Variation of Influenza A, B and C viruses
1) Ans.) Serologically cross-reactive matrix protein and nucleoprotein, are used in differentiating type-A Influenza virus from the internal proteins of type-B and type-C strains.
2) Ans.) Nucleotide sequencing of the 5-prime and 3-prime ends of the genomic RNA of strains of types A, B, and C revealed a high degree of conservation of the terminal 10 to 20 nucleotides which act as the common progenitor for all the three viral subtypes.
3) Ans.) 1.Infrequent association of virus-C with disease in man, frequent association of virus-B with the disease in man was observed and virus A is associated with pandemic influenza in man.
2. A virus has wide host range (man, pigs, horses and birds).
3. Un-like influenza-A viruses, type-B and type-C do not appear to undergo rapid major genetic or antigenic changes. Virus-C does not have neuraminidase gene.
4) Ans.) Recombination of the genes that lead to the synthesis of surface proteins (which play important role in giving antigenic specificity to the virus in causing disease) occurring between different strains of subtypes is called re-assortment. If this recombination occurs significantly and suddenly it is called as antigenic shift. But if recombination of genes leading to non-surface proteins occurs non-significantly within the viral subtypes it is called as antigenic drift.
Re-assortment of genes Recombination of genes between different strains of same subtype or between different subtypes either leading to synthesis of surface proteins or non-surface proteins is called re-assortment of genes.
Antigenic shift Dramatic occurrence of recombination of genes that code for surface antigen proteins among different strains of the viral subtypes.
5) Ans.) If the serum samples of people suffered from a recent pandemic caused by a strain has cross-reacted with another strain that has appeared quiet a few years back, it is an indirect evidence to say that there are a limited number of viral antigens (hemagglutinin genes) that have potential to cause epidemics and pandemic, which keep recycling in the strains although the years. This is called antigen recycling.
6) Ans.) Three basic features appear to be conserved in type-A Influenza virus hemagglutinin which shows great degree of variability. The regions of amino-acid sequence that are conserved are amino-terminal end of Hemagglutinin2 subunit, carboxyl terminal hydrophobic tail of Hemagglutinin2 subunit and many cysteine residues along the entire sequence. These three regions are found to be conserved which means not variable or retained in the evolutionary process. These conserved sequences are responsible to carryout series of common important biological functions and penetration of virus into cells.
7) Ans.) Three HemagglutininA subtype viruses H1, H2 and H3 can infect humans and two NeuraminidaseA subtype viruses N1 and N2 can infect humans.
8) Ans.) Yes, The nucleotide substitutions that were studied by sequence data in the hemagglutinin genes of different H3 subtypes were found to be roughly linear and cumulative. If the nucleotide substitutions occurred are conserved although the evolution of the strains one after the other it is called as linear. If the substitutions continue to increase and get conserved then it is called cumulative.
9) Ans.) 1. Immune response in some patients that led to favor the selection of variants through several cycles of virus replication.
2. The sheer number of people infected with influenza viruses during an epidemic may result in the formation of large numbers of variants.
3. The change of one or more amino acids in the influenza virus hemagglutinin may change the antigenic character of the virus sufficiently, whereas a similar number of amino acid changes may not provide such a selective advantage for other kinds of viruses.
10) Ans.) With the help of Recombinant DNA techniques effective vaccines are generated. Stable attenuated (half killed) virus that can be used as live virus vaccine can be constructed. Influenza virus strains may be constructed which contain altered antigenic determinants (antigenic sites). Viral antigens or portions of viral antigens in bacterial or yeast cells for use as killed virus vaccines can be produced by genetic engineering methods. Synthesis of modified antigens that exhibit determinants cross-reactive among strains (of various subtypes) may lead to preparation of vaccines that represent a significant improvement over conventional vaccines. Effective influenza virus vaccine takes advantage of the chemical synthesis of specific peptides. These peptides have been shown to be immunogenic and synthesis of the right amino acid stretch derived from known viral sequences or construction of a suitable cross-reactive immunogen containing multiple determinants may lead to the ultimate influenza virus vaccine.
