Selection of antigenic variants of the influenza virus on the cells of different hosts]

Antigenic differences were found in influenza B virus variants isolated and propagated in different systems: chick embryos (E variants) and MDCK cell culture (M variants). The antigenic differences in M and E variants were detected in HI tests with polyclonal mouse sera and monoclonal antibodies as well as in biological neutralization tests in chick embryos and MDCK cell culture, and confirmed when M and E variants were used as antigens for antibody detection in human sera. By protein mobility in PAGE, M and E variants did not differ from each other and were also identical with the reference B/Victoria/87 strain.     

Comparative studies on influenza B virus. II. The effect of host cells on biological properties and polypeptide composition

The reproduction patterns of various influenza B virus strains isolated in 1970-1976 using roller cultures of MDCK cells and chick embryos (CE) were compared. The cultural and allantoic virus populations did not differ in their sensitivity to non-specific inhibitors from mammalian sera and in their reactivity with specific haemagglutinins (HA). The content of infectious virus and HA in the harvested allantoic fluids as compared to medium fluids were 94- and 8-fold higher, respectively, even though the fluids did not differ in the titres of complement-fixing (CF) antigen. The mol. weight (MW) of HA1 polypeptide of the B/Len/75 virus prepared in culture was higher than that Of the allantoic virus (57.5 K and 55.5 K, respectively). Under reducing conditions, the HA of the virus from culture was represented mostly by the uncleaved HA0 polypeptide, while that of the allantoic virus by the HA1 and HA2 subunits. Under non-reducing conditions, the virus from medium fluid was found to contain glycopeptide D with MW of 90 K.     

Partial change of the ts-phenotype of cold-adapted influenza virus strain grown in canine kidney cells in the presence of trypsin

The cold-adapted temperature-sensitive (ts) influenza virus strain A/Leningrad/134/17/57 (H2N2) multiplied well at 32 degrees C (optimal temperature); lower titres of infectious virus were obtained in developing chick embryos at 40 degrees C. In a canine kidney (MDCK) cell line and in primary calf kidney (CK) cells an increased reproduction of the virus was found at 40 degrees C especially in the presence of trypsin. The ratios of virus titres obtained at optimal versus higher temperatures (RCT40) were by 1,000 times lower than those found in chick embryos. Polyacrylamide gel electrophoresis revealed a comparable synthesis of the cold-adapted influenza virus strain polypeptides HA, NP, M and NS in MDCK cells, regardless whether they were incubated at optimal or non-permissive temperatures.     

Permanent canine kidney (MDCK) cells for isolation and plaque assay of influenza B viruses

A wide range of influenza B virus strains with various passage histories uniformly formed well-defined clear plaques with high efficiency in cultures of an established line of canine kidney cells (MDCK). PFU titers of the viruses assayed in MDCK exceeded the titers assayedin ovo. With recently isolated strains such as B/Hong Kong/5/72 and Gifu/2/73, the PFU/EID₅₀ ratios were as high as 100 to 400. MDCK cells have been successfully employed for primary isolation of influenza B viruses from throat washings of patients by direct plaquing.     

Epidemic strains influenza viruses A and B in the 2005-2006 season in Russia

Investigations indicated that the epidemic upsurge of influenza morbidity in the 2005-2006 season in Russia was caused by the active circulation of influenza viruses A and B. The Center for Ecology and Epidemiology of Influenza, D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, studied 182 epidemic strains. A hundred and thirteen influenza viruses A(H3N2) were similar to the reference A/California/07/2004 or were its antigenic variants. Thirteen influenza virus A(H1N1) strains that were antigenic variants of the reference A/New Caledonia/20/99 were isolated in sporadic cases. Influenza viruses B were similar to B/Malaysia/2506/2004--lineage B/Victoria/2/87). All the strains were isolated in the MDCK cell culture. Comparative study of the sensitivity of the chicken embryo (CE) and MDCK isolation system to the 1999-2006 epidemic strains showed that CE tropism was least pronounced in influenza viruses A(H3N2). Analysis of the 2002-2006 strains demonstrated that influenza viruses A reacted actively with human erythrocytes of the blood groups 0(I) and A(II) and very slightly with chicken ones. Eighty-five influenza virus A(H3N2) strains from the 2005-2006 epidemic season were investigated for rimantadine susceptibility. The frequency of rimantadine-resistant influenza virus A(H3N2) strains was 38.0%. Studies of 79 paired sera from patients revealed a rise of antibodies to influenza viruses A(H3N2) and B in 25.9-33.3 and 20.7-23.8% of cases, respectively. There was an increase in antibodies to influenza viruses A and B in the sera collected from donors in Moscow and its region in September 2005 to June 2006.     

