Impact of amino acid substitution T203I in hemagglutinin on growth characteristics in vitro and hemagglutinin thermostability of A/H3N2 influenza viruses

2021 ◽  
Vol 21 (3) ◽  
pp. 85-90
Author(s):  
Ekaterina A. Bazhenova ◽  
Ekaterina A. Stepanova ◽  
Tatiana S. Kotomina ◽  
Nataliya V. Larionova ◽  
Irina V. Kiseleva ◽  
...  
Keyword(s):  
Cell Culture ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Influenza A ◽  
Biological Properties ◽  
Growth Characteristics ◽  
Influenza Viruses ◽  
Influenza Vaccines ◽  
Amino Acid Substitutions ◽  
H3n2 Influenza

Background: Russian live attenuated influenza vaccines are a three-component preparations that contain vaccine strains based on current epidemic influenza A/H1N1, A/H3N2 and B strains. Recent influenza viruses A/H3N2 are most susceptible to drift antigenic changes, and therefore, this component of the live attenuated influenza vaccines must be constantly updated. Current vaccine strains of live attenuated influenza vaccines are obtained by the method of classical reassortment using selective factors in developing chicken embryos. During the process of preparation of live attenuated influenza vaccines strains, single reassortants can acquire various egg-adaptive amino acid substitutions in hemagglutinin and neuraminidase the genes responsible for the antigenic correspondence of the vaccine strain to the epidemic parent. These amino acid substitutions can affect the biological properties of the vaccine strain, thereby reducing the effectiveness of this component of live attenuated influenza vaccines. AIM: The aim of the study was to explore the effect of amino acid substitution T203I in hemagglutinin of A/H3N2 influenza viruses on growth characteristics and hemagglutinin thermostability. MATERIALS AND METHODS: For the study, three pairs of A/H3N2 vaccine reassortants were prepared. Reassortants differed from each other by amino acid Thr or Ile at position 203 in the hemagglutinin. The growth properties of vaccine strains were assessed by titration in eggs at 2640C and in a MDCK cell culture at 33C. The thermostability of the hemagglutinin of studied influenza viruses was assessed by determining their ability to agglutinate 1% erythrocytes after exposure to elevated temperatures in the range of 3770C. RESULTS: The amino acid substitution T203I in hemagglutinin in reassortants obtained on the basis of current influenza A/H3N2 viruses acquired during the preparation of vaccine strains does not affect the temperature sensitivity of viruses. It was shown that viruses with an egg-adaptive substitution T203I in hemagglutinin have more pronounced cold-adapted phenotype and a higher reproductive activity in MDCK cell culture, compared to strains without this mutation. It was found that hemagglutinin of reassortants with 203 Ile is more thermostable than with 203 Thr. CONCLUSIONS: Our data indicate that the amino acid substitution of T203I in hemagglutinin in current influenza A/H3N2 viruses does not have a negative effect on biological properties, but improves growth characteristics in eggs and MDCK cells, as well as the thermostability of viruses.

scholarly journals Evolution and Adaptation of the Avian H7N9 Virus into the Human Host

2020 ◽  
Vol 8 (5) ◽  
pp. 778
Author(s):  
Andrew T. Bisset ◽  
Gerard F. Hoyne
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Amino Acid Substitutions ◽  
Upper Respiratory Tract ◽  
Viral Polymerase ◽  
Evolutionary Changes ◽  
Avian Origin

Influenza viruses arise from animal reservoirs, and have the potential to cause pandemics. In 2013, low pathogenic novel avian influenza A(H7N9) viruses emerged in China, resulting from the reassortment of avian-origin viruses. Following evolutionary changes, highly pathogenic strains of avian influenza A(H7N9) viruses emerged in late 2016. Changes in pathogenicity and virulence of H7N9 viruses have been linked to potential mutations in the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA), as well as the viral polymerase basic protein 2 (PB2). Recognizing that effective viral transmission of the influenza A virus (IAV) between humans requires efficient attachment to the upper respiratory tract and replication through the viral polymerase complex, experimental evidence demonstrates the potential H7N9 has for increased binding affinity and replication, following specific amino acid substitutions in HA and PB2. Additionally, the deletion of extended amino acid sequences in the NA stalk length was shown to produce a significant increase in pathogenicity in mice. Research shows that significant changes in transmissibility, pathogenicity and virulence are possible after one or a few amino acid substitutions. This review aims to summarise key findings from that research. To date, all strains of H7N9 viruses remain restricted to avian reservoirs, with no evidence of sustained human-to-human transmission, although mutations in specific viral proteins reveal the efficacy with which these viruses could evolve into a highly virulent and infectious, human-to-human transmitted virus.

