Predicting antigenic variants of influenza A/H3N2 viruses

Current inactivated influenza vaccines provide protection when vaccine antigens and circulating viruses share a high degree of similarity in hemagglutinin protein. Five antigenic sites in the hemagglutinin protein have been proposed, and 131 amino acid positions have been identified in the five antigenic sites. In addition, 20, 18, and 32 amino acid positions in the hemagglutinin protein have been identified as mouse monoclonal antibody-binding sites, positively selected codons, and substantially diverse codons, respectively. We investigated these amino acid positions for predicting antigenic variants of influenza A/H3N2 viruses in ferrets. Results indicate that the model based on the number of amino acid changes in the five antigenic sites is best for predicting antigenic variants (agreement = 83%). The methods described in this study could be applied to predict vaccine-induced cross-reactive antibody responses in humans, which may further improve the selection of vaccine strains.     

Laboratory-based surveillance of influenza A(H1N1) and A(H3N2) viruses in 1980-81: antigenic and genomic analyses

During 1981, the A/Brazil/11/78-like strains of influenza virus that had been prevalent from 1978 to 1980 were displaced by a new set of heterogeneous, but closely related, variants (reference strain, A/England/333/80). Genomic analysis revealed that these new variants were almost exclusively nonrecombinant H1N1 viruses, i.e., they contained no genes of H3N2 origin. However, a few recombinant viruses containing the new variant HA and genes of H3N2 origin were identified. Antigenic analysis of H3N2 viruses indicated that they were also heterogeneous. The majority of these virus isolates were antigenically intermediate between A/Texas/1/77 and A/Bangkok/1/79, but additional variants were detected. Genomic analysis revealed that the H3N2 viruses isolated in the winter of 1980-81 were quite similar to H3N2 viruses isolated from 1977-79 in their T₁ oligonucleotide maps. No H1N1 genes were detected in H3N2 virus isolates. Comparison of pairs of oligonucleotide maps of total virus RNA indicated that a similar rate of genetic change had occurred for nonrecombinant H1N1 viruses, for recombinant H1N1 viruses, and for H3N2 viruses and that, in general, pairs of viruses exhibited increasing numbers of changes in their oligonucleotide maps as the time interval between isolation of the viruses increased.     

Identifying potential immunodominant positions and predicting antigenic variants of influenza A/H3N2 viruses

Human influenza viruses cause annual epidemics due to antigenic drifts in the hemagglutinin protein. Five antigenic sites in the influenza H3 hemagglutinin protein have been proposed and 131 amino acid positions have been identified in the five antigenic sites. A previous study had documented that a model based on the 131 positions in the five antigenic sites could moderately predict antigenic variants of influenza A/H3N2 viruses (agreement=83%). In this study, prediction models combining serology, bioinformatics and statistics were developed to predict antigenic variants of influenza A/H3N2 viruses. Amino acid sequences of hemagglutinin protein of 45 A/H3N2 viruses isolated during 1971-2002 and 181 pairwise antigenic distances determined by antibody cross-reactivity among the 45 viruses were analyzed as training dataset. In addition, 57 pairwise antigenic distances from 12 A/H3N2 viruses isolated during 1999-2004 were used as validation dataset. Multivariate regression models were employed to identify potential immunodominant positions and predict antigenic variants. Seventeen amino acid positions were identified as potential immunodominant positions in the training dataset. Prediction models based on the potential immunodominant positions have improved performance on predicting antigenic variants in the training (agreement=91%) and validation (agreement=93%) datasets. The model could be readily integrated to the global influenza surveillance system.     

Antigenic heterogeneity within influenza A (H3N2) virus strains

On the basis of their antigenic properties, influenza virus strains are classified into types and subtypes, which are further subdivided into variants that differ to various degrees in haemagglutination-inhibition assays. Evidence is presented that during infection with an influenza A(H3N2) virus the respiratory tract of a human patient often harbours more than one antigenic virus variant. These variants are frequently propagated by embryonated fowl eggs and monkey cells with different efficiencies, and this may lead to the selection of different variants by either of these host systems. Also, passage of virus by a given host is sometimes attended by changes in reactivity in haemagglutination-inhibition tests. In some cases the heterogeneity described also affects the specific immunogenicity of the virus in ferrets. Virus strains cloned in monkey kidney cell cultures gave variants that were stable upon further passage. These results may have implications for antigenic and biochemical investigations of epidemiologically relevant virus variants. It is argued that the antigenic drift of influenza A(H3N2) viruses is best characterized by analyses, both with post-infection ferret antisera and with panels of monoclonal antibodies, of virus strains isolated and passaged in monkey kidney cell cultures only.     

