乙型流感病毒河北株的HA1基因序列及种系发生的分析

对１９８９年春在河北保定分离的两株乙型流感病毒进行了抗原性、ＨＡ＿１基因序列和种系发生分析,与不同期的国内外代表株比较结果表明,自１９８８年以来乙型流感病毒变异较快,Ｂ／河北／５３／８９株的ＨＡ＿１基因序列与Ｂ／挪威／１／８５株相比,其氨基酸的同源性为９４．５２％,与同期的Ｂ／香港／２０／８９株同源性为９７．１２％。种系发生分析结果,从１９８８至１９８９年乙型流感病毒出现了５个支系,同期在保定分离的两株病毒,在同源替代中分别属于两个支系。日本国基本每隔两年出现一次乙型流感的流行优势型,其发生频度与甲型流感相似。国内少见乙型的流行优势型,可能和使用的分离病毒材料有关,日本用ＭＤＣＫ细胞比国内用鸡胚对乙型流感病毒的分离阳性率高。     

DNA疫苗预防B型流感病毒感染

用基因枪和电穿孔法将B型流感病毒DNA疫苗免疫BALB/C小鼠 ,实验证明B型流感病毒HA ,NADNA疫苗能有效抵御致死性同源B型病毒感染。同时实验表明 ,基因枪方法较电穿孔法诱导抗体水平略低。     

Microarray Multiplex Assay for the Simultaneous Detection and Discrimination of Influenza A and Influenza B Viruses

In this study, we present a microarray approach for the typing of influenza A and B viruses, and the subtyping of H1 and H3 subtypes. We designed four pairs of specific multiplex RT-PCR primers and eight specific oligonucleotide probes and prepared microarrays to identify the specific subtype of influenza virus. Through amplification and fluorescent marking of the multiplex RT-PCR products on the M gene of influenza A and B viruses and the HA gene of subtypes H1 and H3, the PCR products were hybridized with the microarray, and the results were analyzed using a microarray scanner. The results demonstrate that the chip developed by our research institute can detect influenza A and B viruses specifically and identify the subtypes H1 and H3 at a minimum concentration of 1 × 10² copies/μL of viral RNA. We tested 35 clinical samples and our results were identical to other fluorescent methods. The microarray approach developed in this study provides a reliable method for the monitoring and testing of seasonal influenza.     

Isolation and Classification of Temperature-Sensitive Mutants of Influenza B Virus

We isolated 25 temperature-sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5-fluorouracil and classified them into seven recombination groups by pair-wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10^–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis-positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10^–2 and belonged to recombination group VII.     

The Evolutionary Dynamics of Human Influenza B Virus

Despite their close phylogenetic relationship, type A and B influenza viruses exhibit major epidemiological differences in humans, with the latter both less common and less often associated with severe disease. However, it is unclear what processes determine the evolutionary dynamics of influenza B virus, and how influenza viruses A and B interact at the evolutionary scale. To address these questions we inferred the phylogenetic history of human influenza B virus using complete genome sequences for which the date (day) of isolation was available. By comparing the phylogenetic patterns of all eight viral segments we determined the occurrence of segment reassortment over a 30-year sampling period. An analysis of rates of nucleotide substitution and selection pressures revealed sporadic occurrences of adaptive evolution, most notably in the viral hemagglutinin and compatible with the action of antigenic drift, yet lower rates of overall and nonsynonymous nucleotide substitution compared to influenza A virus. Overall, these results led us to propose a model in which evolutionary changes within and between the antigenically distinct 'Yam88' and 'Vic87' lineages of influenza B virus are the result of changes in herd immunity, with reassortment continuously generating novel genetic variation. Additionally, we suggest that the interaction with influenza A virus may be central in shaping the evolutionary dynamics of influenza B virus, facilitating the shift of dominance between the Vic87 and the Yam88 lineages.     

Cleavage Activation of Human-adapted Influenza Virus Subtypes by Kallikrein-related Peptidases 5 and 12

A critical step in the influenza virus replication cycle is the cleavage activation of the HA precursor. Cleavage activation of influenza HA enables fusion with the host endosome, allowing for release of the viral genome into the host cell. To date, studies have determined that HA activation is driven by trypsin-like host cell proteases, as well as yet to be identified bacterial proteases. Although the number of host proteases that can activate HA is growing, there is still uncertainty regarding which secreted proteases are able to support multicycle replication of influenza. In this study, we have determined that the kallikrein-related peptidases 5 and 12 are secreted from the human respiratory tract and have the ability to cleave and activate HA from the H1, H2, and H3 subtypes. Each peptidase appears to have a preference for particular influenza subtypes, with kallikrein 5 cleaving the H1 and H3 subtypes most efficiently and kallikrein 12 cleaving the H1 and H2 subtypes most efficiently. Cleavage analysis using HA cleavage site peptide mimics revealed that the amino acids neighboring the arginine cleavage site affect cleavage efficiency. Additionally, the thrombolytic zymogens plasminogen, urokinase, and plasma kallikrein have all been shown to cleave and activate influenza but are found circulating mainly as inactive precursors. Kallikrein 5 and kallikrein 12 were examined for their ability to activate the thrombolytic zymogens, and both resulted in activation of each zymogen, with kallikrein 12 being a more potent activator. Activation of the thrombolytic zymogens may therefore allow for both direct and indirect activation of the HA of human-adapted influenza viruses by kallikrein 5 and kallikrein 12.     

