基于第12节段基因序列的版纳病毒分子遗传进化分析

目的 了解自1980-2012年在世界各地分离的版纳病毒分子遗传进化特征。方法 采用多种生物信息学分析方法,对1980-2012年我国及世界各地分离的版纳病毒进行基于第12节段基因序列的系统进化、分子溯源分析。结果 版纳病毒的共同进化祖先出现时间为315（95% HPD：63～619）年前,第12节段的进化速率为2.33×10^-3（95% HPD：2.84×10^-4～8.52×10^-3）碱基替换/位点/年,提示版纳病毒属于新发快速进化的虫媒病毒。结论 版纳病毒属于快速进化的新发虫媒病毒,其地域分布范围进一步扩大且已经分化出新的病毒变种,应加强对该病毒在自然界分布及其致病性的监测。     

Dating the origin and dispersal of Human Papillomavirus type 16 on the basis of ancestral human migrations

A major limitation when reconstructing the origin and evolution of HPV-16 is the lack of reliable substitution rate estimates for the viral genes. On the basis of the hypothesis of human HPV-16 co-divergence, we estimated a mean evolutionary rate of 1.47 × 10^− 7 (95% HPD = 0.64–2.47 × 10^− 7) subs/site/year for the viral LCR region. The results of a Bayesian phylogeographical analysis suggest that the currently circulating HPV-16 most probably originated in Africa about 110 thousand years ago (Kya), before giving rise to four known geographical lineages: the Asian/European lineage, which most probably originated in Asia a mean 38 Kya, and the Asian/American and two African lineages, which probably respectively originated about 33 and 27 Kya. These data closely reflect current hypotheses concerning modern human expansion based on studies of mitochondrial DNA phylogeny. The correlation between ancient human migration and the present HPV phylogeny may be explained by the co-existence of modes of transmission other than sexual transmission.     

Levels and sources of volatile organic compounds including carbonyls in indoor air of homes of Puertollano,the most industrialized city in central Iberian Peninsula. Estimation of health risk

Twenty nine organic air pollutants including carbonyl compounds, alkanes, aromatic hydrocarbons and terpenes were measured in the indoor environment of different houses together with the corresponding outdoor measurements in Puertollano, the most industrialized city in central Iberian Peninsula. VOCs were sampled during 8 weeks using Radiello^® passive samplers, and a questionnaire on potential VOCs sources was filled out by the occupants. The results show that formaldehyde and hexanal was the most abundant VOCs measured in indoor air, with a median concentration of 55.5 and 46.4 μg m⁻³, respectively followed by butanal (29.1 μg m⁻³), acetone (28.4 μg m⁻³) and acetaldehyde (21.4 μg m⁻³). After carbonyls, n-dodecane (13.1 μg m⁻³) and terpenes (α-pinene, 13.4 μg m⁻³ and limonene, 13.4 μg m⁻³) were the compounds with higher median concentrations. The indoor/outdoor (I/O) ratios demonstrated that sources in the indoor environment are prevailing for most of the investigated VOCs especially for limonene, α-pinene, hexanal, formaldehyde, pentanal, acetaldehyde, o-xylene, n-dodecane and acetone with I/O ratio >6. Multiple linear regressions were applied to investigate the indoor VOC determinants and Spearman correlation coefficients were used to establish common sources between VOCs. Finally, the lifetime cancer risk associated to formaldehyde, acetaldehyde and benzene exposure was estimated and they varied from 7.8 × 10⁻⁵ to 4.1 × 10⁻⁴ for formaldehyde, from 8.6 × 10⁻⁶ to 3.5 × 10⁻⁵ for acetaldehyde and from 2.0 × 10⁻⁶ to 1.5 × 10⁻⁵ for benzene. For formaldehyde, the attributed risk in most sampled homes was two orders of magnitude higher than the one (10⁻⁶) proposed as acceptable by risk management bodies.     

Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China

In China, several subtype avian influenza viruses consistently circulate in poultry. Numerous studies have focused on the evolution of the hemagglutinin gene; however, studies on the evolution of the matrix (M) gene are limited. In this study, a large-scale phylogenetic analysis of M gene sequences of avian influenza viruses isolated in China revealed that the M gene has evolved into six different lineages denoted as I–VI. The majority of lineages I and IV were isolated in terrestrial birds, while the majority of lineages II, III, V and VI were isolated in aquatic birds. Lineage I included 148 H9N2 subtype viruses (72.2%), lineage II comprised of 63 H6 subtype viruses (100%), and lineage IV included 157 H5 subtype viruses (97.5%). The mean substitution rates of different lineages ranged from 1.32 × 10⁻³ (lineage III) to 3.64 × 10⁻³ (lineage IV) substitutions per site per year. According to the most recent common ancestor of all lineages, lineage III was the oldest lineage, formed in 1981 or even earlier. And lineage V was the most recent, established around the year 2000. Selective pressure on M2 was stronger than that on M1. The strongest selection pressure was observed in lineage IV. In addition, site-by-site analyses of each lineage identified 8 positive selection sites, all in M2. Most of the sites (5 out of 8) were located in the extracellular domain, which is an antigen for vaccine development. The positive selection sites (amino acid positions 66, 82 and 97) are likely associated with virus budding. This study enhanced our knowledge of M gene evolution of avian influenza viruses, and is expected to improve the early detection of new viruses and lead to vaccine development.     

Voltammetric determination of vitamin B6 in food samples and dietary supplements

A simple and rapid voltammetric method based on the use of disposable screen-printed electrodes is proposed for the determination of vitamin B₆. The influence of the pH on the voltammetric response was analyzed. Estimation of the linear range (2.0 × 10⁻⁶/7.2 × 10⁻⁵ M), calibration function, limit of detection (1.5 × 10⁻⁶ M) and reproducibility was performed along with the determination of possible interferences from species present in real samples. The proposed analytical system was successfully applied for the determination of pyridoxine in multivitamin supplements, energy drinks and breakfast cereals by using the standard addition method.     

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.     

Evolution and dispersal of St. Louis encephalitis virus in the Americas

Using a Bayesian coalescent approach on a dataset of 73 envelope gene sequences we estimated substitution rates and dates of divergence for St. Louis encephalitis virus (SLEV) in the Americas. We found significant rate heterogeneity among lineages, such that “relaxed” molecular clock models were much better supported than a strict molecular clock. The mean substitution rate estimated for all SLEV was 4.1 × 10^?4 substitutions/site/year (95% HPD 2.5–5.7)—higher than previous estimates that relied on the less well-suited strict clock. Mean substitution rates for individual lineages varied from 3.7 × 10^?4 to 7.2 × 10^?4 substitutions/site/year. For the first time we also assessed the magnitude and direction of viral gene flow within the Americas. The overall direction of gene flow during the period represented by the phylogeny is from South to North, and the region between 15°N and 30°N latitude appears to be the major source of virus for the rest of North America, which is consistent with migratory birds returning to their northern breeding grounds having acquired infection while wintering in the region of the Gulf of Mexico.     

Molecular epidemiology and evolutionary dynamics of Echovirus 3 serotype

Echovirus 3 (E3) serotype has been related with several neurologic diseases, although it constitutes one of the rarely isolated serotypes, with no report of epidemics in Europe. The aim of the present study was to provide insights into the molecular epidemiology and evolution of this enterovirus serotype, while an E3 strain was isolated from sewage in Greece, four years after the initial isolation of the only reported E3 strain in the same geographical region.Phylogenetic analysis of the complete VP1 genomic region of that E3 strain and of those available in GenBank suggested three main genogroups that were further subdivided into seven subgenogroups. Further evolutionary analysis suggested that VP1 genomic region of E3 was dominated by purifying selection, as the vast majority of genetic diversity presumably occurred through synonymous nucleotide substitutions and the substitution rate for complete and partial VP1 sequences was calculated to be 8.13 × 10⁻³ and 7.72 × 10⁻³ substitutions/site/year respectively. The partial VP1 sequence analysis revealed the composite epidemiology of this serotype, as the strains of the three genogroups presented different epidemiological characteristics.     