Table2
This table describes about the total length of nucleotides in hemagglutinin gene, amino-acid length in hemagglutinin1 subunit and hemagglutinin2 subunit of H1 (1934), H2 (1957) and H3 (1968) viral Influenza-A subtypes. So the nucleotide lengths were 1778, 1773 and 1765 in H1, H2 and H3 viral Influenza-A subtypes respectively. In the same way, amino-acid lengths of hemagglutinin1 subunit in all the three subtypes were 326, 324 and 328 respectively. Again the amino-acid lengths of hemagglutinin2 subunits in the three subtypes were 222, 222 and 221 respectively.
Table3
Percentage nucleotide conservation in hemagglutinin1 and hemagglutinin2 subunits in
H1 and H2 viral Influenza-A subtypes 61 72
H1 and H3 viral Influenza-A subtypes 45 58
H2 and H3 viral Influenza-A subtypes 45 57
Percentage amino-acid conservation in hemagglutinin1 and hemagglutinin2 subunits in
H1 and H2 viral Influenza-A subtypes 58 79
H1 and H3 viral Influenza-A subtypes 35 53
H2 and H3 viral Influenza-A subtypes 36 50
Nucleotide sequence of the haemagglutinin gene of a human influenza virus H1 subtype
Abstract
1.H1 subtype of human influenza virus found to be dominant in this century was found to be co-circulating with variants of H3 subtype.
2.The sequencing of haemagglutinin gene of an early H1 subtype (strain APR834) was done by recombinant DNA methods and di-deoxy methods.
3.The antigenic site was defined at amino-acid residue 160.
4.After comparison of amino-acid residues among the different subtypes H1 and H2 were found to homologous to one another than all others.
-Influenza virus consists of 8 RNA genes ( 10 proteins, in which band 4 is meant for haemagglutinin gene.
-Methodology of sequencing 1. Generate restriction fragments of double-stranded DNA from RNA of gene4 by reverse transcriptase. 2. These fragments were cloned into the bacteriophage M13 vector and sequenced by sangers method. 3. Then all the sequences of isolated clones were overlapped to deduce unique sequence for the haemagglutinin gene. 4. An indirect RNA sequencing method was also used.
-Haemagglutinin gene, in its mRNA sense coded into amino-acid sequence consisted of 17-residue hydrophobic signal peptide, a haemagglutinin1 subunit (326 residues) and a haemagglutinin2 subunit (222 residues) separated by Arginine residue.
-Haemagglutinin2 subunit contains 14-residue N-terminal hydrophobic sequence and hydrophobic sequence.
-Disulphide bridges or bonds existed within the subunits but at C14 and C466 they are present between the subunits.
-A common antigenic variant of influenza-A, APR834 was identified to have changed serine to leucine at residue position 160 which is C to U transition at nucleotide position 553.
- The antigenic variance at position 160 of H1 subtype virus was homologous to 157 position of H3 subtype.
Table1
Percent amino-acid conservation in haemagglutinin1 haemagglutinin2 subunits in
H2 and H1 viral sub-types 58 79
H3 and H1 35 53
H3 and H2 36 50
H7 and H1 33 51
H7 and H2 35 65
Percent nucleotide conservation in haemagglutinin1 haemagglutinin2 subunits in
H1 and H2 viral subtypes 61 72
H3 and H1 45 58
H7 and H1 44 58
H3 and H2 45 57
H7 and H2 46 59
H7 and H3 45 66
-Amino-acid conservation was more between H1 and H2 viral subtypes than between all other subtypes.
-Homology of all haemagglutinin subtypes indicates that they might have evolved from common ancestor.
- From the extent of antigenic drift of haemagglutinin1 subunit observed in H3 series ANT6068,
AMemphis10272 and AVictoria275, it was estimated that 1 amino-acid change per year occurred.
- So by applying this drift, 42 difference in amino-acid sequence of haemagglutinin1 subunit of H1 and H2 subtypes might have been derived from the assumption that H2 subtype has evolved from H1 subtype in a 42-year period.
- Drift of H2 subtype from H1 subtype (1933) took place at 1957 and no other strains were found to be existing between these two affecting man. Even in avians and pigs also no intermediate strains are found in the last 48years.
- H1 and H2 viral subtypes were identified to be more evolutionarily related than to H3 and H7 subtypes though the evolutionary distance was not clearly estimated.
Future
It would be more informative further for everyone in this regard to sequence completely some of eight other known mammalian or avian haemagglutinin molecules because subtypes that are distantly related which may have been undetected by traditional methods used to classify haemagglutinin subtypes can also be checked for homology.