Biochemical and antigenic analysis using monoclonal antibodies of a series of of influenza A (H3N2) and (H1N1) virus reassortants

Reassortant influenza A viruses with high growth capacity in eggs and suitable as candidate vaccine strains or as standard reagents for influenza HA quantification were prepared using the high yielding A/PR/8/34 (H1N1) as one parent and a number of 'wild' strains of influenza A (H1N1) or (H3N2) viruses as the other parent. The genetic and antigenic composition of the reassortants was determined. The parental derivation of genes in the reassortants was established by electrophoretic analysis of virus RNA and virus induced polypeptides. The haemagglutinin (HA) antigens of the three H1N1 viruses (NIB-6, NIB-7 NIB-12) were found to resemble those of the parental viruses when tested against a panel of monoclonal antibodies and using the HI test. A similar correspondence between the antigenic characteristics of the HA of the influenza A (H3N2) reassortants (NIB-1, NIB-4, NIB-5, NIB-8 and NIB-11) and parental viruses was noted. Therefore laboratory manipulations to produce the reassortants did not result in the selection of significant antigenic variants.     

Swine-origin influenza A (H3N2) virus infection in two children--Indiana and Pennsylvania, July-August 2011

Influenza A viruses are endemic in many animal species, including humans, swine, and wild birds, and sporadic cases of transmission of influenza A viruses between humans and animals do occur, including human infections with avian-origin influenza A viruses (i.e., H5N1 and H7N7) and swine-origin influenza A viruses (i.e., H1N1, H1N2, and H3N2). Genetic analysis can distinguish animal origin influenza viruses from the seasonal human influenza viruses that circulate widely and cause annual epidemics. This report describes two cases of febrile respiratory illness caused by swine-origin influenza A (H3N2) viruses identified on August 19 and August 26, 2011, and the current investigations. No epidemiologic link between the two cases has been identified, and although investigations are ongoing, no additional confirmed human infections with this virus have been detected. These viruses are similar to eight other swine-origin influenza A (H3N2) viruses identified from previous human infections over the past 2 years, but are unique in that one of the eight gene segments (matrix [M] gene) is from the 2009 influenza A (H1N1) virus. The acquisition of the M gene in these two swine-origin influenza A (H3N2) viruses indicates that they are "reassortants" because they contain genes of the swine-origin influenza A (H3N2) virus circulating in North American pigs since 1998 and the 2009 influenza A (H1N1) virus that might have been transmitted to pigs from humans during the 2009 H1N1 pandemic. However, reassortments of the 2009 influenza A (H1N1) virus with other swine influenza A viruses have been reported previously in swine. Clinicians who suspect influenza virus infection in humans with recent exposure to swine should obtain a nasopharyngeal swab from the patient for timely diagnosis at a state public health laboratory and consider empiric neuraminidase inhibitor antiviral treatment to quickly limit potential human transmission.     

武汉市儿童甲型H3N2亚型流感病毒基因特性分析

目的 研究甲型H3N2亚型流感病毒（简称A（H3N2）流感病毒）的血凝素（hemagglutinin,HA）基因序列,与流感疫苗株基因序列进行对比分析,了解武汉市2017年儿童中A（H3N2）流感病毒基因变异及3C.2a1次分支病毒的流行状况。方法 采用聚合酶链式反应（polymerase chain reaction,PCR）方法对30株A（H3N2）流感病毒HA基因全长进行扩增,测序后进行基因序列和进化分析。结果 2017年,武汉市1 041份儿童流感样病例标本中,流感病毒阳性标本219份,其中A（H3N2）流感病毒阳性106份（占阳性标本的48.4%）,高峰主要集中在7~9月。HA基因进化分析显示,30株A（H3N2）流感病毒均属于3C.2a分支,其中14株属于3C.2a1次分支。与A/Hong Kong/4801/2014（2017-2018年度北半球流感疫苗推荐H3N2组分）HA氨基酸序列比较,出现K92R、N121K、T131K、T135K/N、R142K/G、N171K、R261Q、H311Q、I406V、G484E等氨基酸位点变异,其中出现在3C.2a1次分支内的变异位点包括K92R、T135N、N171K、H311Q、I406V和G484E。结论 与当季的流感病毒疫苗内组分序列比较,2017年武汉市儿童中流行的A（H3N2）流感病毒HA抗原决定簇内出现多个位点变异,属于3C.2a1次分支的病毒接近50%。     

