H5N1 virus-like particle vaccine elicits cross-reactive neutralizing antibodies that preferentially bind to the oligomeric form of influenza virus hemagglutinin in humans

Transmission of pathogenic avian influenza viruses (AIV) from wild birds to domestic poultry and humans is continuing in multiple countries around the world. In preparation for a potential AIV pandemic, multiple vaccine candidates are under development. In the case of H5N1 AIV, a clear shift in transmission from clade 1 to clade 2 viruses occurred in recent years. The virus-like particle (VLP) represents an economical approach to pandemic vaccine development. In the current study, we evaluated the humoral immune response in humans vaccinated with H5N1 A/Indonesia/05/2005 (clade 2.1) VLP vaccine manufactured in Sf9 insect cells. The VLPs were comprised of the influenza virus hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1) proteins. In an FDA-approved phase I/II human clinical study, two doses of H5N1 VLPs at 15, 45, or 90 μg HA/dose resulted in seroconversion and production of functional antibodies. Moreover, cross-reactivity against other clade 2 subtypes was demonstrated using virus neutralization assays. H5N1 whole-genome fragment phage display libraries (GFPDL) were used to elucidate the antibody epitope repertoire in postvaccination human sera. Diverse epitopes in HA1/HA2 and NA were recognized by postvaccination sera from the two high-dose groups, including large segments spanning the HA1 receptor binding domain. Importantly, the vaccine elicited sera that preferentially bound to an oligomeric form of recombinant HA1 compared with monomeric HA1. The oligomeric/monomeric HA1 binding ratios of the sera correlated with the virus neutralizing titers. Additionally, the two high-dose VLP vaccine groups generated NA-inhibiting antibodies that were associated with binding to a C-terminal epitope close to the sialic acid binding site. These findings represent the first report describing the quality of the antibody responses in humans following AIV VLP immunization and support further development of such vaccines against emerging influenza virus strains.     

H9N2禽流感病毒反向遗传系统转录／表达载体的构建和验证

设计带有BsmBI、BsaI或AarI酶切位点的引物,用RT-PCR扩增H9N2亚型禽流感病毒(AIV)的8个基因全长片段,克隆入双向转录/表达载体pHW2000,并在PB2、PB1和NA基因中共引入了3个沉默突变标签。将其2个表面基因(HA和NA基因)加上任意1个内部基因,而其它5个内部基因来自A/WSN/33,进行了6种3 5组合形式的基因重排,把相应组合的转录/表达质粒共转染COS-1细胞,均产生了预期组合、有感染性的H9N2亚型流感病毒,表明亲缘关系遥远的流感病毒可以互相获取基因片段产生重组病毒,提示表面结构基因和单个内部基因不足以限制H9N2AIV在哺乳动物细胞上的宿主范围,同时也验证了构建的8个转录/表达载体均能有效工作,为进一步研究H9N2亚型AIV基因结构与功能、AIV与宿主之间的关系打下了基础。     

禽流感病毒H7N2血凝素HA1基因在大肠杆菌中的表达

目的　表达H7N2亚型禽流感病毒 (AIV)HA1基因 ,用于感染H7亚型禽流感病毒抗体的检测和HA1蛋白功能研究。方法　采用RT PCR方法对H7N2亚型AIVHA1基因进行扩增 ,将PCR产物克隆于pGEM T Easy载体 ,将该基因插入pGEX 4T 2中构建HA1基因原核表达载体 ,转化BL2 1大肠杆菌后 ,在IPTG诱导下表达HA1蛋白 ,Westernblot鉴定表达HA1蛋白。电洗脱方法纯化表达HA1蛋白 ,建立间接ELISA方法 ,对感染AIVH7、H9、H5亚型AIV阳性血清进行检测。结果　成功克隆H7N2亚型AIV的HA1基因 ,其核苷酸序列长度 96 6bp ,编码 32 2个氨基酸残基。构建HA1基因原核表达载体在大肠杆菌内表达出约 6 1× 10 3的HA1融合蛋白。Westernblot和ELISA方法鉴定表明 :表达HA1蛋白与感染H7亚型AIV鸡血清有反应 ,与H5、H9亚型AIV阳性血清没有反应。结论　本研究在大肠杆菌中成功表达了H7N2亚型AIVHA1基因蛋白 ,具有与感染H7亚型AIV阳性血清反应原性 ,不与H5和H9亚型AIV感染阳性血清发生反应。     

