Bioinformatics for Genomics_CTS_Research Topic of Interest Disease: Influenza (Flu)

1. Dr. Obumneke Amadi-Onuoha_Scripts
Bioinformatics for Genomics_CTS_Research Topic of Interest
Disease: Influenza (Flu)
Gene of interest: “H275Y”.
ResearchQuestion:
Would the presence of the changes in H275Y neuraminidase gene be a reliable predictor of
reduced susceptibility and antiviral resistance of influenza infection (Flu Virus) in affected
individuals or not?
Background:
There are four types of influenza viruses (A to D). Human influenza A and B viruses cause
seasonal epidemics in the United States. The influenza virus continues to progress, and new
antigenic variants (drift strains) emerge continuously, giving rise to annual epidemics/pandemic
(Webster, 1998, p.436). Influenza A viruses are subdivided into two based on two proteins on
the surface of the virus: the hemagglutinin (H) and the neuraminidase (N) and are broken down
into different strains. The existing subtypes of influenza A viruses found in people are influenza
A (H1N1) and influenza A (H3N2) viruses. In the year 2009, a new influenza A (H1N1) virus
emerged to cause pandemic in USA and globally, leading to severe illness requiring
hospitalization, including pneumonia and death (Jain et al., 2009, p.1934), this virus was very
different from the human influenza A (H1N1) viruses spreading at that time. The influenza
A(H1N1) pdm09 has now replaced the H1N1 virus that was previously spreading in humans, the
risk of a NA inhibitor-resistant strain developing is of concern (CDC, 2017). The resistance in
influenza A(H1N1) pdm09 has been described to be associated with the histidine-to-tyrosine
(H275Y) mutation in the NA gene (Pawestri, Nugraha, Hariastuti, & Setiawaty, 2018, p.1-
2). This virus can become a major global, clinical and public health issue. It is important for
the constant monitoring for NA inhibitor resistant viruses to predict any increases in the number
of resistant viruses since NA inhibitors are used largely for treatment and outbreak interventions
(Pawestri, Nugraha, Hariastuti, & Setiawaty, 2018, p.1-2).
Short justification: Influenza (flu) is a contagious respiratory illness caused by influenza
viruses. Influenza virus A (HINI) occasionally change their strain in the course of flue seasons,

2. the reason being that flu virus replicates. These changes genetic makeup may change the ways
of treatment/therapy adherence and the effectiveness of the antiviral drugs against these viruses
(Illinois Department of Public Health, 2014, p. I), among the vaccines used in the treatment for
the infection, oseltamivir is the most commonly prescribed and recommended in the United
States. A gene the “H275Y” is conferred to resist oseltamivir in 2009 H1N1 flu viruses
(Brookes, Miah, Lackenby, Hartgroves, & Barclay, 2010, p.466; CDC, 2019). H275Y
substitution in the active site of the NA-1 gene indicates’ reduced binding affinity of the
neuraminidase inhibitor oseltamivir (Hindiyeh, 2010, p.1884). Detection of reduced
susceptibility and antiviral resistance involves local and advanced laboratory tests, such as
specific functional assays and molecular techniques (sequencing and pyrosequencing) to identify
the genetic changes linked with reduced antiviral susceptibility (cdc, 2019). According to Grund
et al. (2015), to access spread and clinical impact of these viruses, it is vital to conduct antiviral
susceptibility testing and reporting of viruses carrying amino acid substitutions in order to confer
their antiviral drug resistance strength (Grund, et al., 2015). There are genes that make up this
influenza virus, this study seeks to examine the specific targeted genes and its components for
the changes that occur and what types of changes occurs, it implications that supports the virus
adherence and vaccine resistance. The purpose of this study is to investigate the genomic aspect
of the viral infections H1N1pdm09 strain and the mutation in NA gene H275Y to confirm its
responsibility for reduced susceptible and antiviral drug resistance in affected people and
examine if there is a difference in the gene expression of the virus infection in 2 groups: normal
and affected people (children, adults and immunocompromised). The outcome of the study will
give a greater insight of the gene responsibility for reduced susceptibility and antiviral resistance
that may guide clinicians as a reliable predictor or biomarkers to identify presence of the virus
for support towards efficient and effective vaccine productions, intervention & treatment. It may
help providers towards diagnosis/planning during spontaneous change in flu season. Also, the
study will confirm previous research knowledge of the gene capability to reliably predict
changes in H275Y mutation in the NA gene to monitor a potential antiviral developing inhibitor-
resistant strain.
Hypothesis: Presence of mutations is sufficient to develop a change in gene expression that are
responsible for immune response against influenza virus and vaccine.
Aims:

3. 1) Investigate the functional outcome of the mutations - what changes in A(H1N1)pdm09
functions occur when each H275Ymutation is present
2) Examine the implications of the changes that supports the virus adherence and vaccine
resistance.
Summary of the selectedgenes:
A. Gene symbol : NA
Gene description : neuraminidase
Locus tag : UJ99_s6gp1
Gene type: protein coding
RefSeq status: PROVISIONAL
Organism: Influenza A virus (A/California/07/2009(H1N1)) (strain:
A/California/07/2009, serotype: H1N1, nat-host: Homo sapiens; gender M;
age 54)
Lineage: Viruses; ssRNA viruses; ssRNA negative-strand viruses; Orthomyxoviridae;
Influenzavirus A
Location: segment: 6
Sequence: NC_026434.1 (1..1410)
SNPs: mRNA and Protein(s):
YP_009118627.1 neuraminidase [Influenza A virus (A/California/07/2009(H1N1))]
UniProtKB/TrEMBL : C3W6G3
Conserved Domains (1) summary : cd15483
Location:85 → 465 : Influenza_NA; Sialidase or neuraminidase (EC 3.2.1.18) of
Influenza viruses A and B
B. Gene symbol: NA
Gene description: neuraminidase
Locus tag: FLUAVs6gp1
Gene type: protein coding
RefSeq status: PROVISIONAL
Organism: Influenza A virus (A/Puerto Rico/8/1934(H1N1)) (strain: A/Puerto
Rico/8/1934(H1N1))
Lineage: Viruses; ssRNA viruses; ssRNA negative-strand viruses; Orthomyxoviridae;

4. Influenzavirus A
Location: segment: 6
Sequence: NC_002018.1 (21..1385)
SNPs: mRNA and Protein(s):
NP_040981.1 neuraminidase [Influenza A virus (A/Puerto Rico/8/1934(H1N1))]
UniProtKB/Swiss-Prot : P03468
Conserved Domains (1) summary: cd15483
Location:68 → 450 Influenza_NA; Sialidase or neuraminidase (EC 3.2.1.18) of Influenza
viruses A and B
Method:
1. Investigating activities of potential promoter RNA, gene polymorphism, and gene
expression in people with and without influenza A (H1N1) infection from peripheral
blood collected prior and post antiviral
immunization and the host’s transcriptome will be sequenced for identification of
differentially expressed genes post manipulation and between individuals or groups.
2. To compare the gene of the present influenza virus with older one, to determine their
primary structures and properties and how they respond to antiviral drugs.
Experimental Protocol development:
1. Literature review: Examination of primary research and systematic reviews of H275Y
and oseltamivir-resistant data and link with influenza A(H1N1)pdm09 viruses and
Influenza A virus (A/Puerto Rico/8/193.
2. Bioinformatics: Collection of genomic data on H275Y and influenza A(H1N1)pdm09
and Influenza A virus (A/Puerto Rico/8/193 viruses disease (genomes from sequenced
isolates), alignment of certain strains, and annotation of nucleotides and variations; b)
collection and analysis of the genomic profiles from available resources.
3. Development of experimental procedures to be included in the final protocol.
Reference

5. Brookes, D. W., Miah, S., Lackenby, A., Hartgroves, L., & Barclay, W. S. (2010). Pandemic
H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase
mutation shows a small compromise in enzyme activity and viral fitness. Journal of
antimicrobial chemotherapy, 66(3), 466-470.
CDC (2019). Influenza (FLU): Antiviral Drug Resistance. Retrieved from
https://www.cdc.gov/flu/treatment/antiviralresistance.htm
CDC (2017). Types of Influenza Viruses. Retrieved from
https://www.cdc.gov/flu/about/viruses/types.htm
Grund, S., Gkioule, C., Termos, T., Pfeifer, N., Kobbe, G., Verheyen, J., & Adams, O. (2015).
Case report Primarily oseltamivir-resistant influenza A (H1N1pdm09) virus evolving into
a multidrug-resistant virus carrying H275Y and I223R neuraminidase substitutions.
Antiviral therapy, 20, 97-100.
Hindiyeh, M., Ram, D., Mandelboim, M., Meningher, T., Hirsh, S., Robinov, J., &
Mendelson, E. (2010). Rapid detection of influenza A pandemic (H1N1) 2009 virus
neuraminidase resistance mutation H275Y by real-time reverse transcriptase PCR.
Journal of clinical microbiology, 48(5), 1884-1887.
Illinois Department of Public Health (2014). Pandemic Influenza Preparedness and Response
Plan. Retrieved from
http://www.idph.state.il.us/pandemic_flu/Illinois_Pandemic_Flu_Plan.pdf
Jain, S., Kamimoto, L., Bramley, A. M., Schmitz, A. M., Benoit, S. R., Louie, J., & Jasuja, S.
(2009). Hospitalized patients with 2009 H1N1 influenza in the United States, April–June
2009. New England journal of medicine, 361(20), 1935-1944.
Pawestri, H. A., Nugraha, A. A., Hariastuti, N. I., & Setiawaty, V. (2018). Detection of
neuraminidase inhibitor-resistant influenza A (H1N1) pdm09 viruses obtained from
influenza surveillance in Indonesia. SAGE open medicine, 6, 2050312118818293
Webster, R. G. (1998). Influenza: an emerging disease. Emerging infectious diseases, 4(3), 436.