Transmission of avian influenza virus (H3N2) to dogs

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) isolate. The beagles shed virus through nasal excretion, seroconverted, and became ill with severe necrotizing tracheobronchitis and bronchioalveolitis with accompanying clinical signs (e.g., high fever). Consistent with histologic observation of lung lesions, large amounts of avian influenza virus binding receptor (SAalpha 2,3-gal) were identified in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for direct transmission of avian influenza virus (H3N2) from poultry to dogs. Our data provide evidence that dogs may play a role in interspecies transmission and spread of influenza virus.     

Host cell-mediated selection of influenza A (H3N2) virus variant subpopulations: lack of association between antigenic and receptor-binding properties

During the outbreak of influenza due to A (H3N3) viruses in Finland in 1985/6 virus pairs were isolated from the same clinical specimens in embryonated hens' eggs (CE) and in canine kidney cell cultures (MDCK). Some of these isolates, the E and M pairs, were distinguished by their reactions in haemagglutination inhibition (HI) tests carried out using polyclonal antisera, and by receptor-binding properties, as evidenced by differences in their elution activity from erythrocytes. Passage of the E- and M-virus isolates in the foreign host affected their serological characteristics, but the E virus did not convert to an M-like virus and the M virus did not convert to an E-like virus. Returning the viruses to grow in the host used for their isolation changed the serological reactions so that they were once more close to the reactions of the original isolates. This contrasts with the changes in receptor-binding properties. Rapid elution from hen erythrocytes, which has been described as a property of viruses binding to the SA alpha 2,3Gal sequence in preference to SA alpha 2,6Gal, characterized the virus passages grown solely in MDCK cell cultures. Cultivation of the M virus in CE, at any stage of its passage history, made the virus irreversibly incapable of elution. The M virus was more sensitive than the E virus to HI antibodies against heterologous viruses of the H3N2 subtype, and, when used as an antigen in HI serology, it more frequently (90% vs. 69%; P less than 0.01) detected diagnostic antibody responses in patients infected with viruses of this subtype in 1985/6. Use of antigens with a different passage history in HI serology provided evidence that this superiority, which may be due to the ability of the virus to pick out anamnestic antibody responses, is unrelated to the receptor-binding peculiarity of the M virus under consideration. The results support the concept that the host cell can select a diversity of virus variant subpopulations from a single clinical specimen during isolation and subsequent cultivation procedures. Moreover, the MDCK-grown influenza viruses may correspond better than the egg-grown isolates to the natural epidemic viruses.     

市场外环境禽流感监测数据的分析与利用

目的 为更好地利用市场外环境禽流感监测数据指导防控工作。方法 用生态学趋势研究方法对中山市2014 - 2017年的市场常规外环境监测数据进行描述性分析,结合人感染H7N9的病例数进行等级相关分析和logistics回归分析。结果 中山市市场外环境样本总计H7亚型禽流感病毒的阳性率（下简称H7阳性率）为5.37%（397/7396）;月度市场外环境的H7阳性率与发生人感染H7N9病例数存在正相关（r = 0.52, P＜0.05）,H7污染情况为人感染H7N9病例的危险因素（RR = 5.92, P＜0.05, 95%CI:1.43～24.56）;综合市场H7的阳性率6.72%要显著高于禽类批发市场的 1.85%（χ2 = 68.16,P＜0.05）;砧板、刀具及台面的H7阳性率为7.43%,高于禽类笼具及冰箱物表的4.28%和禽类粪便、污水及地面的2.73%（χ2 = 68.96,P＜0.05）。结论 综合市场的活禽交易区仍应是禽流感防控的重点区域之一,但小范围的短期的活禽交易限制对减少人们暴露的作用有限;单纯依靠市场外环境监测结果开展预警、预测远远不够,需要进一步规范和扩大外环境禽流感病毒的监测。     

