Quantitative characteristics of the process of solubilizing hemagglutinin isolated from influenza viruses H1N1 and H3N2

Quantitative characteristics of the process of solubilization of influenza H1N1 and H3N2 virus hemagglutinin treated with bromeline enzyme were studied. After a long-time contact of viruses with the enzyme the hemagglutinin was found to be destroyed significantly. Certain conditions should be observed in order to increase the amount of hemagglutinin separated by the enzyme and its final yield. First, the quality of the original virus concentrate should be evaluated by electron microscopy. Second, the process of hemagglutinin solubilization should be monitored by single radial immunodiffusion method and electron microscopy to determine the optimal period of incubation of the virus material with bromeline. Finally, several (usually 2-3) incubations of optimal duration ("fractionated" incubation) should be carried out.     

Reassortment and evolution of current human influenza A and B viruses

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.     

Internal epidemic influenza virus proteins: isolation and investigation

The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.     

Influenza viruses which preconditioned the epidemic rise in Russia in 2002-2003. A resumed circulation of influenza viruses similar to V/Victoria/2/87

According to research, the epidemic rise of influenza was preconditioned, during 2002-2003, in Russia by the circulation of influenza A(H1N1), A(H3N2) and B viruses. The Center of Influenza Ecology and Epidemiology undertook a study of 178 epidemic strains: 41 strains A(H1N1), 116 strains A(H3N2) and 21 strains of influenza B were among them. All strains were isolated in the MDCK cell culture. A simultaneous isolation in embryonated eggs as well as changing of the isolation system from MDCK to embryonated eggs were found to be effective only for influenza A(H1N1) viruses. According to the antigenic analysis, all A(H1N1) viruses were variants of the etalon A/New Caledonia/20/99. The A(H3N2) viral strains' population was heterogeneous by its antigenic properties: among its isolates, there were variants similar to the etalons of A/Moscow/10/99 and of A/Panama/200/99 as well as strains, which weakly reacted with sera of both above etalons; possibly the latter were close to the etalon of A/Fujian/411/02. All epidemic strains of influenza B virus belonged, according to the antigenic properties of hemagglutinin, to the virus group of B/Victoria/2/87-like and were antigenic variants of the etalon of B/Hong Kong/22/01. This confirmed that influenza B viruses with the antigenic hemagglutinin structure of the virus group of B/Victoria/2/87-like, which were not present in Russia for more than 10 years, re-entered the active circulation. An analysis of antigenic properties of neuraminidases (NA) of the mentioned epidemic strains showed their different degrees of relationship with the NA etalons of both evolutionary groups, i.e. B/Victoria/2/87 and B/Yamagata/16/88-like. A study of paired sera obtained from patients showed a growth of antibodies to the etalons of influenza A(H1N1), A(H3N2) and B viruses of the season in question, which confirmed the virology data.     

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.     

Spread and properties of epidemic influenza A and B virus strains accountable for morbidity in Russia during 1999-2002

The strains of influenza A -A(H1N1), A(H3N2) and B viruses were shown in the paper to have been circulating in Russia in 1999-2002. A co-circulation of viruses of 2 to 3 types was detected in all epidemic seasons. A majority of strains was isolated on the culture of cells MDCK. A study of epidemic strains revealed the predominance of viruses A(H3N2) in 1999-2000, the predominance of viruses A(h1N1) in 2000-2001, and the predominance of influenza B viruses in 2001-2002. According to the conducted antigenic analysis, all A(H1N1) isolates were similar to the etalon A/New Caledonia/20/99. The antigenic drift of hemagglutinin of A(H3N2) epidemic strains was oriented towards the etalons of A/Sydney/5/97--A/Moscow/10/99; while in influenza B viruses it was oriented towards the etalons B/Beejing/184/93--B/Yamanashi/166/98--B/Sichuan/379/99. Sequencing of hemagglutinin gene HA1 showed certain difference in the gene structure of epidemic strains A(H1N1) and A(H3N2) versus the etalon ones, which were registered, including at antigen sites. An analysis of paired sera obtained from patients confirmed the virologic findings, i.e. it detected a growth of antibodies to viruses that circulated during an actual season.     