流感病毒H3N2血凝素基因和神经氨酸酶基因在大肠杆菌中的表达

克隆、表达和鉴定流感病毒H3N2 HA,NA基因序列,为制备抗体和基因工程疫苗打下基础。在成功克隆流感病毒H3N2全长HA、NA基因并测序的基础上,将部分基因序列克隆到表达载体pET32a(+)上,全基因序列克隆到表达载体pGEX4T-1上,构建了重组表达质粒pET32a(+)/HA(截短),pET32a(+)/NA(截短),pGEX4T-1/HA,转化大肠杆菌BL21/Rosetta,IPTG诱导表达,利用Ni2+亲和层析柱和GSTrap 4B亲和层析柱对重组蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。结果显示,重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致。ELISA和Western blotting试验证实,重组蛋白具有良好的抗原性。成功克隆和表达了流感病毒H3N2 HA、NA基因序列,可为流感病毒H3N2诊断试剂和疫苗的开发等进一步的研究提供参考。     

Estimating phylogenetic trees from pairwise likelihoods and posterior probabilities of substitution counts

The field of phylogenetic tree estimation has been dominated by three broad classes of methods: distance-based approaches, parsimony and likelihood-based methods (including maximum likelihood (ML) and Bayesian approaches). Here we introduce two new approaches to tree inference: pairwise likelihood estimation and a distance-based method that estimates the number of substitutions along the paths through the tree. Our results include the derivation of the formulae for the probability that two leaves will be identical at a site given a number of substitutions along the path connecting them. We also derive the posterior probability of the number of substitutions along a path between two sequences. The calculations for the posterior probabilities are exact for group-based, symmetric models of character evolution, but are only approximate for more general models.     

Maximum Likelihood Estimation on Large Phylogenies and Analysis of Adaptive Evolution in Human Influenza Virus A

Algorithmic details to obtain maximum likelihood estimates of parameters on a large phylogeny are discussed. On a large tree, an efficient approach is to optimize branch lengths one at a time while updating parameters in the substitution model simultaneously. Codon substitution models that allow for variable nonsynonymous/synonymous rate ratios (ω=d _N/d _S) among sites are used to analyze a data set of human influenza virus type A hemagglutinin (HA) genes. The data set has 349 sequences. Methods for obtaining approximate estimates of branch lengths for codon models are explored, and the estimates are used to test for positive selection and to identify sites under selection. Compared with results obtained from the exact method estimating all parameters by maximum likelihood, the approximate methods produced reliable results. The analysis identified a number of sites in the viral gene under diversifying Darwinian selection and demonstrated the importance of including many sequences in the data in detecting positive selection at individual sites. Received: 25 April 2000 / Accepted: 24 July 2000     

Likelihood Analysis of the Chalcone Synthase Genes Suggests the Role of Positive Selection in Morning Glories (<Emphasis Type="Italic">Ipomoea</Emphasis>)

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoides, which are important for the pigmentation of flowers and act as attractants to pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. In morning glories (Ipomoea), five functional CHS genes (A–E) have been described. Phylogenetic analysis of the Ipomoea CHS gene family revealed that CHS A, B, and C experienced accelerated rates of amino acid substitution relative to CHS D and E. To examine whether the CHS genes of the morning glories underwent adaptive evolution, maximum-likelihood models of codon substitution were used to analyze the functional sequences in the Ipomoea CHS gene family. These models used the nonsynonymous/synonymous rate ratio ( = d_N/d_S) as an indicator of selective pressure and allowed the ratio to vary among lineages or sites. Likelihood ratio test suggested significant variation in selection pressure among amino acid sites, with a small proportion of them detected to be under positive selection along the branches ancestral to CHS A, B, and C. Positive Darwinian selection appears to have promoted the divergence of subfamily ABC and subfamily DE and is at least partially responsible for a rate increase following gene duplication.     