Klebsiella pneumoniae blaKPC-3 nosocomial epidemic: Bayesian and evolutionary analysis

K. pneumoniae isolates carrying blaKPC-3 gene were collected to perform Bayesian phylogenetic and selective pressure analysis and to apply homology modeling to the KPC-3 protein. A dataset of 44 bla_kpc-3 gene sequences from clinical isolates of K. pneumoniae was used for Bayesian phylogenetic, selective pressure analysis and homology modeling. The mean evolutionary rate for bla_kpc-3 gene was 2.67 × 10^− 3 substitution/site/year (95% HPD: 3.4 × 10^− 4–5.59 × 10^–3). The root of the Bayesian tree dated back to the year 2011 (95% HPD: 2007–2012). Two main clades (I and II) were identified. The population dynamics analysis showed an exponential growth from 2011 to 2013 and the reaching of a plateau. The phylogeographic reconstruction showed that the root of the tree had a probable common ancestor in the general surgery ward. Selective pressure analysis revealed twelve positively selected sites. Structural analysis of KPC-3 protein predicted that the amino acid mutations are destabilizing for the protein and could alter the substrate specificity. Phylogenetic analysis and homology modeling of blaKPC-3 gene could represent a useful tool to follow KPC spread in nosocomial setting and to evidence amino acid substitutions altering the substrate specificity.     

Cost-effectiveness of rabies post-exposure prophylaxis in the context of very low rabies risk: A decision-tree model based on the experience of France

IntroductionBenefit-risk of different anti-rabies post-exposure prophylaxis (PEP) strategies after scratches or bites from dogs with unknown rabies status is unknown in very low rabies risk settings.Design and settingA cost-effectiveness analysis in metropolitan France using a decision-tree model and input data from 2001 to 2011.PopulationA cohort of 2807 patients, based on the mean annual number of patients exposed to category CII (minor scratches) or CIII (transdermal bite) dog attacks in metropolitan France between 2001 and 2011.InterventionsFive PEP strategies: (A) no PEP for CII and CIII; (B) vaccine only for CIII; (C) vaccine for CII and CIII; (D) vaccine+ rabies immunoglobulin (RIG) only for CIII; and (E) vaccine for CII and vaccine+ RIG for CIII.Main outcomes measuresThe number of deaths related to rabies and to traffic accidents on the way to anti-rabies centers (ARC), effectiveness in terms of years of life gained by reducing rabies cases and avoiding traffic accidents, costs, and incremental cost-effectiveness ratios (ICER) associated with each strategy.ResultsStrategy E led to the fewest rabies cases (3.6 × 10⁻⁸) and the highest costs (€1,606,000) but also to 1.7 × 10⁻³ lethal traffic accidents. Strategy A was associated with the most rabies cases (4.8 × 10⁻⁶), but the risk of traffic accidents and costs were null; therefore, strategy A was the most effective and the least costly. The sensitivity analysis showed that, when the probability that a given dog is rabid a given day (P_A) was >1.4 × 10⁻⁶, strategy D was more effective than strategy A; strategy B became cost-effective (i.e. ICER vs strategy A <3 × French Gross Domestic Product per capita) when P_A was > 1.4 × 10⁻⁴.ConclusionsIn the metropolitan France's very low rabies prevalence context, PEP with rabies vaccine, administered alone or with RIG, is associated with significant and unnecessary costs and unfavourable benefit-risk ratios regardless to exposure category.     

Molecular evolution of fever,thrombocytopenia and leukocytopenia virus (FTLSV) based on whole-genome sequences

FTLSV is a novel bunyavirus that was discovered in 2007 in the Henan province of China and has reported case fatality rates of up to 30%. Despite the high case fatality rate, knowledge of the evolution and molecular epidemiology of FTLSV is limited. In this study, detailed phylogenetic analyses were performed on whole-genome sequences to examine the virus's evolutionary rates, estimate dates of common ancestry, and determine the population dynamics and selection pressure for FTLSV. The evolutionary rates of FTLSV were estimated to be 2.28 × 10^− 4, 2.42 × 10^− 4 and 1.19 × 10^− 4 nucleotide substitutions/site/year for the S, M and L segments, respectively. The most recent ancestor of the viruses existed approximately 182–294 years ago. Evidence of RNA segment reassortment was found in FTLSV. A Bayesian skyline plot showed that after a period of genetic stability following high variability, the FTLSV population appeared to have contracted it. Selection pressures were estimated and revealed an abundance of negatively selected sites and sparse positively selected sites. These data will be valuable in understanding the evolution and molecular epidemiology of FTLSV, eventually helping to determine mechanisms of emergence and pathogenicity and the level of the virus's threat to public health.     

