A novel M2e-multiple antigenic peptide providing heterologous protection in mice

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.     

H9N2亚型禽流感疫苗株的筛选及其免疫效果评价

通过交叉血凝试验,疫苗备用毒株免疫SPF鸡后再用同源和异源毒株攻毒,评价疫苗备用毒株对SPF鸡保护效果。结果显示,H9N2疫苗备用株免疫SPF鸡后,再用H9亚型流感病毒流行株攻毒,SPF鸡咽喉和泄殖腔排毒量大大降低。结果表明,本试验筛选的疫苗备用毒株对H9N2亚型流感病毒具有一定的保护率,完全符合疫苗株要求。     

An H9N2 influenza virus vaccine prepared from a non-pathogenic isolate from a migratory duck confers protective immunity in mice against challenge with an H9N2 virus isolated from a girl in Hong Kong

H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.     

Development of vaccine strains of H5 and H7 influenza viruses

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.     

Evaluation of the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 influenza viruses in chickens

Despite the long-term vaccination programs implemented in China, H9N2 avian influenza viruses (AIVs) continue to persist in chicken populations, even in vaccinated flocks. We previously demonstrated that H9N2 AIV isolated from chickens in China also underwent antigenic drift and evolved into distinct antigenic groups (C, D and E). To understand whether antigenic drift of viruses away from the vaccine strain partially contributed to the circulation of H9N2 AIV in China, we evaluated the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 AIV. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group C viruses, but not those of the more recent groups D and E. Vaccinated chickens, even those with vaccine-induced HI titers of 1:1024, shed virus after being infected with A/chicken/Shandong/ZB/2007, a representative virus of antigenic group D. Genetic analysis showed that the representative viruses of antigenic groups D and E possessed greater numbers of amino acid substitutions in the hemagglutinin protein compared to the vaccine strain and the antigenic group C virus, and many of which were located in antigenic sites. Our results indicated that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection, and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection. Furthermore, the avian influenza vaccination policy also needs to be re-assessed, and increased veterinary biosecurity on farms, rather than vaccine application alone, should be implemented to prevent and control avian influenza.     

Antigenic variation among equine H 3 N 8 influenza virus hemagglutinins

To provide information on the antigenic variation of the hemagglutinins (HA) among equine H 3 influenza viruses, 26 strains isolated from horses in different areas in the world during the 1963-1996 period were analyzed using a panel of monoclonal antibodies recognizing at least 7 distinct epitopes on the H 3 HA molecule of the prototype strain A/equine/Miami/1/63 (H 3 N 8). The reactivity patterns of the virus strains with the panel indicate that antigenic drift of the HA has occurred with the year of isolation, but less extensively than that of human H 3 N 2 influenza virus isolates, and different antigenic variants co-circulate. To assess immunogenicity of the viruses, antisera from mice vaccinated with each of the 7 representative inactivated viruses were examined by neutralization and hemagglutination-inhibition tests. These results emphasize the importance of monitoring the antigenic drift in equine influenza virus strains and to introduce current isolates into vaccine. On the basis of the present results, equine influenza vaccine strain A/equine/Tokyo/2/71 (H 3 N 8) was replaced with A/equine/La Plata/1/93 (H 3 N 8) in 1996 in Japan. The present results of the antigenic analysis of the 26 strains supported the results of a phylogenetic analysis, that viruses belonging to each of the Eurasian and American equine influenza lineages have independently evolved. However, the current vaccine in Japan consists of two American H 3 N 8 strains; A/equine/Kentucky/1/81 and A/equine/La Plata/1/93. It is also therefore recommended that a representative Eurasian strain should be included as a replacement of A/equine/Kentucky/1/81.     