Complete Genome Sequence of a Novel Reassortant H11N2 Avian Influenza Virus Isolated from a Live Poultry Market in Eastern China

A/chicken/Nanjing/908/2009(H11N2) (CK908) was isolated from a live poultry market in Nanjing, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the CK908 virus. The sequence analysis demonstrated that this H11N2 virus was a novel reassortant AIV whose PB1, PB2, PA, HA, NP, NA, M, and NS genes originated from H9N2, H7N7, H5N2, H11N8, H3N6, H6N2, H1N1, and H5N1, respectively. Knowledge regarding the complete genome sequences of the CK908 virus will be useful for epidemiological surveillance.     

NA Proteins of Influenza A Viruses H1N1/2009, H5N1, and H9N2 Show Differential Effects on Infection Initiation,Virus Release,and Cell-Cell Fusion

Two surface glycoproteins of influenza virus, haemagglutinin (HA) and neuraminidase (NA), play opposite roles in terms of their interaction with host sialic acid receptors. HA attaches to sialic acid on host cell surface receptors to initiate virus infection while NA removes these sialic acids to facilitate release of progeny virions. This functional opposition requires a balance. To explore what might happen when NA of an influenza virus was replaced by one from another isolate or subtype, in this study, we generated three recombinant influenza A viruses in the background of A/PR/8/34 (PR8) (H1N1) and with NA genes obtained respectively from the 2009 pandemic H1N1 virus, a highly pathogenic avian H5N1 virus, and a lowly pathogenic avian H9N2 virus. These recombinant viruses, rPR8-H1N1NA, rPR8-H5N1NA, and rPR8-H9N2NA, were shown to have similar growth kinetics in cells and pathogenicity in mice. However, much more rPR8-H5N1NA and PR8-wt virions were released from chicken erythrocytes than virions of rPR8-H1N1NA and rPR8-H9N2NA after 1 h. In addition, in MDCK cells, rPR8-H5N1NA and rPR8-H9N2NA infected a higher percentage of cells, and induced cell-cell fusion faster and more extensively than PR8-wt and rPR8-H1N1NA did in the early phase of infection. In conclusion, NA replacement in this study did not affect virus replication kinetics but had different effects on infection initiation, virus release and fusion of infected cells. These phenomena might be partially due to NA proteins’ different specificity to α2-3/2-6-sialylated carbohydrate chains, but the exact mechanism remains to be explored.     

Genome sequencing and phylogenetic analysis of three avian influenza H9N2 subtypes in Guangxi

Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.     

Influenza-A viruses in ducks in northwestern Minnesota: fine scale spatial and temporal variation in prevalence and subtype diversity

Waterfowl from northwestern Minnesota were sampled by cloacal swabbing for Avian Influenza Virus (AIV) from July-October in 2007 and 2008. AIV was detected in 222 (9.1%) of 2,441 ducks in 2007 and in 438 (17.9%) of 2,452 ducks in 2008. Prevalence of AIV peaked in late summer. We detected 27 AIV subtypes during 2007 and 31 during 2008. Ten hemagglutinin (HA) subtypes were detected each year (i.e., H1, 3-8, and 10-12 during 2007; H1-8, 10 and 11 during 2008). All neuraminidase (NA) subtypes were detected during each year of the study. Subtype diversity varied between years and increased with prevalence into September. Predominant subtypes during 2007 (comprising ≥5% of subtype diversity) included H1N1, H3N6, H3N8, H4N6, H7N3, H10N7, and H11N9. Predominant subtypes during 2008 included H3N6, H3N8, H4N6, H4N8, H6N1, and H10N7. Additionally, within each HA subtype, the same predominant HA/NA subtype combinations were detected each year and included H1N1, H3N8, H4N6, H5N2, H6N1, H7N3, H8N4, H10N7, and H11N9. The H2N3 and H12N5 viruses also predominated within the H2 and H12 subtypes, respectively, but only were detected during a single year (H2 and H12 viruses were not detected during 2007 and 2008, respectively). Mallards were the predominant species sampled (63.7% of the total), and 531 AIV were isolated from this species (80.5% of the total isolates). Mallard data collected during both years adequately described the observed temporal and spatial prevalence from the total sample and also adequately represented subtype diversity. Juvenile mallards also were adequate in describing the temporal and spatial prevalence of AIV as well as subtype diversity.     