The reproduction of reassortant influenza A and B viruses with a known genome composition in different cell systems]

Reproduction of parental strains and reassortants (with known genome composition) of influenza A and B viruses was studied in chick embryos (CE) and in different cell lines (SPEV, MDCK, BHK-21, M22, etc.). The results agree with the concept that the yield of influenza A virus in CE depends on its M-gene. At the same time, the experimental results suggest that reproduction of influenza B virus in the same system is not determined by M-gene. Reproduction (hr-phenotype) of influenza A and B viruses in cell cultures was shown to be determined not only by the gene coding for hemagglutinin but also by other virus genes, the reproduction level being dependent on different genes in different cell systems.     

北京市门头沟区2015—2016年Victoria系乙型流感病毒HA1基因特征分析

目的 了解北京市门头沟区2015—2016年分离的Victoria系乙型流感病毒血凝素HA1基因变异特征,分析流行株与我国疫苗株的匹配情况,为乙型流感防控提供依据.方法 对狗肾传代细胞(MDCK)培养分离得到的14株Victoria系乙型流感病毒进行核酸提取,采用逆转录-聚合酶链反应(RT-PCR)扩增病毒HA1基因后进行核苷酸序列测定,采用邻接法进行遗传进化树分析.结果 2015—2016年北京市门头沟区流行的乙型流感病毒以Victoria系为主.分离并测序的14株Victoria系乙型流感病毒HA1基因与WHO推荐的2016—2017年流感疫苗株B/Brisbane/60/2008(FJ766842)和国内代表株B/JilinNanguan/1223/2016(EPI768805)亲缘性更近.与B/Brisbane/60/2008和B/JilinNanguan/1223/2016(EPI768805)的HA1区的氨基酸相比,所有毒株都在2个位点发生氨基酸替换,个别毒株也会在其他个别位点发生点突变,变异涉及1个抗原决定簇.而与WHO推荐的2015—2016年Yamagata系疫苗株B/Phuket/3073/2013(EPI608074)亲缘关系稍远一些.结论 在2015—2016年流感监测季中,北京市门头沟区乙型流感病毒以Victoria系为优势流行株.而WHO推荐的乙型流感疫苗株为Yamagata系,可见疫苗株与本区流行株匹配性不佳.     

Replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line

Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.     

Variability of hemagglutinin from strains of influenza virus A (H3N2), isolated in Russian from 1989 to 1999

Analysis of 154 strains isolated in Russia and CIS countries in 1989-1999 showed that influenza virus A(H3N2) caused epidemics and epidemic rises 8 times, circulating together with A(H1N1) and B viruses. Antigenic drift was revealed using polyclonal and monoclonal antibodies. Analysis of antigenic properties of the viruses in the population showed that strains isolated during the same year were usually variants of one or rarely two reference strains. A drop of isolation rate of A(H3N2) strains on chick embryos in recent years was associated with increase in these strains' sensitivity to MDCK culture. Differences in amino acid sequences of epidemic and reference strain hemagglutinins were detected. The number of positions in which the changes were detected varied from 6 to 16 in all antigenic sites: A, B, C, D. and E.     

清远市流行性感冒病原学监测分析

目的分析2012年清远市流感病原学监测结果,了解流感病毒的流行特征以及流行优势毒株。为流感防控工作提供科学依据。方法对清远市监测哨点医院就诊的流感样病例鼻咽拭子用RT-PCR法检测流感病毒核酸．同时用狗肾传代细胞（MDCK细胞）法分离流感病毒并进行毒株鉴定。结果2012年共检测流感样病例鼻咽拭子标本489份,RT-PCR检出阳性115份,阳性率为23．52％,其中季节性H3型、B型和A未分型阳性率分别为45．22％、43．48％、11．30％。细胞培养分离出毒株97株,阳性率为19．84％,其中季节性H3N2型、Bv型、By型分离率分别为61．86％、35．05％和3．09％。1-2月份以B型流感病毒为主要优势毒株,3～7月份流感流行的优势毒株以季节性H3N2型为主。同时也伴随B型流感病毒流行,而8—12月份均未检测到流感阳性病例。结论2012年清远市流感流行优势毒株为季节性H3N2型和B型,流行季节主要在春夏季。RT—PCR检测流感病毒的特异性和敏感性均较细胞培养法高。但细胞培养法作为流感病原学监测的基础,仍然是实验室诊断的金标准。     

MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventional MDCK cells

The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. The suitability of MDCK-SIAT1 cells, engineered to express increased levels of alpha-2,6-linked sialic acid receptors, as an alternative to conventional MDCK cells for isolation of circulating influenza virus was assessed. A greater number of influenza A (H1N1 and H3N2) and B viruses from stored human clinical specimens collected between 2005 and 2007 were isolated following inoculation in MDCK-SIAT1 cells than in MDCK cells. In addition, a higher titer of virus was recovered following culture in MDCK-SIAT1 cells. All A(H1N1) viruses recovered from MDCK-SIAT1 cells were able to agglutinate both turkey and guinea pig red blood cells (RBC), while half of the A(H3N2) viruses recovered after passage in MDCK-SIAT1 cells lost the ability to agglutinate turkey RBC. Importantly, the HA-1 domain of the hemagglutinin gene was genetically stable after passaging in MDCK-SIAT1 cells, a feature not always seen following MDCK cell or embryonated chicken egg passage of human influenza virus. These data indicate that the MDCK-SIAT1 cell line is superior to conventional MDCK cells for isolation of human influenza virus from clinical specimens and may be used routinely for the isolation and propagation of current human influenza viruses for surveillance, diagnostic, and research purposes.     

Pandemic influenza 2009 in Russia. Characteristics of the isolation and biological properties of viruses

Research Institute of Influenza, Ministry of Health and Social Development of Russia, Saint Petersburg The characteristics of the isolation of pandemic influenza A(H1N1)v viruses were studied on chick embryos (CE) and MDCK cell culture. The materials (nasal swabs and autopsies) were collected in different regions Russia in the period from 20 July to 30 December 2009. The paper gives the data of the antigenic analysis of isolates, their capacity to multiply in different species-specific and tissue cell cultures. The viruses isolated on CE were shown to have higher hemagglutination titers and to be more stable. Isolation from the autopsies was effective only on CE. All the test cell lines other than MDCK were insensitive to the isolated pandemic influenza strains. The antigenic analysis showed no significant antigenic drift of the viruses isolated during the first wave of the pandemic in the Russian Federation.     

甘肃省2000-2007年度流感监测结果分析

目的 掌握我省流感流行情况,为流感防治提供依据.方法 按照国家流感监测方案,在6所国家级哨点医院门诊内、儿科开展流感样病例的监测和在全省开展流感暴发疫情的监测,采集流感样患者的鼻咽拭子标本,用MDCK细胞或鸡胚进行流感病毒分离与鉴定.结果 2000-2007年,哨点医院流感样病例占门诊就诊人数的5.16%,从国家级监测哨点医院及暴发疫情共采集标本6383份,分离出流感病毒943份,分离率14.77%,其中H1N1亚型218株,H3N2亚型352株,B型(Victoria系)312株,B型(Yamagata系)61株;全省发生的61起疑似流感疫情暴发,经实验室检测44起确定为流感暴发,其中38起为B(victoria)亚型流感病毒,3起为H3N2亚型、2起为B型(Yamagata系)、1起为H1N1亚型.结论 流感每年均会在人群中活动,冬季以甲型流感为主;2005-2007年,每年3-6月份B型(Victoria系)是造成学校、幼儿园等集体单位暴发的主要病毒.     

Host range recombinants of fowl plague (influenza A) virus

Recombinants between the influenza virus strains fowl plague virus (FPV, Hav1N1) and Hong Kong (H3N2) have been isolated which form plaques on MDCK cells but not on chick embryo cells, although they carry the hemagglutinin of FPV. These host range recombinants have been characterized and one of them has been used for a second recombination with virus N (Hav2Neq1) or equi 2 (Heq2Neq2) on chick embryo cells. In this way, recombinants were obtained with a mixed genome which have regained the natural host range of FPV and some pathogenic properties for chicken. The results are discussed as a possible mechanism for a pandemic influenza strain to survive in an animal reservoir by changing its host range by recombination, and to regain the original host range by a second recombination but always keeping the same hemagglutinin.     

Specificity of neuraminidase activity from influenza viruses isolated in different hosts tested with novel substrates

Summary. Influenza A and B viruses isolated in Vero and Madin Darby canine kidney (MDCK) cells as well as in fertilised hen eggs were tested for the specificity of their neuraminidase (NA) activity. Novel glycoconjugates with variations of terminally bound sialic acid mimicking the three main receptor types for influenza viruses were synthesised. These new substrates together with the lectin from Ricinus communis were used in a solid phase microtitre assay for the detection of NA specificity. Egg or MDCK isolated virus strains tended to exhibit highest NA activity against 3sialyl-bound sialic acid whereas Vero isolated strains favoured 6sialyl-(N-acetyllactosamine)-bound sialic acid. Differences were more pronounced for influenza A than for influenza B strains.     

