discussed.     

Serum antibody response to Moraxella catarrhalis proteins OMP CD,OppA, Msp22, Hag,and PilA2 after nasopharyngeal colonization and acute otitis media in children

BackgroundThere is no licensed vaccine for Moraxella catarrhalis (Mcat), which is a prominent bacterium causing acute otitis media (AOM) in children and lower respiratory tract infections in adults. Nasopharyngeal (NP) colonization caused by respiratory bacteria results in natural immunization of the host. To identify Mcat antigens as vaccine candidates, we evaluated the development of naturally induced antibodies to 5 Mcat surface proteins in children 6–30 months of age during Mcat NP colonization and AOM.MethodsHuman serum IgG against the recombinant Mcat proteins, outer membrane protein (OMP) CD, oligopeptide permease (Opp)A, hemagglutinin (Hag), Moraxella surface protein (Msp)22, and PilA clade 2 (PilA2) was quantitated by using an ELISA assay.ResultsThere were 223 Mcat NP colonization episodes documented in 111 (60%) of 184 children in the study. Thirty five Mcat AOM episodes occurred in 30 (16%) of 184 children. All 5 Mcat candidate vaccine antigens evaluated stimulated a significant rise in serum IgG levles over time from 6 to 36 months of age (P < 0.001), with a rank order as follows: Msp22 = OppA > OMP CD = Hag = PilA2. Children with no detectable Mcat NP colonization showed a higher serum IgG level against OppA, Hag, and Msp22 compared to those with Mcat NP colonization (P < 0.05). Individual data showed that some children responded to AOM with an antibody increase to one or more of the studied Mcat proteins but some children failed to respond.ConclusionsSerum antibody to Mcat candidate vaccine proteins OMP CD, OppA, Msp22, Hag, and PilA2 increased with age in naturally immunized children age 6–30 months following Mcat NP colonization and AOM. High antibody levels against OppA, Msp22, and Hag correlated with reduced carriage. The results support further investigation of these vaccine candidates in protecting against Mcat colonization and infection.     

HCV dispersal patterns among intravenous drug users (IDUs) in Athens metropolitan area

BackgroundMost of the HCV transmission the recent years in Greece was among IDUs. Our aim was to estimate the prevalence of HCV genotypes and to investigate the patterns of HCV dispersal among IDUs in Athens using current state of the art molecular epidemiology methods.MethodsHCV sequences were determined from 238 HIV-negative IDUs collected on the basis of the “ARISTOTLE” prevention program carried out in Athens between 2012 and 2013. Phylogenetic trees were inferred on HCV sequences isolated from IDUs in Athens for the most prevalent HCV clades (subtypes 1a and 3a). Phylogenetic analysis was performed by Neighbor-Joining and Bayesian methods using GTR + G as nucleotide substitution model. HCV dispersal patterns were estimated using as references, all globally available HCV sequences for subtypes 1a and 3a.ResultsThe prevalence of HCV subtypes was: 3a (59.2%), 1a (21.9%), 4 (13.0%), 1b (5.4%) and 2 (0.5%). Phylogenetic analyses revealed that most sequences (63.5%) οf subtypes 1a and 3a fell within IDU-specific monophyletic groups. The proportion of sequences in monophyletic clades was similar for subtype 3a (62.9%) and 1a (65.3%). For the latter group, monophyletic clades were smaller in size. Multivariable logistic regression analyses showed that monophyletic clustering was marginally associated recent onset of injecting ([AOR] = 1.44; 95% CI (0.97–2.13), p = 0.068).ConclusionsThe high proportions of HCV sequences within IDU-specific monophyletic clusters suggest that transmissions occurred locally among IDUs in Greece. The numerous clusters for both 1a and 3a provide evidence that both sub-epidemics were the result of multiple introductions among the IDUs. Higher regional clustering was probably associated with a more recent onset of drug use.     

Hepatitis B virus intergenotypic recombinants worldwide: An overview

Novel variants generated by recombination events between different hepatitis B virus (HBV) genotypes have been increasingly documented worldwide, and the role of recombination in the evolutionary history of HBV is of significant research interest. In the present study, large-scale data retrieval and analysis on HBV intergenotypic recombinant genomes were performed. The geographical distribution of HBV recombinants as well as the molecular processes involved in recombination were examined. After review of published data, a total of 436 complete HBV sequences, previously identified as recombinants, were included in the recombination detection analysis. About 60% of HBV recombinants were B/C (n = 179) and C/D (n = 83) hybrids. A/B/C, A/C, A/C/G, A/D, A/E, A/G, B/C/U (U = unknown genotype), C/F, C/G, C/J, D/E, D/F, and F/G hybrids were additionally identified. HBV intergenotypic sequences were reported in almost all geographical regions with similar circulation patterns as their original genotypes, indicating the potential for spreading in a wide range of human populations and developing their own epidemiology. Recombination breakpoints were non-randomly distributed in the genome, and specific favored sites detected, such as within nt 1700–2000 and 2100–2300 regions, which displayed a statistically significant difference in comparison with the remaining genome. Elucidation of the effects of recombination events on the evolutionary history of HBV is critical to understand current and future evolution trends.     

Human bocavirus infection, People's Republic of China

A newly identified parvovirus, human bocavirus (HBoV), was found in 21 (8.3%) of 252 nasopharyngeal aspirates from hospitalized children with lower respiratory tract infection in Hunan Province, People's Republic of China. Viral loads were 10(4) to 10(10) copies/mL. Phylogenetic analysis of the VP1 gene showed a single genetic lineage of HBoV worldwide.