2) Ans.) Nucleotide sequencing of the 5-prime and 3-prime ends of the genomic RNA of strains of types A, B, and C revealed a high degree of conservation of the terminal 10 to 20 nucleotides which act as the common progenitor for all the three viral subtypes.
3) Ans.) 1.Infrequent association of virus-C with disease in man, frequent association of virus-B with the disease in man was observed and virus A is associated with pandemic influenza in man.
2. A virus has wide host range (man, pigs, horses and birds).
3. Un-like influenza-A viruses, type-B and type-C do not appear to undergo rapid major genetic or antigenic changes. Virus-C does not have neuraminidase gene.
4) Ans.) Recombination of the genes that lead to the synthesis of surface proteins (which play important role in giving antigenic specificity to the virus in causing disease) occurring between different strains of subtypes is called re-assortment. If this recombination occurs significantly and suddenly it is called as antigenic shift. But if recombination of genes leading to non-surface proteins occurs non-significantly within the viral subtypes it is called as antigenic drift.
Re-assortment of genes Recombination of genes between different strains of same subtype or between different subtypes either leading to synthesis of surface proteins or non-surface proteins is called re-assortment of genes.
Antigenic shift Dramatic occurrence of recombination of genes that code for surface antigen proteins among different strains of the viral subtypes.
5) Ans.) If the serum samples of people suffered from a recent pandemic caused by a strain has cross-reacted with another strain that has appeared quiet a few years back, it is an indirect evidence to say that there are a limited number of viral antigens (hemagglutinin genes) that have potential to cause epidemics and pandemic, which keep recycling in the strains although the years. This is called antigen recycling.
6) Ans.) Three basic features appear to be conserved in type-A Influenza virus hemagglutinin which shows great degree of variability. The regions of amino-acid sequence that are conserved are amino-terminal end of Hemagglutinin2 subunit, carboxyl terminal hydrophobic tail of Hemagglutinin2 subunit and many cysteine residues along the entire sequence. These three regions are found to be conserved which means not variable or retained in the evolutionary process. These conserved sequences are responsible to carryout series of common important biological functions and penetration of virus into cells.
7) Ans.) Three HemagglutininA subtype viruses H1, H2 and H3 can infect humans and two NeuraminidaseA subtype viruses N1 and N2 can infect humans.
8) Ans.) Yes, The nucleotide substitutions that were studied by sequence data in the hemagglutinin genes of different H3 subtypes were found to be roughly linear and cumulative. If the nucleotide substitutions occurred are conserved although the evolution of the strains one after the other it is called as linear. If the substitutions continue to increase and get conserved then it is called cumulative.
9) Ans.) 1. Immune response in some patients that led to favor the selection of variants through several cycles of virus replication.
2. The sheer number of people infected with influenza viruses during an epidemic may result in the formation of large numbers of variants.
3. The change of one or more amino acids in the influenza virus hemagglutinin may change the antigenic character of the virus sufficiently, whereas a similar number of amino acid changes may not provide such a selective advantage for other kinds of viruses.
10) Ans.) With the help of Recombinant DNA techniques effective vaccines are generated. Stable attenuated (half killed) virus that can be used as live virus vaccine can be constructed. Influenza virus strains may be constructed which contain altered antigenic determinants (antigenic sites). Viral antigens or portions of viral antigens in bacterial or yeast cells for use as killed virus vaccines can be produced by genetic engineering methods. Synthesis of modified antigens that exhibit determinants cross-reactive among strains (of various subtypes) may lead to preparation of vaccines that represent a significant improvement over conventional vaccines. Effective influenza virus vaccine takes advantage of the chemical synthesis of specific peptides. These peptides have been shown to be immunogenic and synthesis of the right amino acid stretch derived from known viral sequences or construction of a suitable cross-reactive immunogen containing multiple determinants may lead to the ultimate influenza virus vaccine.
Table2
This table describes about the total length of nucleotides in hemagglutinin gene, amino-acid length in hemagglutinin1 subunit and hemagglutinin2 subunit of H1 (1934), H2 (1957) and H3 (1968) viral Influenza-A subtypes. So the nucleotide lengths were 1778, 1773 and 1765 in H1, H2 and H3 viral Influenza-A subtypes respectively. In the same way, amino-acid lengths of hemagglutinin1 subunit in all the three subtypes were 326, 324 and 328 respectively. Again the amino-acid lengths of hemagglutinin2 subunits in the three subtypes were 222, 222 and 221 respectively.