Molecular phylogeny and evolutionary dynamics of influenza A nonstructural (NS) gene

While the nonstructural gene (NS) of the influenza A virus plays a crucial role in viral virulence and replication, the complete understanding of its molecular phylogeny and evolutionary dynamics remains lacking. In this study, the phylogenetic analysis of 7581 NS sequences revealed ten distinct lineages within alleles A and B: three host-specific (human, classical swine and equine), two reassortment-originated (A(H1N1)pdm09 and triple reassortment swine), one transmission-originated (Eurasian swine), and two geographically isolated avian (Eurasian/Oceanian and North American) for allele A and two geographically isolated avian (Eurasian/Oceanian and North American) for allele B. The average nucleotide substitution rates of the lineages range from 1.24 × 10⁻³ (equine) to 4.34 × 10⁻³ (A(H1N1)pdm09) substitutions per site per year. The selection pressure analysis demonstrated that the d_N/d_S ratio of the NS gene in A(H1N1)pdm09 lineage was higher than its closely related triple reassortant swine, which could be attributed to the adaptation to the new host and/or intensive surveillance after the inter-species transmission from swine to human. The positive selection sites were found in all lineages except the equine lineage and mostly in the NS1 region. The positive selection sites 22, 26, 226, 227 and 230 of the human lineage are significant because these residues participate in either forming the dimerization of the two RNA binding domain (RBD) monomers or blocking the replication of host genes. Residues at position 171 provide hydrophobic interactions with hydrophobic residues at p85β and thus induce viral cell growth. The lineages and evolutionary dynamics of influenza A NS gene obtained in this study, along with the studies of other gene segments, are expected to improve the early detection of new viruses and thus have the potential to enhance influenza surveillance.     

Time scale evolution of avipoxviruses

Avipoxviruses are divided into three clades: canarypox-like viruses, fowlpox-like viruses, and psittacinepox-like viruses. Several molecular clock and demographic models available in the BEAST package were compared on three avipoxvirus genes (P4b, cnpv186 and DNA polymerase genes), which enabled to determine that avipoxviruses evolved at a rate of 2–8 × 10⁻⁵ substitution/site/year, in the range of poxviruses previously reported evolution rates. In addition, the date of mean time of divergence of avipoxviruses from a common ancestor was extrapolated to be about 10,000–30,000 years ago, at the same period as modern poxvirus species. Our findings will facilitate epidemiological investigations on avipoxviruses’ spread, origin and circulation.     

Emergence of a novel lineage genetically divergent from the predominant Ind2001 lineage of serotype O foot-and-mouth disease virus in India

In India, emergence of Ind2001 lineage of foot-and-mouth disease virus (FMDV) serotype O was recorded in the year 2001. After causing sporadic incidences, the Ind2001 lineage that re-surged in 2008 out-competed PanAsia from the field during 2009 and continued its dominance during 2010 and 2011 as well. The lineage has diversified in due course of time, leading to two sub-lineages (Ind2001a and Ind2001b). The sub-lineage Ind2001a include isolates collected during 2001–2002 and sub-lineage Ind2001b is constituted largely by isolates collected during 2008–2012. The nucleotide substitution rate of sub-lineage Ind2001b was estimated at 6.58 × 10⁻³ substitutions/site/year. The most stable PanAsia lineage is restricted only to few outbreaks. During 2011, emergence of a new genetic group with >9% nucleotide divergence from rest of the lineages circulating in the country was detected and named as lineage Ind2011. Two specific amino acid substitutions at positions VP1–36 (F) and VP2–133 (T) were observed in the Ind2011 lineage. The new lineage at present is restricted only to southern states of the country. It is uncertain whether the emergence was triggered by immune pressure or due to a bottleneck in transmission or selected for higher fitness value. Six sites (4, 68, 83, 135, 138 and 209) in VP1 protein were identified to undergo episodic diversifying selection in serotype O field isolates. Both emerging and re-emerging lineages had appropriate antigenic match with currently used vaccine strain, INDR2/1975. Irrespective of genetic variability, the field isolates showed remarkable conservation at antigenically critical residues that might contribute to the observed antigenic stability. With the emergence of a new genetic group after a span of 10 years, the overall epidemiological scenario in the region is expected to change in the coming years.     