Clade2.3.2 H5N1亚型禽流感疫苗候选株的构建

近年来Clade2.3.2H5N1亚型的禽流感病毒逐渐成为我国及越南等其他一些国家流行的优势毒株,并呈现出一定变化规律的氨基酸进化趋势。本研究以A/Puerto Rico/8/34(H1N1)(PR8)AIV为内部基因供体,以Clade2.3.2H5N1亚型AIV A/chicken/Yangzhou/1117/2011(YZC3)为表面抗原血凝素(HA)和神经氨酸酶(NA)基因供体,通过反向遗传操作,在符合人类疫苗生产标准的COS-1细胞中救获低致病性的疫苗毒株。结果成功拯救出1株重组病毒rYZC3,该病毒在鸡胚和MDCK细胞上均具有较好的繁殖能力,对SPF鸡和鸡胚无致病性。本研究为防控当代流行的H5亚型禽流感提供了良好的疫苗候选株。     

H9N2亚型禽流感病毒抗原性变异的研究

对1998—2002年间在河南省豫北地区分离到的5株H9N2亚型禽流感病毒的抗原性变异进行了研究。经HI试验、鸡胚中和试验、细胞中和试验及攻毒保护试验证明，5株H9N2亚型间已经发生了抗原性漂移。98A5和99S毒株间的保护力接近100％，HI试验、鸡胚中和试验、细胞中和试验的相关性均在0．74以上。表明2毒株间的抗原性相近；用00Y毒株攻击其他4株免疫的鸡，其保护率仅为60％～80％；而02Y株对除00Y株外的4株的免疫保护率分别为60％、75％、80％、100％，与分离年代呈负相关性，HI、鸡胚中和试验、细胞中和试验也取得类似结果，说明2000年后的毒株间已发生抗原性变异。     

A single dose of inactivated oil-emulsion bivalent H5N8/H5N1 vaccine protects chickens against the lethal challenge of both highly pathogenic avian influenza viruses

In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 10⁸/EID₅₀ /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log₂). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.     

Molecular characterization of human influenza viruses--a look back on the last 10 years

Influenza A (H3N2) viruses and influenza B viruses have caused more than 90% of influenza infections in Germany during the last then years. Continuous and extensive antigenic variation was evident for both the hemagglutinin (HA) and neuraminidase (NA) surface proteins of H3N2 and influenza B viruses. Molecular characterisation revealed an ongoing genetic drift even in years when the antigenic profiles of circulating strains were indistinguishable from those of the previous season. Retrospective phylogenetic studies showed that viruses similar to vaccine strains circulated one or two years before a given strain was recommended as vaccine strain. New drift variants of H3N2 viruses with significantly changed antigenic features appeared during the seasons 1997/1998 and 2002/2003. Most influenza seasons were characterised by a co-circulation of at least two different lineages of H3N2 viruses. Genetic reassortment between H3N2 viruses belonging to separate lineages caused the different evolutionary pathways of the HA and viruses was responsible for the occurrence of H1N2 viruses during the season 2001/02. This new subtype has been detected only sporadically in Germany. The evolution of influenza B viruses was characterised by the re-emergence of B/Victoria/2/87-lineage viruses and their co-circulation with viruses of the B/Yamagata/16/88-lineage. Reassortant B viruses possessing a Victoria/87-lineage HA and a Yamagata/88-like NA were predominant in Germany during 2002/03 and 2004/05.     

Evaluation of a protective immunity induced by an inactivated influenza H3N2 vaccine after an intratracheal challenge of pigs.

A challenge study was conducted to evaluate the safety and efficacy of an inactivated influenza H3N2 virus vaccine combined with Quil A/Alhydrogel mixture under controlled conditions in piglets. Twenty-four piglets from 12 sows were allocated to 2 groups; injected intramuscularly with 2 doses of the tested vaccine or with PBS at 2 wk intervals and challenged intratracheally with 105TCID50 of the H3N2 swine influenza virus 6 d after the 2nd immunization. Clinical and virological parameters were recorded for 4 d after the challenge. The use of the tested vaccine produced high serum hemagglutination-inhibition titers against the swine H3N2 strain virus. This strong immune response suppressed all clinical signs and viral shedding and reduced pulmonary lesions due to the challenge in the vaccinated group, without causing any secondary effects. Our results suggest that the serum HI titers correlated with the degree of protection induced by an inactivated swine influenza H3N2 vaccine.     