H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析

血凝素(hemagglutinin,HA)蛋白是禽流感病毒(avian influenza virus,AIV)的一个重要表面抗原性蛋白,在疾病诊断和防治上有重要意义。本研究为了探讨一种更为简便有效的HA重组蛋白表达途径,利用生物信息学软件,对H5N1亚型AIVHA基因编码的氨基酸序列进行分析,在分析其在大肠杆菌中的密码子偏好性、稀有密码子分布情况及有关蛋白的抗原性等重要特性后,构建了HA抗原表位重组表达质粒pET-32a(+)-HA。经测试,该重组质粒在1mmol/LIPTG诱导剂作用下诱导过夜,能在大肠杆菌Rosetta-gami B(DE3)中高效表达,并得到48.1kD大小的目的重组表达蛋白。重组蛋白用6×His-tagged protein纯化试剂盒纯化后,与福氏佐剂等量混合制备成抗原,以200μg/鸡的剂量皮下注射2月龄SPF鸡3次,采血分离血清。Western-Blot试验结果表明,该重组表达蛋白能分别与所制备的高免鸡血清及H5N1亚型AIV阳性血清发生特异性反应,在硝酸纤维素膜上出现特异性杂交带。说明本试验研究的HA抗原重组表达蛋白具有良好的免疫原性和反应原性,保留了HA蛋白的抗原活性,提示该重组蛋白在H5亚型AIV的防治技术研究中具有重要的实际应用价值。     

一株鹅源H5N2亚型高致病性禽流感病毒的分离鉴定及生物学特性分析

分离到一株鹅源 H5N2亚型高致病性禽流感病毒,SPF鸡静脉接种致病指数为2.99,但鸭子对该病毒不敏感．病毒感染小鼠后不致病,但能够在肺内有效复制,表明其具有感染哺乳动物的潜在风险．血凝素(hemagglutinin, HA)蛋白裂解位点上插入有多个连续的碱性氨基酸(-RRRKKR-),从分子上证实这是一株高致病性禽流感病毒．核酸序列比较分析表明,分离的流感病毒HA基因与A/chicken/Hubei/489/2004 (H5N1)同源率达到99.4%,神经氨酸酶(neuraminidase, NA)基因与A/chicken/Jilin/53/01(H9N2)同源率达到99.8%;氨基酸水平上,HA与2004年分离到的A/chicken/Hubei/489/2004(H5N1)、A/swan/Guangxi/307/2004(H5N1)、A/wildduck/Guangdong/314/　2004(H5N1)和A/chicken/Henan/210/2004(H5N1)同源率均为99.3%,NA 与A/chicken/Jilin/53/01(H9N2)同源率为99.6%．进化树分析结果表明,该流感病毒分离株可能是由H5N1和H9N2两个亚型病毒重排而来．     

2017年河北省蛋鸡养殖场H9N2亚型禽流感病毒的遗传演化和抗原性分析

【背景】H9N2亚型禽流感病毒在鸡群中广泛流行,引起巨大损失。【目的】了解河北省蛋鸡养殖场H9N2亚型禽流感病毒(avian influenza virus,AIV)的基因序列和抗原性的变异情况,为该病原的科学防控提供理论依据。【方法】于2017年从河北省部分蛋鸡养殖场分离鉴定出7株H9N2亚型AIV,对其HA基因进行序列测定,并进行遗传演化、关键氨基酸位点及抗原性分析。【结果】7株分离毒株HA基因同源性在95.5%?97.2%之间;与2016年前的流行毒株相比,分离病毒HA裂解位点均为典型低致病性AIV特征,在受体结合区域出现变异,潜在糖基化位点无明显差异;抗原分析结果显示分离毒株与早期分离株相比抗原性发生了变异,形成了新的抗原群;抗原性相关位点分析显示,分离毒株在9个位点发生了较为明显的突变,可能是导致抗原性变异的分子基础。【结论】河北省蛋鸡养殖场H9N2亚型AIV中的流行毒株在关键功能区发生基因突变,并且抗原性发生变异,提示应持续监测H9N2亚型AIV的遗传变异情况,并及时更换疫苗株。     