Molecular evolution of respiratory syncytial virus subgroup A genotype NA1 and ON1 attachment glycoprotein (G) gene in central Vietnam

We performed molecular evolutionary analyses of the G gene C-terminal 3rd hypervariable region of RSV-A genotypes NA1 and ON1 strains from the paediatric acute respiratory infection patients in central Vietnam during the 2010–2012 study period. Time-scaled phylogenetic analyses were performed using Bayesian Markov Chain Monte Carlo (MCMC) method, and pairwise distances (p-distances) were calculated. Bayesian Skyline Plot (BSP) was constructed to analyze the time-trend relative genetic diversity of central Vietnam RSV-A strains. We also estimated the N-glycosylation sites within G gene hypervariable region. Amino acid substitutions under positive and negative selection pressure were examined using Conservative Single Likelihood Ancestor Counting (SLAC), Fixed Effects Likelihood (FEL), Internal Fixed Effects Likelihood (IFEL) and Mixed Effects Model for Episodic Diversifying Selection (MEME) models. The majority of central Vietnam ON1 strains detected in 2012 were classified into lineage 1 with few positively selected substitutions. As for the Vietnamese NA1 strains, four lineages were circulating during the study period with a few positive selection sites. Shifting patterns of the predominantly circulating NA1 lineage were observed in each year during the investigation period. Median p-distance of central Vietnam NA1 strains was wider (p-distance = 0.028) than that of ON1 (p-distance = 0.012). The molecular evolutionary rate of central Vietnam ON1 strains was estimated to be 2.55 × 10^− 2 (substitutions/site/year) and was faster than NA1 (7.12 × 10^− 3 (substitutions/site/year)). Interestingly, the evolutionary rates of both genotypes ON1 and NA1 strains from central Vietnam were faster than the global strains respectively. Furthermore, the shifts of N-glycosylation pattern within the G gene 3rd hypervariable region of Vietnamese NA1 strains were observed in each year. BSP analysis indicated the rapid growth of RSV-A effective population size in early 2012. These results suggested that the molecular evolution of RSV-A G gene detected in central Vietnam was fast with unique evolutionary dynamics.     

Molecular evolution of hepatitis B vaccine escape variants in China,during 2000–2016

Hepatitis B vaccine escape variants are the main threat to hepatitis B virus (HBV) infection in vaccination era worldwide. With 215 genotype B HBV and 313 genotype C HBV vaccine escape variants isolated from China during 2000–2016, we reported that genotype B HBV vaccine escape strains diverged in ∼1997 (95% HPD; 1987–2005), while genotype C HBV vaccine escape strains diverged in ∼1976 (95% HPD; 1955–2003). Additionally, the p-distance of genotype C HBV vaccine escape strains was 0.0291 ± 0.0169, which was significantly higher than that in the genotype B HBV (t = 131.02, p < 0.05). However, genotype B HBV vaccine escape strains evolved more rapidly than genotype C HBV (2.103 × 10⁻³ vs 1.083 × 10⁻³ substitutions/site/year). Bayesian skyline plot analysis showed that the populations of genotype C HBV vaccine escape strains fluctuated more than those in genotype B HBV. Four sites (A5T/S, L21S, T/A126S and T/N131I/A) and 13 sites (N3S, T5A, G10Q/R/E, L21S, T47K/A/V, L98V/P, I/S126N/V/T, Q129H/R/L, T131P/I/N/A, G145A/R, L175S/F, L213I/S, V224A/G) were found to be under positive selection in genotype B and C HBV vaccine escape strains, respectively. More importantly, N3S, L21S, T47K, L98V, I/S126T and L213I mutations were detected in 1 (2.5%), 1 (2.5%), 1 (2.5%), 3 (7.5%), 1 (2.5%), 1 (2.5%) genotype C HBV infected Chinese younger with neonatal HBV vaccination, respectively. Therefore, our results should be valuable in further understanding the molecular evolution of HBV and providing new ideas for the elimination of HBV infection.     

