Viral Fusion, Stability and Infectivity Affected by a Single Amino Acid Mutation in H1N1 Influenza Virus.

Flu or Influenza is a seasonal infectious disease caused by an RNA virus that belongs to Orthomyxoviridae family. A seasonal epidemic of this disease causes millions of deaths. Influenza virus mutates to produce a new strain every time. This process occurs by blending of genes of humans and birds mostly. In the year 2009 a novel strain appeared and was named as swine flu or A/H1N1. This strain also caused Spanish flu of 1918. Strain A is most virulent and has many identified serotypes like H1N1, H2N2 (Asian Flu), H3N2, H5N1 (Bird Flu), H7N7, H1N2, H9N2, H7N9 etc.

RNA genome is segmented into seven to eight pieces of RNA and encodes proteins like neuraminidase(NA), hemagglutinin (HA), nucleoprotein(NP), nuclear export protein (NS1 and 2) etc. HA is indispensable for target binding and NA for release of progeny virus. HA has a stalk region and a head region. It specifically binds sialic acid residues on host cell surfaces and is known to be major determinant of viral host tropism and pathogenicity. After viral fusion and endocytosis, the low pH inside endosome is known to be encouraging for irreversible structural changes for HA subunits viz., HA1 and HA2. Scrutiny of data suggests that in the HA2 stalk there is a mutation (E47K). This mutation of H1N1 isolates has been exploited by the authors to contrast two H1N1 viruses that differ at HA2 position 47 of their HA proteins.

Original strain of H1N1 A/California/7/2009 (Cal/09) and a later strain A/Brisbane/10/2010 (Bris/10) were used in this study. Cal/09-like strains contain E47 in the HA2 stalk. However since 2009, an E47K mutation emerged and became dominant globally. Membrane fusion activity as determined by transient expression assay and viral fusion assay revealed that the HA2-47 in H1N1 HA stalk region affects the threshold pH for membrane fusion. Cal/09 HA fusion triggers at pH 5.4 and that of Bris/10 at pH 5, indicating a difference of 0.4 units. Moreover HA2-E47K substitution (glutamic acid to lysine substitution) lowered threshold pH of Cal/09 HA fusion, indicating HA2 47th residues regulatory role. It is to note that higher threshold pH for fusion means a lower viral thermal stability.

Since it is known both for balance of viral acidic stability in invaded tissues and for fusion activation, optimal pH is required. Hence pH affects host-specific replication and pathogenicity even in narrow pH range differences. Thus for transmission and virus host tropism, pH of HA fusion activation could be an important factor. In order to account for such statements vero cells (Verda Reno Cells) were used. Vero cells are known to have higher endosomal pH than MDCK cells; they were exploited for comparing pH and viral replication support. Experiments revealed that replication support for viruses with a fusion pH threshold of 5.4 is more as compared to those with pH 5. Moreover it has been demonstrated that Cal/09 HA2-E47 virus that has low acid stability (high threshold pH for fusion) also displays lower thermal stability compared to HA2-K47 virus. Compared to Cal/09, the H1N1pdm strain of 2012 has lower fusion pH, indicating that lower fusion pH might be coupled with adapting to human environment.

Most effective approach to prevent influenza is vaccination. Vaccinations against flu usually contains antigens from three viral strains (two A strains and one B strain) that are inactivated and is called as trivalent influenza vaccine(TIV). Each year a new vaccine has to be formulated as the virus is mutating rapidly to render previously formulated vaccines as ineffective. For the 2009 pandemic, a monovalent live attenuated vaccine (LAIV) was produced. In this study it has been found that E47 mutation of HA2 in Cal/09 vaccine awards more stability from 4°C to about 57.5°C.

In summary, this study adds to the better understanding of influenza infection and might aid in speedy reaction to future epidemics. In-fact genetic signature identified in the HA stalk has noteworthy implication for production of vaccines for future pandemics.


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