Ultrastructure and some biological properties of influenza A virus. IV. Effect of proteolytic enzymes on viral particle

Influenza virus strain A/USSR/053/74/H3N2 was subjected to the effect of soluble and nonsoluble trypsin. Both enzymes appeared to affect the activity of neuraminidase. The viruses altered in this way did not induce interferon (IFN) in mice. Treatment of viral particles with soluble bromeline brought about almost complete inactivation of hemagglutinin and slight suppression of neuraminidase activity. This virus was capable of IFN induction. On the other hand, when nonsoluble bromeline was used as the treatment agent, complete inactivation of neuraminidase and reduction of hemagglutinin titer were observed. This virus was not capable of IFN induction. Viral preparations treated with pronase, trypsin and bromeline for a total extraction of the surface antigens, did not induce IFN. The viruses treated with these enzymes were examined under the electron microscope. Trypsin-treated viruses showed no changes on their surface while bromeline and pronase totally extracted the spikes of surface antigens.     

Monoclonal antibodies with high virus-neutralizing activity against pandemic influenza virus A/llV-Moscow/01/2009 (H1N1)swl

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.     

The properties of the epidemic influenza viruses A and B strains circulating in Russia in the 2004-2005 epidemic season

The epidemic upsurge of influenza morbidity in Russia in 2004-2005 was caused by the active circulation of influenza A(H3N2) and B viruses. A hundred and sixty-six epidemic strains were studied. All the strains were isolated in the MCK cell culture. Influenza A(H3N2) viruses (n=77) were antigenic variants of the reference A/Fujian/411/ 2002 and A/California/7/2004 strains. Three influenza A(H1N1) viral strains that were antigenic variants of A/New Caledonia/20/99 strains were isolated in sporadic cases. Influenza B virus strains (n=83) were antigenic variants of the reference B/Shanghai/361/02--lineage B/Yamagata/l6/88. In addition, 3 antigenic variants of B/Hong Kong/ 330/2002 (lineage B/Victoria/2/87) strains were isolated. Nine (20%) strains resistant to rimantadine at a concentration of 5 microg/ml were identified. Chromatographic analysis of B/Shanghai/361/02 and BIHong Kong/330/01 viral protein M1 trypsin hydrolysates revealed differences in the profiles of chromatograms of influenza A virus proteins M1. Examination of 121 pair sera from patients revealed an increase in antibodies to influenza A(H3N2) viruses in 10-21% of cases and to influenza B viruses in 20-36% of cases.     

Analysis of etiology of influenza-like morbidity and monitoring influenza epidemic of 1998-1999 by laboratory diagnosis methods

The etiological structure of influenza-like was analyzed in the population in cities and towns and in Russia as a whole in November 1998 to April 1999 by the findings of immunofluorescence and serological surveys of patients with acute respiratory viral infections (ARVI). By the results of both tests, the proportion of the incidence of influenza A (H3N2) was largest, the decreasing order in their significance was as follows: adenoviruses, type 3 parainfluenza virus, RSV, influenza B virus, influenza A(H1N1), types 2 and 1 parainfluenza virus. All influenza viruses A(H1N1) were isolated in Samara in February 1999. Three of them were similar to the reference strain A/Johannesburg/82/96 in antigenic properties, two strains appeared to be its drift variants. No A/Beijing/262/95 (H1N1)-like viruses recommended for incorporation as part of vaccines were detected. All influenza A(H3N2) viruses were drift variants of strain A/Sydney/05/97, and all influenza B viruses were similar to the reference strain B/Harbin/07/94 in antigenic structure.     

Molecular characterization of influenza viruses circulating in Northern Italy during two seasons (2005/2006 and 2006/2007) of low influenza activity