中国流行的2009甲型H1N1猪源流感病毒HA和NA基因的分子和遗传特征

目前流行的甲型H1N1流感病毒是一个复杂的基因重配病毒。对病毒的分子生物学研究,尤其是病毒囊膜蛋白血凝素（haemagglutini,HA）基因和神经氨酸酶（neuraminidase,NA）基因的研究,为控制和预防H1N1流感病毒具有重要的意义。本研究对中国流行的2009甲型H1N1猪源流感病毒的HA和NA基因与疫苗株A/California/07/2009（H1N1）,以及不同国家和地区的病毒株进行核苷酸和氨基酸序列分析。从NCBI的GenBank数据库下载所需要毒株的序列,采用Lasergene 6.0软件包中的EditSeq和MegAlign进行序列分析,进化树分析采用MEGA4.1软件。进化分析表明,中国流行的2009 H1N1流感病毒与疫苗株的核苷酸同源率分别在98.8%~99.7%和98.6%~99.6%之间;裂解位点处为I/VPSIQSR↓G,不具备高致病性流感病毒的特征;有1株NA抗性病毒。尽管与疫苗株相比,中国流行株2009甲型H1N1猪源流感病毒的HA和NA基因有部分突变,但这些突变并不是重要的。本研究首次详细分析了中国流行的2009甲型H1N1猪源流感病毒株与疫苗株的HA和NA基因的分子特征,对实时监测流感病毒HA和NA基因的变化具有重要意义。     

Efficacy of a pandemic (H1N1) 2009 virus vaccine in pigs against the pandemic influenza virus is superior to commercially available swine influenza vaccines

In April 2009 a new influenza A/H1N1 strain, currently named "pandemic (H1N1) influenza 2009" (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5-3 log(10) by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log(10)TCID(50) per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is able to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.     

Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Expression of the C-type lectins DC-SIGN or L-SIGN alters host cell susceptibility for the avian coronavirus, infectious bronchitis virus

Despite extensive vaccination, H9N2 subtype influenza A viruses (IAVs) have prevailed in chicken populations in China. H9N2 IAVs have been a major cause of respiratory disease and reduced egg production, resulting in great economic losses to the Chinese poultry industry. In attempt to find reasons for lack of adequate protection by commercial vaccines, 41 H9N2 viruses isolated from chicken flocks in various regions of China through surveillance between 1998 and 2007 were systemically analyzed using molecular and serological methods in comparison to IAV Ck/Shandong/6/96 and Ck/Shanghai/F/98 that have been used in a majority of commercial vaccines for H9N2 in China since 1998. The analyses showed that the field isolates were predominantly of Beijing/94 lineage and underwent rapid genetic and antigenic changes, forming several antigenic groups. Comparisons between the field isolates and vaccine strains revealed that a majority of the field isolates examined were antigenically distinct from the vaccine strains to some extent. Therefore, the rapid antigenic evolution of H9N2 IAV and resulting antigenic difference from the earlier vaccine strains appears to be a key factor for suboptimal control of H9N2 IAV in China, emphasizing that the vaccine strain should be updated in a timely manner through surveillance and accompanying laboratory evaluation of contemporary viruses for antigenic similarity with existing vaccine strains.     

Generation and evaluation of reassortant influenza vaccines made by reverse genetics for H9N2 avian influenza in Korea

The prevalence and continuous evolution of H9N2 avian influenza viruses in poultry have necessitated the use of vaccines in veterinary medicine. Because of the inadequate growth properties of some strains, additional steps are needed for producing vaccine seed virus. In this study, we generated three H9N2/PR8 reassortant viruses using a total cDNA plasmid-transfection system, as an alternative strategy for developing an avian influenza vaccine for animals. We investigated the vaccine potency of the reassortant viruses compared with the existing vaccine strain which was adapted by the 20th serial passages in embryonated eggs with A/Ck/Kor/01310/01 (H9N2). The H9N2/PR8 reassortant viruses, containing the internal genes of the high-yielding PR8 strain and the surface gene of the A/Ck/Kor/01310/01 strain, could be propagated in eggs to the same extent as existing vaccine strain without additional processing. Similar to vaccine strain, the H9N2/PR8 reassortant viruses induced hemagglutination-inhibiting antibodies in chickens and prevented virus shedding and replication in multiple organs in response to homologous infection. However, due to the continuing evolution and increasing biologic diversity of H9N2 influenza in Korea, the vaccine provided only partial protection against currently isolates. Taken together, our results suggest that the H9N2/PR8 reassortant virus can be used as a seed virus for avian influenza vaccines in poultry farm. Considering the constant genetic changes in H9 strains isolated in Korea, this reverse genetic system may offer a prompt and simple way to change the vaccine seed virus and mitigate the impact of unexpected influenza outbreaks.     