Acridine derivatives (PANHs,azaarenes) in raw,fried or grilled pork from different origins,and PANH formation during pork thermal processing

The aim of this work was to determine level of azaarenes (PANHs) in raw pork and to investigate their formation during meat frying or grilling, in particular to verify a suggestion that endogenous vitamin E might inhibit production of azaarenes. Azaarene concentration in raw pork samples from various origins ranged from 2.75 ng g⁻¹ to 3.69 ng g⁻¹ (2.75–2.93 ng g⁻¹ in meat of Polish Landrace pigs, 3.00–3.69 ng g^–1 in meat of hybrid Duroc × Polish Landrance pigs). PANH formation during frying of pork meat was not confirmed. On the other hand, PANHs were indeed formed during grilling; their levels ranged from 6.21 ng g⁻¹ to 8.08 ng g^–1. No inhibition influence of vitamin E on formation of PANHs on was found either in fried or grilled pork meat.     

Molecular evolution of human respiratory syncytial virus attachment glycoprotein (G) gene of new genotype ON1 and ancestor NA1

We conducted a comprehensive genetic analysis of the C-terminal 3rd hypervariable region of the attachment glycoprotein (G) gene in human respiratory syncytial virus subgroup A (HRSV-A) genotype ON1 (93 strains) and ancestor NA1 (125 strains). Genotype ON1 contains a unique mutation of a 72 nucleotide tandem repeat insertion (corresponding to 24 amino acids) in the hypervariable region. The Bayesian Markov chain Monte Carlo (MCMC) method was used to conduct phylogenetic analysis and a time scale for evolution. We also calculated pairwise distances (p-distances) and estimated the selective pressure. Phylogenetic analysis showed that the analyzed ON1 and NA1 strains formed 4 lineages. A strain belonging to lineage 4 of ON1 showed wide genetic divergence (p-distance, 0.072), which suggests that it might be a candidate new genotype, namely ON2. The emergence of genotype NA1 was estimated to have occurred in 2000 (95% of highest probability density, HPD; 1997–2002) and that of genotype ON1 in 2005 (95% HPD; 2000–2010) based on the time-scaled phylogenetic tree. The evolutionary rate of genotype ON1 was higher than that of ancestral genotype NA1 (6.03 × 10⁻³ vs. 4.61 × 10⁻³ substitutions/site/year, p < 0.05). Some positive and many negative selection sites were found in both ON1 and NA1 strains. The results suggested that the new genotype ON1 is rapidly evolving with antigenic changes, leading to epidemics of HRSV infection in various countries.     

Molecular evolution of the hypervariable region of the attachment glycoprotein gene in human respiratory syncytial virus subgroup B genotypes BA9 and BA10

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8 × 10^− 3 substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance > 0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.     

Cathodic adsorptive stripping voltammetric determination of rutin in soybean cultivars

A highly sensitive and selective cathodic adsorptive stripping voltammetric method for determination of rutin is presented. The method relies on the accumulation of a Cu(II)–rutin complex at a hanging mercury drop electrode (HMDE), followed by its reduction during a differential pulse voltammetric scan. The electrochemical behavior of the Cu(II)–rutin complex at HMDE was investigated by cyclic voltammetry. Results show that the electrode process is adsorption-controlled and gradually becomes less reversible at high scan rates where peak separation grows. Under the optimized conditions (phosphate buffer pH 6, ?1.000 V accumulation potential, 180 s accumulation time, 70 mV pulse amplitude, 50 mV s^?1 scan rate and 1.6 × 10^?6 M Cu(II) concentration), the reduction peak current (I_pc) of the Cu(II)–rutin complex is linear (I_pc (nA) = 10.070 + 1.9 × 10⁸ [Rutina]) to rutin concentration in the range from 2.0 × 10^?7 to 1.4 × 10^?6 M, with a correlation coefficient of 0.999. The detection and quantification limits obtained were 7.0 × 10^?9 M and 2.2 × 10^?8 M, respectively. The method was successfully applied to the determination of rutin in soybean cultivars, with recoveries of 94–105%.