The influenza activity and circulation of influenza viruses in Lombardy (the most populous Italian region) were observed during two consecutive seasons (2005/2006 and 2006/2007) characterized by low influenza activity by the Italian Influenza Surveillance Network. The molecular characteristics of circulating viruses were analyzed to evaluate the introduction of new variants and emergence of vaccine-escape viruses. In both seasons, the epidemic in Lombardy was sustained almost exclusively by influenza A viruses, accounting for 80.5% and 93.6% of total detections, respectively, and the co-circulation of A/H3 viruses belonging to distinct phylogenetic groups was observed. The A/H1N1 viruses isolated during the 2005/2006 season were closely related to A/New Caledonia/20/99, while the hemagglutinin (HA) sequences of the A/H1N1 viruses from the 2006/2007 season exhibited a greater diversity. These viruses were A/Solomon Islands/3/2006-like and showed several variants. All B isolates were similar to B/Malaysia/2506/2004 belonging to the B/Victoria/2/87-lineage. Influenza B virus was the dominant virus in Europe in the 2005/2006 season and accounted for the 20% of total detections in Lombardy. Overall, the viruses studied presented heterogeneity in their HA sequences suggesting the circulation of a miscellaneous set of variants during the two seasons notwithstanding the medium-low activity of influenza. The importance of virological surveillance of influenza viruses is recognized widely and the molecular characterization of the viruses, especially in vaccinated subjects, is of particular importance to evaluate the introduction and circulation of new variants.     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the influenza B antibodies detected influenza B reference viruses collected between 1940 and 1984. Pools of either influenza A or influenza B monoclonal antibodies were used to detect influenza viruses reisolated from clinical specimens in tissue culture. At 48 h after inoculation, the influenza A monoclonal antibodies detected 64% of H1N1 and 94% of H3N2 influenza A specimens, and the influenza B monoclonal antibodies detected 79% of the influenza B specimens. The results of this study suggest that the monoclonal antibodies described should provide useful diagnostic reagents for workers in virology laboratories who wish to isolate and identify influenza virus but have been unable to obtain consistent supplies of animal sera specific for influenza A or B viruses.     

Reassortants in recent human influenza A and B isolates from South East Asia and Oceania

From 2000 to 2002, human influenza A and B viruses that were genetic reassortants of contemporary circulating human strains, were isolated in South East Asia and Oceania. Similar to reports from other regions, A(H1N2) isolates were found to be reassortants of circulating A(H3N2) viruses that had acquired only the haemagglutinin gene of an A(H1N1) virus. Some of these reassortants from Thailand and Singapore predate those previously recorded during the winter of 2001-2002 in Europe and the Middle East and may be precursors of these viruses. The B reassortants had a haemagglutinin similar to an earlier B strain, B/Shangdong/7/97 (B/Victoria/2/87-lineage) and a neuraminidase similar to the recently circulating B/Sichuan/379/99 virus (B/Yamagata/16/88-lineage). Despite the early occurrences of A(H1N2) reassortants and the extensive circulation of A(H1) viruses in South East Asia and Oceania during 2000-2001, these reassortant influenza A viruses have to date not been prominent unlike Europe and the Middle East where they were common in the 2001-2002 winter. In contrast the reassortant B viruses, which first emerged in this region in early 2002, rapidly became the predominant strains isolated from patients with influenza B in South East Asia and Oceania.     

Influenza surveillance during the 2009-2010, 2010-2011, 2011-2012, and 2012-2013 seasons in Algeria

We report the activity and circulation of influenza viruses in Algeria during four influenza seasons, from a national surveillance study carried out from 2009-2010 to 2012-2013. A total of 2766 samples from in- and outpatients, with no age restriction, were collected. The overall proportion of specimens that tested influenza positive was 46.0%. Overall, 96.6% of influenza A viruses were subtyped, and A/H1 subtypes accounted for 57.3% of influenza A viruses. Influenza A/H1 and A/H3 virus subtypes cocirculated in 2009-2010. In 2010-2011, a high proportion of type B viruses (66.2%) was observed. The subtype H3N2 was identified in 99% of cases typed in 2011-2012. Influenza A/H3N2 and B virus cocirculated in 2012-2013. A remarkably low influenza vaccination rate of 2.4% was observed among all age groups. Antibiotics were prescribed for 926 (41.3%) patients, and no difference was observed between patients with confirmed influenza and patients with influenza-like illness not related to influenza. The burden of influenza is largely undocumented in Algeria and strategies to expand this surveillance across the country are needed. Strategies to increase vaccination coverage are warranted to control and prevent influenza in individuals at risk of complications as well as in the general population.     