猪流感病毒的分离与鉴定

从广东某猪场表现发热和流鼻涕等呼吸道症状的猪群采集28份样品,用鸡胚分离到9株病毒。将病毒传代至第6代,经血清学鉴定、理化特性检查、电镜观察、生物学特性检查和人工致病试验,结果表明,4株分离株为H1N1亚型猪流感病毒,5株为H3N2亚型猪流感病毒。     

水禽用H5亚型禽流感灭活疫苗对高致病性禽流感病毒流行毒株的免疫保护效果研究

为评价水禽用禽流感灭活疫苗(H5N2亚型,D7株)对2010年以后分离的高致病性禽流感病毒流行毒株的免疫保护效果,将该疫苗免疫3周龄SPF鸭后,21 d采血、分离血清测定HI抗体效价,同时用5株2010年以后分离的高致病性禽流感流行毒进行攻毒保护试验,攻毒后5d采集所有试验鸭喉头和泄殖腔拭子进行病毒分离.结果显示,该疫苗免疫SPF鸭21 d后的HI抗体效价的几何平均滴度达7.4log2,对5株高致病性禽流感病毒的攻击均可产生良好的免疫保护,并有效阻止病毒排泄.该疫苗的推广使用将对我国水禽高致病性禽流感的防控发挥重要作用.     

Novel H1N2 swine influenza reassortant strain in pigs derived from the pandemic H1N1/2009 virus

Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.     

H9N2 AIV HA蛋白S145N变异毒株的抗原性和免疫原性

在研究1998-2008年中国H9N2亚型禽流感病毒（AIV）分离株HA基因的进化时,发现在25个毒株中有2个致病性最强的毒株因HA基因第145位氨基酸的突变导致产生1个潜在的糖基化位点,从而使其不与单抗H6、F6等反应。为进一步探究这类变异毒株HA基因变异对H9亚型AIV的抗原性和免疫原性的影响,本试验对12株HA蛋白S145N变异的H9N2AIV进行了交叉中和试验和交叉攻毒试验。结果显示,不同H9N2S145N变异株与疫苗株间在抗原性上变化不大,或无显著差异（0.5≤R≤0.67）。但参照现有的H9灭活疫苗效力检验方法对HP疫苗免疫鸡进行攻毒,用HP株攻毒对照组0/5保护,免疫组保护≥9/10,达到了H9灭活疫苗质量标准要求;但用S145N变异株N3攻毒,仅保护2/10～6/10,且随免疫量剂量的增加,抗体水平的提高,攻毒保护也依次升高。对H9变异株疫苗（N1、N2、N3、N8）免疫鸡用N3攻毒,仅保护2/5～4/5,N3同源抗体也不能有效地阻止其攻毒后的排毒。用N3、N6 2个变异株交叉攻毒,采用与疫苗株攻毒相同的剂量作攻毒试验也得到类似结果。表明高于6log2的抗体能抵抗疫苗株和大多数流行毒株攻毒后的排毒,但不能抵抗S145N变异株攻毒后的排毒。这类毒株免疫原性上的变化与病毒HA基因的变异密切相关。因HA基因145～147aa位增加了1个NGT,导致三维空间构象的变化,并影响其邻近的受体结合位点,从而使这类毒株致病性提高,免原性发生改变。虽然这一类变异株或免疫逃逸毒株仅占当前流行毒株总数的5%～7%,但在强大的免疫压力和自然选择下有可能逐步成为优势毒株,造成更大的危害,这为该病的防控提出了新的挑战。