scholarly journals MOLECULAR AND GENETIC CHARACTERISTICS OF SURFACE AND NONSTRUCTURE PROTEINS OF PANDEMIC INFLUENZA VIRUSES A(H1N1)PDM09 IN 2015-2016 EPIDEMIC SEASON

2016 ◽  
Vol 72 (2) ◽  
pp. 44-48
Author(s):  
O. Smutko ◽  
L. Radchenko ◽  
A. Mironenko
Keyword(s):  
Amino Acid ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Phylogenetic Trees ◽  
Influenza Viruses ◽  
Amino Acid Substitutions ◽  
Ns1 Protein ◽  
Genetic Characteristics ◽  
Epidemic Season ◽  
Influenza A H1n1

The aim of the present study was identifying of molecular and genetic changes in hemaglutinin (HA), neuraminidase (NA) and non-structure protein (NS1) genes of pandemic influenza A(H1N1)pdm09 strains, that circulated in Ukraine during 2015-2016 epidemic season. Samples (nasopharyngeal swabs from patients) were analyzed using real-time polymerase chain reaction (RTPCR). Phylogenetic trees were constructed using MEGA 7 software. 3D structures were constructed in Chimera 1.11.2rc software. Viruses were collected in 2015-2016 season fell into genetic group 6B and in two emerging subgroups, 6B.1 and 6B.2 by gene of HA and NA. Subgroups 6B.1 and 6B.2 are defined by the following amino acid substitutions. In the NS1 protein were identified new amino acid substitutions D2E, N48S, and E125D in 2015-2016 epidemic season. Specific changes were observed in HA protein antigenic sites, but viruses saved similarity to vaccine strain. NS1 protein acquired substitution associated with increased virulence of the influenza virus.

scholarly journals Biological properties of influenza A virus mutants with amino acid substitutions in the HA2 glycoprotein of the HA1/HA2 interaction region

2019 ◽  
Vol 100 (9) ◽  
pp. 1282-1292
Author(s):  
L. Jakubcová ◽  
M. Vozárová ◽  
J. Hollý ◽  
K. Tomčíková ◽  
M. Fogelová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Biological Properties ◽  
Interaction Region ◽  
Amino Acid Substitutions

scholarly journals Functional Constraints of Influenza A Virus Epitopes Limit Escape from Cytotoxic T Lymphocytes

2005 ◽  
Vol 79 (17) ◽  
pp. 11239-11246 ◽  
Author(s):  
E. G. M. Berkhoff ◽  
E. de Wit ◽  
M. M. Geelhoed-Mieras ◽  
A. C. M. Boon ◽  
J. Symons ◽  
...  
Keyword(s):  
Amino Acid ◽  
T Lymphocytes ◽  
Influenza A Virus ◽  
Cytotoxic T Lymphocytes ◽  
Influenza A ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Amino Acid Substitutions ◽  
Functional Constraints ◽  
Ctl Epitopes

ABSTRACT Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M158-66. We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M158-66 epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.

scholarly journals Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells

2016 ◽  
Vol 90 (13) ◽  
pp. 5928-5938 ◽  
Author(s):  
Benjamin Mänz ◽  
Miranda de Graaf ◽  
Ramona Mögling ◽  
Mathilde Richard ◽  
Theo M. Bestebroer ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Avian Influenza ◽  
Mammalian Cells ◽  
Influenza A ◽  
Polymerase Activity ◽  
Host Adaptation ◽  
Influenza Viruses ◽  
Amino Acid Substitutions ◽  
Avian Influenza A Virus