Evidence of selection pressures of neuraminidase gene (NA) of influenza A virus subtype H5N1 on different hosts in Guangxi Province of China

In the present study, the possible evidence of positive selection was analyzed for the neuraminidase (NA) sequences of Guangxi H5N1 strains of China. Based on an overall site-specific positive selection analysis, it was found that NA gene of H5N1 Guangxi strains underwent purifying selection and no significant positively selected sites were identified. For the branch-specific positive selection analysis, there was no positive selection evidence for the branches leading to different poultry hosts (chicken, duck and goose). Conclusively, positive selection seems not possible (if not rare) for the NA gene in influenza H5N1 subtype, at least for the samples found in Guangxi Province of China.     

Analytic solutions of maximum likelihood on forks of four taxa

This work deals with symbolic mathematical solutions to maximum likelihood on small phylogenetic trees. Maximum likelihood (ML) is increasingly used as an optimality criterion for selecting evolutionary trees, but finding the global optimum is a hard computational task. In this work, we give general analytic solutions for a family of trees with four taxa, two state characters, under a molecular clock. Previously, analytical solutions were known only for three taxa trees. The change from three to four taxa incurs a major increase in the complexity of the underlying algebraic system, and requires novel techniques and approaches. Despite the simplicity of our model, solving ML analytically in it is close to the limit of today's tractability. Four taxa rooted trees have two topologies--the fork (two subtrees with two leaves each) and the comb (one subtree with three leaves, the other with a single leaf). Combining the properties of molecular clock fork trees with the Hadamard conjugation, and employing the symbolic algebra software Maple, we derive a number of topology dependent identities. Using these identities, we substantially simplify the system of polynomial equations for the fork. We finally employ the symbolic algebra software to obtain closed form analytic solutions (expressed parametrically in the input data).     

基于多源异构大数据挖掘的流感病毒防控预测预警平台构建研究 *

流感传播速度快,病原变异频繁,影响范围广,对其快速反应与防范对全球来说仍然是一个严重的挑战。医疗卫生和高通量测序技术的组合产生了非常复杂可变的海量的异质异构数据集,对其实现整合挖掘分析是个重要任务。现有逐级上报方式的流感监测体系存在分析结果滞后(1~2周)的问题,与流感病毒变异和传播速度快形成尖锐矛盾。因此,实时而全面的了解其流行动态非常必要。基于以上原因,建立统一的大数据平台,整合不同来源、不同结构的监测数据和特定算法及模型,对信息加以分析利用,并对病毒的流行、发展、变异、控制和反馈进行全生命周期的监控,通过时间、地域、环境和病毒之间的关联性等综合分析,形成一个高效安全、快速稳定,且准确及时的流感地理信息图谱预测预警系统,成为提升流感中心信息管理水平的必由之路。     

Exploring variation in the d(N)/d(S) ratio among sites and lineages using mutational mappings: applications to the influenza virus

We use a likelihood-based method for mapping mutations on a phylogeny in a way that allows for both site-specific and lineage-specific variation in selection intensity. The method accounts for many of the potential sources of bias encountered in mapping of mutations on trees while still being computationally efficient. We apply the method to a previously published influenza data set to investigate hypotheses about changes in selection intensity in influenza strains. Influenza virus is sometimes propagated in chicken cells for several generations before sequencing, a process that has been hypothesized to induce mutations adapting the virus to the lab medium. Our analysis suggests that there are approximately twice as many replacement substitutions in lineages propagated in chicken eggs as in lineages that are not. Previous studies have attempted to predict which viral strains future epidemics may arise from using inferences regarding positive selection. The assumption is that future epidemics are more likely to arise from the strains in which positive selection on the so-called “trunk lineages” of the evolutionary tree is most pervasive. However, we find no difference in the strength of selection in the trunk lineages versus other evolutionary lineages. Our results suggest that it may be more difficult to use inferences regarding the strength of selection on mutations to make predictions regarding viral epidemics than previously thought. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Willie Swanson     

Molecular evolution of the hepatitis B virus genome

The hepatitis B virus (HBV) has a circular DNA genome of about 3,200 base pairs. Economical use of the genome with overlapping reading frames may have led to severe constraints on nucleotide substitutions along the genome and to highly variable rates of substitution among nucleotide sites. Nucleotide sequences from 13 complete HBV genomes were compared to examine such variability of substitution rates among sites and to examine the phylogenetic relationships among the HBV variants. The maximum likelihood method was employed to fit models of DNA sequence evolution that can account for the complexity of the pattern of nucleotide substitution. Comparison of the models suggests that the rates of substitution are different in different genes and codon positions; for example, the third codon position changes at a rate over ten times higher than the second position. Furthermore, substantial variation of substitution rates was detected even after the effects of genes and codon positions were corrected; that is, rates are different at different sites of the same gene or at the same codon position. Such rates after the correction were also found to be positively correlated at adjacent sites, which indicated the existence of conserved and variable domains in the proteins encoded by the viral genome. A multiparameter model validates the earlier finding that the variation in nucleotide conservation is not random around the HBV genome. The test for the existence of a molecular clock suggests that substitution rates are more or less constant among lineages. The phylogenetic relationships among the viral variants were examined. Although the data do not seem to contain sufficient information to resolve the details of the phylogeny, it appears quite certain that the serotypes of the viral variants do not reflect their genetic relatedness. Correspondence to: Z. Yang     