鸭源H9N2AIV血凝素基因序列比较

为明确国内外鸭源H9N2亚型禽流感病毒(Avian influenza virus,AIV)血凝素基因(hemagglutinin,HA)的遗传进化关系、血凝素蛋白裂解位点的氨基酸结构特征和血凝素蛋白受体结合位点的氨基酸变异特征,本研究选取GenBank中登录鸭源H9N2亚型AIV HA基因,通过MEGA4.1进行比对和分析,并绘制其遗传进化树。结果表明,鸭源H9N2亚型AIV在遗传进化上分为2大谱系:即Ck-Bj-1-94-like和North-Ame-like,中国大陆鸭源H9N2亚型AIV和亚欧美其它国家鸭源H9N2亚型AIV在遗传进化上分居完全不同的谱系,相互之间遗传进化关系较远。从血凝素受体结合位点看,亚欧美国家鸭源H9N2亚型AIV在第183、190和226位点的氨基酸均为鸭源AIV经典H、E和Q,且高度保守。但中国大陆地区H9N2亚型AIV第183位为N;第190位为A or V or T,与中国大陆鸡源H9N2亚型AIV一致;第226位中国鸭源H9N2亚型AIV有相当一部分为L,且近年福建省H9N2亚型AIV分离株在此处均为L。提示我们,中国大陆地区H9N2亚型AIV鸭鸡和鸡鸭相互交叉感染较为普遍。     

Highly conserved influenza A virus epitope sequences as candidates of H3N2 flu vaccine targets

This study focused on identifying the conserved epitopes in a single subtype A (H3N2)-as candidates for vaccine targets. We identified a total of 32 conserved epitopes in four viral proteins [22 HA, 4PB1, 3 NA, 3 NP]. Evaluation of conserved epitopes in coverage during 1968-2010 revealed that (1) 12 HA conserved epitopes were highly present in the circulating viruses; (2) the remaining 10 HA conserved epitopes appeared with lower percentage but a significantly increasing trend after 1989 [p<0.001]; and (3) the conserved epitopes in NA, NP and PB1 are also highly frequent in wild-type viruses. These conserved epitopes also covered an extremely high percentage of the 16 vaccine strains during the 42 year period. The identification of highly conserved epitopes using our approach can also be applied to develop broad-spectrum vaccines.     

Genetic Characterization of a Novel Recombinant H5N2 Avian Influenza Virus Isolated from Chickens in Tibet

In this report, a novel H5N2 avian influenza virus (AIV) was isolated from chickens in Tibet in 2010, western China. Phylogenetic analysis demonstrated that it was a natural reassortant between H9N2 and H5N1 subtypes. It is of note that this virus has an HP genotype with HA, PB2, M, and NS genes homologous to those of A/peregrine falcon/Hong Kong/2142/2008(H5N1)-like HPAIV isolated from dead wild birds. Publishing this genome information will contribute to the investigation of avian influenza epidemiology and to further research of AIV''s biological properties.     

禽流感病毒H9N2血凝素基因和神经氨酸酶因在大肠杆菌中的表达

克隆、表达和鉴定禽流感病毒H9N2 HA,NA基因序列,为制备抗体和基因工程疫苗打下基础。在成功克隆禽流感病毒H9N2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、pGEX4T-1/NA,转化大肠杆菌BL21/rosetta,IPTG诱导表达,利用Ni2+亲和层析柱和GSTrap4B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实验证实,重组蛋白具有良好的抗原性。本研究成功克隆和表达了禽流感病毒H9N2 HA、NA基因序列。为禽流感病毒H9N2诊断试剂和疫苗的开发等进一步的研究奠定了基础。     

Epitope peptides of influenza H3N2 virus neuraminidase gene designed by immunoinformatics

The virus surface protein neuraminidase (NA) is a main subtype-specific antigen in influenza type A viruses. Neuraminidase functions as an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells. In this study, NA genes from the H3N2 subtype virus were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics. Based on this information, three peptides ES8, RR9, and WK7 (covering amino acid residues 221-228, 292-300, and 383-389, respectively) of the NA protein were selected and synthesized artificially. These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera. Results showed that these three peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive manner, detected in vitro by enzyme-linked immunosorbent assay. Furthermore, hemadsorption anti-releasing effects occurred in three antisera mixtures at a dilution of 1:40. Alignment using database software showed that amino acid residues in these three epitope peptides were substituted at specific sites in all the NAs sequenced in this study. We suggest that these NA epitope peptides might be used in conjunction with HA proteins as vaccine antigens.     