ABSTRACTA strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified usingin vitroassays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts.IMPORTANCEInfluenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions—most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2—have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans.

scholarly journals Y155H amino acid substitution in influenza A(H1N1)pdm09 viruses does not confer a phenotype of reduced susceptibility to neuraminidase inhibitors

2014 ◽  
Vol 19 (27) ◽  
Author(s):  
U Perez-Sautu ◽  
F Pozo ◽  
I Cuesta ◽  
S Monzon ◽  
A Calderon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Influenza A ◽  
High Throughput Sequencing ◽  
Biological Significance ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Neuraminidase Inhibitors ◽  
Epidemic Season ◽  
Influenza A H1n1

The Y155H amino acid substitution in the neuraminidase gene (NA) has previously been associated with highly reduced inhibition by neuraminidase inhibitors in the seasonal H1N1 influenza A virus which circulated in humans before the 2009 pandemic. During the 2012/13 epidemic season in Spain, two A(H1N1)pdm09 viruses bearing the specific Y155H substitution in the NA were detected and isolated from two patients diagnosed with severe respiratory syndrome and pneumonia requiring admission to the intensive care unit. Contrary to what was observed in the seasonal A(H1N1) viruses, neither of the Y155H A(H1N1)pdm09 viruses described here showed a phenotype of reduced inhibition by NAIs as determined by the neuraminidase enzyme inhibition assay (MUNANA). High-throughput sequencing of the NA of both Y155H viruses showed that they were composed to >99% of H155 variants. We believe that this report can contribute to a better understanding of the biological significance of amino acid substitutions in the neuraminidase protein with regard to susceptibility of influenza viruses to neuraminidase inhibitors. This is of critical importance for optimal management of influenza disease patients.

scholarly journals Whole genomic analysis of two influenza H3N2 virus strains isolated from Qinghai, China

2019 ◽  
Author(s):  
Huaxiang Rao ◽  
Hong Li ◽  
Nannan Lu ◽  
Youju Lei ◽  
Shengcang Zhao ◽  
...  
Keyword(s):  
Hong Kong ◽  
Amino Acid ◽  
Vaccine Strain ◽  
Amino Acid Substitution ◽  
Antigenic Site ◽  
Influenza Viruses ◽  
H3n2 Virus ◽  
Amino Acid Substitutions ◽  
Qinghai Province ◽  
Influenza H3n2

Abstract Background Influenza H3N2 virus has a faster evolution rate than other types of influenza viruses. This study was performed to better understand the molecular evolution of influenza H3N2 in Qinghai Province, China in 2017.Methods Complete sequences of eight gene segments of two influenza H3N2 isolates in 2017 in Qinghai Province were sequenced and analyzed by MEGA 6.06 software.Results Phylogenetic analysis showed that two Qinghai H3N2 isolates were relatively close to the 2016–2017 vaccine strain, 3C.2a-A/Hong Kong/4801/2014. In HA protein, compared with the 2015-2016 WHO recommended vaccine strain A/Switzerland/971 5293/2013, six amino acid substitutions were observed in epitopes A and B in Qinghai isolates in 2017, however, only two amino acid substitutions were observed in epitopes A and B in Qinghai isolates compared with the A/Hong Kong/4801/2014, which indicated 2016-2017 vaccine strain might have a better protection against the strains circulating in Qinghai Province in 2017 . Besides, amino acid substitution of K160T at the glycosylation site of HA and H75P in PB1-F2 in the two Qinghai isolates might affect the antibodies binding ability and the virulence of influenza virus. And there was no key amino acid substitution in the key sites of segment NA, M, NP, NS, PA and PB2.Conclusions The presence of several antigenic site mutations in Qinghai H3N2 isolates confirms the evolution of circulating H3N2 strains. Enhancing the surveillance of influenza epidemic by whole genome sequencing is important to monitor whether the selected vaccine strains are protective against the circulating strains in Qinghai Province.

scholarly journals PROBLEMS OF ISOLATION, IDENTIFICATION AND ANTIGENIC CHARACTERIZATION OF RECENT HUMAN A(H3N2) INFLUENZA VIRUSES