Egg-grown and tissue-culture-grown variants of influenza A (H3N2) virus with special attention to their use as antigens in seroepidemiology

A field strain of influenza A (H3N2) virus isolated in embryonated eggs during the 1984-5 influenza outbreak (A/Finland/13/85E) was compared in an antigenic analysis with virus from the same clinical specimen isolated in MDCK cell cultures (A/Finland/13/85M). The M-virus appeared to be more sensitive to haemagglutination-inhibiting antibodies against heterologous viruses than did the E-virus. The results of propagation and plaque purification experiments support the hypothesis that a single clinical specimen may consist of distinct antigenic variant subpopulations promoted selectively by the host during isolation procedures. Receptor-binding properties are discussed as a possible explanation for this selectivity. A set of 471 paired sera consisting of pre-epidemic and post-epidemic specimens taken from the same subjects in 1984-5 was studied for haemagglutination-inhibiting antibodies to six influenza A (H3N2) virus strains, including the E-virus and the M-virus from A/Finland/13/85. Of the antigens used, the M-virus detected significant antibody increases more frequently than did the E-virus (10.0 v. 5.9%). The superiority of the M-virus may rest primarily in its ability to pick out anamnestic antibody responses. Irrespective of this cross-reactivity, pre-epidemic antibody to the M-virus was fairly well associated with protection. In the set of sera (230 specimens) collected in summer 1985 to represent different age groups, the antibody status against the M-virus was significantly better than the status against the E-virus. The results suggest that, at least in some instances, antibody to MDCK-grown virus is a more accurate indicator of the immune status of a community than antibodies to egg-grown virus variants.     

Influenza vaccine strain selection: equivalence of two antigenically distinct haemagglutinin variants of 1989 H3N2 influenza A virus in protection of mice.

Precise antigenic analysis with haemagglutinin-inhibition (HI) tests of 1989 H3N2 influenza A viruses with polyclonal ferret, rabbit and mouse antisera has shown, first, significant differences among 1989 wild-type isolates, second, antigenic differences between two high-yield vaccine candidate reassortant viruses, third, significant antigenic differences of one reassortant (X-105) from the wild-type virus (A/Guangdong A/39) from which it was derived, and fourth, dependence of antigenic characterization of viruses upon the host species used in immunization. Nevertheless, the two reassortant viruses (only 43% similar by HI test) were equally protective in preventing homovariant or heterovariant infection in either previously unimmunized or infection-primed mice. These results not only confirm the known antigenic heterogeneity of influenza A viruses, but raise questions about the adequacy of current methods of antigenic characterization of influenza viruses and the basis for decisions on vaccine strain selection.     

2013-2017年河南省H3N2亚型流感病毒流行特征分析

目的 分析河南省2013-2017年H3N2亚型流感病毒流行特征,为流感防控策略的制定提供依据。方法 采集河南省22家流感监测哨点医院的流感样病例(Influenza like illness,ILI)咽拭子标本,进行荧光定量PCR检测和病毒分离,对H3N2亚型流感病毒检测阳性标本进行统计分析。结果 河南省2013-2017年共检测62 307例ILI病例标本,其中H3N2亚型流感病毒核酸检测阳性4 791例,四个监测年度H3N2亚型阳性占流感总阳性的率分别为14.9%、95.68%、12.36%、76.57%,男女发病率差异无统计学意义;有三个监测年度不同年龄组的H3N2感染率差异有统计学意义;H3N2亚型流感病毒感染人群职业分布以散居儿童(24.73%-35.41%)、托幼儿童(21.94%-30.17%)、学生(16.77%-27.66%)检出人数最多;各个监测年度各市核酸阳性率和病毒分离率不同;H3N2病毒分离株以低血凝滴度(1〖DK〗∶16)二代细胞分离毒株为主(62.33%)。结论 2河南省H3N2亚型流感病毒呈年度周期性流行,是近些年流感流行的的主要型别,儿童和学生是H3N2亚型流感防控的重点人群,且该病毒在细胞中不易培养,呈低滴度高代数特点,应加强病毒分离工作。     

Cross-reactivity of influenza A (H3N2) hemagglutination-inhibition antibodies induced by an inactivated influenza vaccine