共表达H5亚型禽流感病毒HA和NA基因的重组鸡痘病毒及其免疫效力

为了构建更为安全有效地抵抗高致病性H5亚型禽流感病毒的基因工程疫苗,将H5亚型禽流感病毒分离株的血凝素(HA)基因和神经氨酸酶(NA)基因定向插入鸡痘病毒转移载体p11S中,H5A和NA基因的启动子分别为PS和PE/L,获得用不同的启动子启动不同的外源基因且两基因盒方向为背向串联的重组转移载体p11SH5ANA。将p11SH5ANA转染至已感染鸡痘病毒282E4疫苗株(wt-FPV)的鸡胚成纤维细胞(CEF)中。p11SH5ANA与wt-FPV基因组DNA之间的同源重组产生了重组鸡痘病毒rFPV-11SH5ANA。通过在含X-Gal的营养琼脂上连续挑选蓝色病毒蚀斑,获得纯化的重组病毒。经传代证实该重组病毒具有良好的遗传稳定性。用105PFU的rFPV-11SH5NA免疫无特定病原体(SPF)鸡,能激发机体产生有效的血凝抑制(HI)抗体。初步的动物试验表明,该重组病毒能使经肌肉注射攻毒的SPF鸡抵抗H5亚型AIV的致死性攻击,保护率为100%,显示出一定的应用前景。     

Novel Genotypes of H9N2 Influenza A Viruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.     

H9N2亚型禽流感病毒基因组的遗传进化分析

2009～2011年从江苏省、湖北省和安徽省等地来源于鸡、鸭、鹌鹑和鸽子的样品中分离鉴定出16株H9N2亚型禽流感病毒。通过反转录聚合酶链式反应(RT-PCR)扩增出分离株的全基因片段,并对其进行测序及遗传进化分析。序列分析显示,16株病毒HA基因裂解位点氨基酸序列为P-S-R/K-S-S-R,符合低致病性禽流感的分子特征;226位均为L,具有与哺乳动物唾液酸α,2-6受体结合的特性。M2基因均出现了对金刚烷胺产生耐药性的N31S突变。不同宿主来源的H9亚型AIV的主要分子特征一致。全基因遗传进化分析表明16株H9N2亚型禽流感病毒全基因发生了3配体重组,即以F98亚系AIV为骨架,HA来源于Y280亚系,PB2和M基因来源于G1亚系,形成了2种新的基因型。因此,要加强对H9N2亚型禽流感病毒的监测,密切关注它的重组趋势。     

利用反向遗传技术研究H9N2亚型AIV传播途径的分子机制

利用反向遗传技术,通过基因重排方法,产生两个表面基因来自A/Chicken/Guangdong/SS/94(H9N2)禽流感病毒(avian influenza virus,AIV)株和其余基因来自A/Chicken/Shanghai/F/98(H9N2)AIV株的3株H9N2亚型重排AIV,动物传播性试验发现A/Chicken/Shanghai/F/98(H9N2)株、A/Chicken/Guangdong/SS/94(H9N2)AIV株和3株H9N2亚型重排AIV都可以经直接接触途径传播;在粪便接触途径下,3株重排AIV都不经粪便接触传播;只有A/Chicken/Shanghai/F/98(H9N2)株和重排AIV RF7/SSHA能经过气溶胶途径传播。HI试验结果进一步证明了以上的结果。实验结果表明H9N2亚型AIV的NA基因与H9N2亚型AIV气溶胶传播途径有重要的关系,即1998年中国大陆H9N2亚型AIV大流行可能是因为病毒获得气溶胶传播途径的特性,推测病毒的NA基因发挥了重要作用。     

H9与H5亚型禽流感病毒血凝素基因的快速鉴别

建立一步法RT-PCR检测方法,对禽流感病毒(Avian influenza virus,AIV)的血凝素(Hemagglutinin,HA)分型进行了研究。参照AIV的HA基因序列设计1对引物,对H9和H5亚型AIV进行了扩增,产物大小分别为579bp和177bp。经测试,该引物不与新城疫病毒等鸡的其它传染性病原及鸡肌肉组织的核酸发生交叉反应。敏感性分析发现,从50pg的AIV总RNA中亦能扩增到目的条带。结果表明,此次利用1对引物建立的一步法RT-PCR方法简便适用,可以在一次反应中同时将H9和H5亚型AIV进行快速检测和分型。另外,两个亚型的扩增产物均包含了HA裂解位点在内的基因序列,可通过测序推导氨基酸顺序以预测H5或H9亚型禽流感病毒的潜在毒力。