2018 ◽  
Vol 63 (4) ◽  
pp. 160-164
Author(s):  
P. A. Petrova ◽  
N. I. Konovalova ◽  
D. M. Danilenko ◽  
A. D. Vasilieva ◽  
M. Yu. Eropkin
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
High Rate ◽  
Influenza Vaccines ◽  
Antigenic Analysis ◽  
Hemagglutination Inhibition Test ◽  
Glycosylation Sites ◽  
Antigenic Properties ◽  
H3n2 Influenza ◽  
Selection Of

Human A (H3N2) influenza viruses are distinguished by a high rate of evolution and regularly cause epidemics around the world. Their ability to adapt and to escape from the host's immune response and to change their receptor specificity is very high. Over the past 20 years, these viruses have lost the ability to agglutinate red blood cells of chickens and turkeys and have practically ceased to propagate in chicken embryos - the main source of influenza vaccines. Isolation of viruses in the MDCK cell culture led to the selection of strains that lose one of the potential glycosylation sites. Many of the A (H3N2) strains have acquired mutations in neuraminidase, which distort the results of antigenic analysis in the hemagglutination inhibition test - the cornerstone method for the analysis of the match between viral isolates circulating in human population to strains selected for the influenza vaccines. In this regard, the characteristics of the antigenic properties of influenza A (H3N2) viruses by traditional methods become poorly informative, and the selection of vaccine strains of this subtype is erroneous, which is reflected in the discrepancy between vaccine and circulating A (H3N2) viruses in recent years (2013-2014, 2014 -2015, 2015-2016). The search, development and implementation of new algorithms for the isolation and antigen analysis of influenza A (H3N2) viruses are extremely urgent.

scholarly journals Genetic diversity of influenza A viruses circulating in Bulgaria during the 2018–2019 winter season

2020 ◽  
Vol 69 (7) ◽  
pp. 986-998
Author(s):  
Neli Korsun ◽  
Rodney Daniels ◽  
Svetla Angelova ◽  
Burcu Ermetal ◽  
Iliyana Grigorova ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Influenza A ◽  
Winter Season ◽  
Vaccine Virus ◽  
Influenza Viruses ◽  
Amino Acid Substitutions ◽  
Influenza A Viruses ◽  
Antigenic Sites

Introduction. Influenza viruses evolve rapidly and change their antigenic characteristics, necessitating biannual updates of flu vaccines. Aim. The aim of this study was to characterize influenza viruses circulating in Bulgaria during the 2018/2019 season and to identify amino acid substitutions in them that might impact vaccine effectiveness. Methodology. Typing/subtyping of influenza viruses were performed using real-time Reverse Transcription-PCR (RT-PCR) and results of phylogenetic and amino acid sequence analyses of influenza strains are presented. Results. A(H1N1)pdm09 (66 %) predominated over A(H3N2) (34 %) viruses, with undetected circulation of B viruses in the 2018/2019 season. All A(H1N1)pdm09 viruses studied fell into the recently designated 6B.1A subclade with over 50 % falling in four subgroups: 6B.1A2, 6B.1A5, 6B.1A6 and 6B.1A7. Analysed A(H3N2) viruses belonged to subclades 3C.2a1b and 3C.2a2. Amino acid sequence analysis of 36 A(H1N1)pdm09 isolates revealed the presence of six–ten substitutions in haemagglutinin (HA), compared to the A/Michigan/45/2015 vaccine virus, three of which occurred in antigenic sites Sa and Cb, together with four–nine changes at positions in neuraminidase (NA), and a number of substitutions in internal proteins. HA1 D222N substitution, associated with increased virulence, was identified in two A(H1N1)pdm09 viruses. Despite the presence of several amino acid substitutions, A(H1N1)pdm09 viruses remained antigenically similar to the vaccine virus. The 28 A(H3N2) viruses characterized carried substitutions in HA, including some in antigenic sites A, B, C and E, in NA and internal protein sequences. Conclusion. The results of this study showed the genetic diversity of circulating influenza viruses and the need for continuous antigenic and molecular surveillance.

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