The antigenic drift of influenza A (H3N2) virus in 2003-2004 necessitated a change in the vaccine from the A/Panama to the A/Wyoming strain for the 2004-2005 season. Using hemagglutination inhibition, we therefore tested antibodies in sera of 39 individuals (mean age 64.6 years) at the end of the 2003-2004 season for cross-reactivity to vaccine strains and H3N2 antigens subject to antigenic drift. Antibodies against both A (H3N2) Panama and Wyoming developed in 5/13 (38.5%) unvaccinated individuals, whereas, 22/26 (84.6%) vaccinees developed antibodies to Panama and 21/26 (80.8%) to Wyoming. None of these individuals suffered an influenza episode that season. The results suggest that the elderly might develop protective levels of cross-reactive A (H3N2) Wyoming HI antibodies following vaccination with the Panama strain. Such strains, like the ones included in the 2003-2004 influenza vaccine, might be expected to provide a broad-spectrum antibody response that could be effective even in the face of single season antigenic drift.     

Laboratory-based surveillance of influenza virus in the United States during the winter of 1977-1978. I. Periods of prevalence of H1N1 and H3N2 influenza A strains, their relative rates of isolation in different age groups, and detection of antigenic variants

Influenza A (H3N2) viruses were isolated from outbreaks and epidemics of disease during the period December 1977 to March 1978. For the last two months of this period, H1N1 strains of influenza A were also responsible for epidemics. In some regions (e.g., Hawaii) co-circulation of H1N1 AND H3N2 strains occurred, whereas in other regions (e.g., Wisconsin) isolation of H3N2 strains had almost ceased prior to isolation of H1N1 strains. Few influenza B isolates were reported. Analysis of the ages of patients from whom specimens were submitted for influenza virus isolation confirmed that, whereas H3N2 strains were isolated from persons of all ages, greater than 97 per cent of H1N1 isolates in six states analyzed were recovered from patients less than 26 years old, although specimens were tested from older persons who were ill during the period of prevalence of H1N1 influenza. The majority of H3N2 isolates tested by hemagglutinin-inhibition reaction were similar to A/Texas/1/77, and the majority of H1N1 isolates were similar to A/USSR/90/77. Antigenic analysis of isolates, however, identified a small number of variants of H3N2 and H1N1 strains.     

新型甲型H1N1流感流行期间大连市郊区流感病毒监测分析

目的:了解2009~2010年新型甲型H1N1流感大流行期间,大连市郊区流感病毒的流行情况。方法:对大连市外各区的流感样病人咽拭子标本采用Real-time RT-PCR方法进行流感病毒核酸检测。结果:2009年11月~12月郊区标本均为新型甲型H1N1流感病毒核酸阳性,2010年1月部分地区标本季节性流感病毒核酸阳性,2010年2月后各区标本均季节性流感病毒核酸阳性。结论:2010年1月为新型甲型H1N1流感病毒和季节性流感病毒交替期,之前流行株为新型甲型H1N1流感病毒,之后的流行株为季节性流感病毒。     

Laboratory characteristics of an attenuated influenza type A (H3N2) virus ('Alice' strain).

The Alice strain of live attenuated influenza virus was obtained by selection of a gamma inhibitor-resistant strain from a virus recombinant between A/PR/8/34 (HON1) and A/England/42/72 (H3N2). Its behaviour in vitro and in vivo was studied. Three marker systems were investigated: resistance to serum inhibitors, growth capacity at high temperature and low sensitivity to amantadine hydrochloride. In ferrets the strain was found to be attenuated and immunogenic. Passages in man, animals and eggs have not affected its resistance to gamma inhibitors.     

Laboratory characteristics of an attenuated influenza type A (H3N2) virus ('Alice' strain).

The Alice strain of live attenuated influenza virus was obtained by selection of a gamma inhibitor-resistant strain from a virus recombinant between A/PR/8/34 (HON1) and A/England/42/72 (H3N2). Its behaviour in vitro and in vivo was studied. Three marker systems were investigated: resistance to serum inhibitors, growth capacity at high temperature and low sensitivity to amantadine hydrochloride. In ferrets the strain was found to be attenuated and immunogenic. Passages in man, animals and eggs have not affected its resistance to gamma inhibitors.