Table3
Percentage nucleotide conservation in hemagglutinin1 and hemagglutinin2 subunits in
H1 and H2 viral Influenza-A subtypes 61 72
H1 and H3 viral Influenza-A subtypes 45 58
H2 and H3 viral Influenza-A subtypes 45 57
Percentage amino-acid conservation in hemagglutinin1 and hemagglutinin2 subunits in
H1 and H2 viral Influenza-A subtypes 58 79
H1 and H3 viral Influenza-A subtypes 35 53
H2 and H3 viral Influenza-A subtypes 36 50
Nucleotide sequence of the haemagglutinin gene of a human influenza virus H1 subtype
Abstract
1.H1 subtype of human influenza virus found to be dominant in this century was found to be co-circulating with variants of H3 subtype.
2.The sequencing of haemagglutinin gene of an early H1 subtype (strain APR834) was done by recombinant DNA methods and di-deoxy methods.
3.The antigenic site was defined at amino-acid residue 160.
4.After comparison of amino-acid residues among the different subtypes H1 and H2 were found to homologous to one another than all others.
-Influenza virus consists of 8 RNA genes ( 10 proteins, in which band 4 is meant for haemagglutinin gene.
-Methodology of sequencing 1. Generate restriction fragments of double-stranded DNA from RNA of gene4 by reverse transcriptase. 2. These fragments were cloned into the bacteriophage M13 vector and sequenced by sangers method. 3. Then all the sequences of isolated clones were overlapped to deduce unique sequence for the haemagglutinin gene. 4. An indirect RNA sequencing method was also used.
-Haemagglutinin gene, in its mRNA sense coded into amino-acid sequence consisted of 17-residue hydrophobic signal peptide, a haemagglutinin1 subunit (326 residues) and a haemagglutinin2 subunit (222 residues) separated by Arginine residue.
-Haemagglutinin2 subunit contains 14-residue N-terminal hydrophobic sequence and hydrophobic sequence.
-Disulphide bridges or bonds existed within the subunits but at C14 and C466 they are present between the subunits.
-A common antigenic variant of influenza-A, APR834 was identified to have changed serine to leucine at residue position 160 which is C to U transition at nucleotide position 553.
- The antigenic variance at position 160 of H1 subtype virus was homologous to 157 position of H3 subtype.
Table1
Percent amino-acid conservation in haemagglutinin1 haemagglutinin2 subunits in
H2 and H1 viral sub-types 58 79
H3 and H1 35 53
H3 and H2 36 50
H7 and H1 33 51
H7 and H2 35 65
Percent nucleotide conservation in haemagglutinin1 haemagglutinin2 subunits in
H1 and H2 viral subtypes 61 72
H3 and H1 45 58
H7 and H1 44 58
H3 and H2 45 57
H7 and H2 46 59
H7 and H3 45 66
-Amino-acid conservation was more between H1 and H2 viral subtypes than between all other subtypes.
-Homology of all haemagglutinin subtypes indicates that they might have evolved from common ancestor.
- From the extent of antigenic drift of haemagglutinin1 subunit observed in H3 series ANT6068,
AMemphis10272 and AVictoria275, it was estimated that 1 amino-acid change per year occurred.
- So by applying this drift, 42 difference in amino-acid sequence of haemagglutinin1 subunit of H1 and H2 subtypes might have been derived from the assumption that H2 subtype has evolved from H1 subtype in a 42-year period.
- Drift of H2 subtype from H1 subtype (1933) took place at 1957 and no other strains were found to be existing between these two affecting man. Even in avians and pigs also no intermediate strains are found in the last 48years.
- H1 and H2 viral subtypes were identified to be more evolutionarily related than to H3 and H7 subtypes though the evolutionary distance was not clearly estimated.
Future
It would be more informative further for everyone in this regard to sequence completely some of eight other known mammalian or avian haemagglutinin molecules because subtypes that are distantly related which may have been undetected by traditional methods used to classify haemagglutinin subtypes can also be checked for homology.
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