Cytokine induction in human cord blood lymphocytes after pulsing with UV-inactivated influenza viruses

Mitogenic activity of UV-inactivated influenza viruses in cord blood lymphocytes (CBL), as measured by cytokine release, was investigated. Using prototype viruses of subtype H3N2 (A/Aichi/68), H2N2 (A/Japan/57), and H1N1 (A/Puerto Rico/34) for influenza A virus, and B/Lee/40 for influenza B virus, the results indicated that both Th1 and Th2 cytokines were induced. Stimulation indices were significantly higher for IFNgamma, IL-4 and IL-10 by influenza A viruses than by influenza B virus. Stimulation indices for IL-2 and IL-6 were lower, as these two cytokines were spontaneously released by cord blood lymphocytes in culture. Alignment of the amino acid sequences of the HA for the viruses used in this study indicated that influenza B virus lacked sequence homology to the antigenic sites identified for influenza A virus. Therefore, the antigenic sites may play a role in the mitogenic property, and cord blood lymphocytes could provide a system to compare this property for clinical isolates of influenza virus.     

Influenza morbidity in the Kazakh SSR 1977-1984

In 1977-1984, 6 epidemic outbreaks of influenza were recorded in the republic of which 2 were due to influenza A (H1N1), 2 to A (H3N2) and 2 to influenza B virus. The epidemics of influenza H1N1 (1977-1978, 1981) and H3N3 (1979-1980, 1983) were characterized by gradual decrease of their intensity as manifested by lowering of the morbidity rate and frequency of virus isolation. On the contrary, epidemics of influenza B showed a certain trend to increase. An important feature of the epidemics etiologically associated with A (H1N1), A (H3N2), and B viruses consisted in the predominant involvement of the child population.     

1994～1997年深圳地区流感病毒活动及H3N2亚型毒 …

目的 了解几年流感病毒在深圳地区活动的特点及甲３（Ｈ３Ｎ２）亚型毒株ＨＡ１基因演变概况。方法 病毒分离采用常规的鸡胚双腔接种,毒株检和常量半加敏ＨＩ测定。新鲜收获含病毒粒的鸡胚尿囊液用来提取ＲＮＡ,经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ）扩增,产物纯化采用双脱氧链末端终止法进行核苷酸序列测定。结果 近几年来深圳地区流感活动概况与全国情况相一致;在人群中仍同时流行Ｈ３Ｎ２,Ｈ１Ｎ１ 型和乙型毒     

Evaluation of live avian-human reassortant influenza A H3N2 and H1N1 virus vaccines in seronegative adult volunteers.

An avian-human reassortant influenza A virus deriving its genes coding for the hemagglutinin and neuraminidase from the human influenza A/Washington/897/80 (H3N2) virus and its six "internal" genes from the avian influenza A/Mallard/NY/6750/78 (H2N2) virus (i.e., a six-gene reassortant) was previously shown to be safe, infectious, nontransmissible, and immunogenic as a live virus vaccine in adult humans. Two additional six-gene avian-human reassortant influenza viruses derived from the mating of wild-type human influenza A/California/10/78 (H1N1) and A/Korea/1/82 (H3N2) viruses with the avian influenza A/Mallard/NY/78 virus were evaluated in seronegative (hemagglutination inhibition titer, less than or equal to 1:8) adult volunteers for safety, infectivity, and immunogenicity to determine whether human influenza A viruses can be reproducibly attenuated by the transfer of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The 50% human infectious dose was 10(4.9) 50% tissue culture infectious doses for the H1N1 reassortant virus and 10(5.4) 50% tissue culture infectious doses for the H3N2 reassortant virus. Both reassortants were satisfactorily attenuated with only 5% (H1N1) and 2% (H3N2) of infected vaccines receiving less than 400 50% human infectious doses developing illness. Consistent with this level of attenuation, the magnitude of viral shedding after inoculation was reduced 100-fold (H1N1) to 10,000-fold (H3N2) compared with that produced by wild-type virus. The duration of virus shedding by vaccines was one-third that of controls receiving wild-type virus. At 40 to 100 50% human infectious doses, virus-specific immune responses were seen in 77 to 93% of volunteers. When vaccinees who has received 10(7.5) 50% tissue culture infectious doses of the H3N2 vaccine were experimentally challenged with a homologous wild-type human virus only 2 of 19 (11%) vaccinees became ill compared with 7 of 14 (50%) unvaccinated seronegative controls ( P < 0.025; protective efficacy, 79%). Thus, three different virulent human influenza A viruses have been satisfactorily attenuated by the acquisition of the six internal genes of the avian influenza A/Mallard/NY/78 virus. The observation that this donor virus can reproducibly attenuate human influenza A viruses indicates that avian-human influenza A reassortants should be further studied as potential live influenza A virus vaccines.