Molecular Evolution of a Small Gene Family of Wound Inducible Kunitz Trypsin Inhibitors in Populus

Maximum likelihood models of codon substitutions were used to analyze the molecular evolution of a Kunitz trypsin inhibitor (KTI) gene family in Populus and Salix. The methods support previous assertions that the KTI genes comprise a rapidly evolving gene family. Models that allow for codon specific estimates of the ratio of nonsynonymous to synonymous substitutions (ω) among sites detect positive Darwinian selection at several sites in the KTI protein. In addition, branch-specific maximum likelihood models show that there is significant heterogeneity in ω among branches of the KTI phylogeny. In particular, ω is substantially higher following duplication than speciation. There is also evidence for significant rate heterogeneity following gene duplication, suggesting different evolutionary rates in newly arisen gene duplicates. The results indicate uneven evolutionary rates both between sites in the KTI protein and among different lineages in the KTI phylogeny, which is incompatible with a neutral model of sequence evolution. [Reviewing Editor: Dr. Willie J. Swanson]     

待孕夫妇乙肝表面抗原及乙肝表面抗体阳性情况分析

目的 分析与探讨待孕夫妇乙肝表面抗原及乙肝表面抗体检测结果,并研究其对临床孕前检查的影响及评价。方法 随机选取2015‒2017年度在我院进行孕前检查的夫妇2440对（4 880例）为研究对象,按照年度将待孕夫妇分为两组,每组2 440例,两组均加强孕前检查中的乙肝表面抗原（HBsAg）及乙肝表面抗体（HBsAb）的检测。A组为2015年3月‒2016年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇;B组为2016年3月‒2017年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇。比较两组待孕夫妇乙肝表面抗原及乙肝表面抗体检测的阳性结果。结果 B组HBsAg阳性率、HBsAb阳性率明显高于A组（6.43% vs 4.63%;62.99% vs 58.44%）,差异有统计学意义（P＜0.05）。B组、A组男性HBsAg阳性率明显高于同组女性（59.87% vs 40.13%;60.18% vs 39.82%）,HBsAb阳性率低于同组女性（46.52% vs 53.48%;47.41% vs 52.59%）,差异均有统计学意义（P＜0.05）。B组、A组高中及以上学历HBsAg阳性率明显低于同组高中以下学历（38.85% vs 61.95%;38.05% vs 61.15%）,高中及以上学历HBsAb阳性率高于同组高中以下学历（53.15% vs 46.84%;51.75% vs 48.25%）,差异均有统计学意义（P＜0.05）结论 目前夫妇乙肝感染仍处于增高趋势,对于进行孕前检查的待孕夫妇加强乙肝表面抗原及乙肝表面抗体的检测,有助于疾病的早期诊断、干预及治疗,能够减少乙肝传播,可有效降低新生儿乙肝发病率,促进优生优育,提高出生人口整体素质。     

Epidemiological Characteristics of Influenza A and B in Macau, 2010–2018

Influenza is one of the major respiratory diseases in humans. Macau is a tourist city with high density of population and special population mobility. The study on the epidemiological characteristics of influenza in Macau should bring great value for preventing influenza in tourist cities like Macau in the world. In this study, we collected a total of 104,874 samples with influenza-like illness (ILI) in Macau from 2010 to 2018. Chi-square test and binary multivariable logistic regression were used to investigate the epidemiological characteristics of influenza A and B in Macau. Among these ILI samples, the overall positive rate is 17.17% for influenza A and 6.97% for influenza B. The epidemics of influenza in three years (i.e., 2012, 2017 and 2018) differ from the remaining years (i.e., normal years). In a normal year, influenza A occurs year-round whereas influenza B is seasonal. Our research shows significant differences in influenza infections between different age groups in normal years. Interestingly, our analysis shows no significant difference between locals and tourists in influenza A and B infection in a normal year, whereas the odds of influenza A in tourists were significantly higher than those in locals in July 2017 and the odds of influenza B in tourists were significantly higher than those in locals in January–February 2012 and January–February 2018. This is possibly attributed by the policy of free vaccination to everyone in Macau. These findings should be valuable for preventing influenza in not only Macau but also the world.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12250-021-00388-6.