Effects of streptovirudin on influenza viruses type A and B: inhibition of the lipid-linked oligosaccharide synthesis of fowl plague virus

Antibiotics of the streptovirudin complex (SV) inhibited the growth of influenza A and B viruses such as influenza A/fowl plague virus (FPV), strain Weybridge (Hav1 Neq1), influenza A/England 42/72 (H3N2), influenza A/Port Chalmers 1/73 (H3N2), influenza B/Leningrad 235/74, influenza B/Tokyo 7/66, and influenza B/Jamagata in chick embryo cell (CEC) cultures, in permanent canine kidney cells (MDCK), and in suspended fragments of chick embryo chorioallantoic membranes (CAM). As revealed by spectrophotometric turbidity measurements, SV completely inhibited the FPV-induced cytopathic effect (CPE). A 99.99% reduction of infectious virus yield was obtained in one-step growth cycle experiments and in the plaque reduction test. The haemagglutination inhibition titres of influenza viruses in suspended CAM fragment cultures in the presence of SV drugs were also substantially reduced. The incorporation assays indicated that SV exhibited no effect on virus-induced RNA synthesis, but influenced virus maturation by inhibition of lipid-linked oligosaccharide synthesis. A partial protection from infection was found in influenza virus A/England infected mice.     

Rimantadine and arbidol sensitivity of influenza viruses that caused epidemic morbidity rise in Russia in the 2004-2005 season

The study of the activity of arbidol against epidemic influenza A and B virus strains (2002-2005) in the cultured MDCK cells showed the higher sensitivity of enzyme immunoassay than that of hemagglutination test. The influenza A virus strains tested, including those resistant to rimantadine (5 microg/ml), were sensitive to arbidol (10 microg/ml). The population of influenza B virus strains was heterogeneous in this indicator, 43% of the strains being less sensitive to arbidol. There was an increase in the number of rimantadine-resistant influenza A(H3N2) virus strains (10-18%) in our country during 3 epidemic seasons. The sequencing analysis of protein M2-endoding gene revealed the amino acid replacement of serine by asparagine in position 31, which is characteristic of rimantadine-resistant strains. Arbidol in combination with rimantadine potentiated the effect of viral reproduction in the cultured cells, as compared with the effect produced by the same concentrations of the drugs used alone.     

Extensive heterogeneity in the hemagglutinin of egg-grown influenza viruses from different patients

We establish that the cultivation of influenza (H3N2) virus from any infected individual in chicken embryos (eggs) can result in the isolation of viruses with antigenic and/or structural heterogeneity in the hemagglutinin (HA) molecule. This variability contrasted sharply with the apparent lack of antigenic alterations in the HA of influenza viruses isolated from patients in Madin Darby canine kidney (MDCK) cells. The most common subpopulation of egg-grown influenza viruses had the same phenotype as MDCK cell-grown virus and may best represent the virus circulating in humans. It should be considered the optimal strain for use in vaccine and epidemiologic studies.     

Detection of type A and B influenza viruses in clinical materials by immunoelectronmicroscopy

Direct immunoelectronmicroscopy (IEM) was used for detecting influenza subtype A(H1N1), A(H3N2) and type B viruses in nasopharyngeal washings or swabs collected during three consecutive periods of enhanced influenza incidence. Virus identification was performed with immune rat sera and in the case of the A(H3N2) subtype also with convalescent human sera. In all the materials examined influenza virus was demonstrated by isolation in chick embryos or by immunofluorescence in infected tissue cultures. IEM detected subtype A(H1N1), A(H3N2) and type B viruses in 6 of 13, all 5 and 3 of 8 washings, respectively. Immune complexes were observed in only those materials from which virus was isolated already in the first chick embryo passage, which was evidence that a positive IEM result depended on the amount of virus present in the material. The use of immune sera against two antigenically distinct A(H1N1) strains, A/Khabarovsk/77 and A/England 403/80, did not considerably influence the IEM result.     

用McAb免疫酶染法和IPA法检测鼻咽脱落细胞内流感病毒抗原

用甲型和乙型流感病毒单克隆抗体间接免疫酶组化法（ＩＰＡ）和间接免疫酶染法（ＩＥ）快速诊断鼻咽脱落细胞内流感病毒抗原８０例，并以传统的病毒分离和过ＭＤＣＫ细胞的直接和间接ＩＥ法作对照，获得满意结果。在８０份标本中，用鸡胚分离法分离出阳性标本４６份，分离率为５７．５％；用ＭｃＡｂ直接或间接ＩＥ法测标本感染ＭＤＣＫ细胞的流感病毒抗原，阳性检出率分别为６１．３％、６７．５％；用ＭｃＡｂＩＰＡ法和ＩＥ法检测鼻咽脱落细胞中流感病毒抗原，阳性检出率分别为５７．５％、５５．６％；两种方法与对照组相比较灵敏性分别为８２．１％和８０％，特异性均为１００％，发病第１天阳性检出率占总检出率的６９％。本方法的特点是直接检测标本，只需４小时即可作出诊断，同时可以直接定型。