225E with Adjacent 226R on Novgorod01 May Signal Instability and Hyper-morphic Behaviour at the Receptor Binding Domain

On 2009-12-30, Russia's Vector labs deposited a novel sequence with dual adjacent HA RBD changes as part of a signal-dense batch of data from 4 additional samples.  All are compelling because each shows a polymorphism at position 225, but A/Novgorod/01/2009 is special and may herald a new phase of maturation within ΣPF11.

Special sequence and special location.  Let's talk about geographic connectedness, location, for one moment before examining the sequence.

Though we cannot be certain which Novgorod is the sample location, we will profile one of the two for connectedness in this initial evaluation.  Nizhny Novgorod, in general, was founded at the confluence of the Volga and Oka rivers and is Russia's fourth largest city with a population of 1.3 million.  The geographic and economic details discussed here are supplied by the Nizhny Novgorod entry from Wikipedia.  This city, that you may recall as "Gorky" (1932-1990), offered an ideal administrative seat for the surrounding oblast.  The Kremlin was built in Nizhny Novgorod between 1508 and 1511. 

Though the area is well-connected via multiple transport hubs including the Trans-Siberian Railway, the city was closed to foreign tourism for most of the Soviet era due to military research and development.  Today direct trains run to many Russian cities and to Beijing.  Airports serve Frankfurt, Germany 3 times per week.  The Ukraine, Belarus and Kazakhstan feature in the top four of import and of export partners for this area.  Additional to the extensive cargo transport, passenger cruise vessels operate on the Volga from this area to various Russian cities including Astrakhan where H5N1 has been active in wild birds and humans.

Information about Veliky Novgorod (Great Novgorod) is also available at Wikipedia.

With those transmission vectors, ingres and egress in mind, let's examine the sequence sampled in Novgorod.

Novgorod01 appears to be the first human sequence in this pandemic to carry the dual adjacent RBD polymorphisms of 225E with 226R.  Though we may expect at some point to see 225G paired with 226R, this 225E+226R combination from Russia is yet another point of data to use in determining randomisation versus patterning.  We personally begin to speculate and revise our opinion that the expressively variant RBD sequences recently from Singapore may perhaps be perfectly valid and not the result of lab error?

Though the analysis is far from complete on this set of lung samples from Russia, a second point of interest does come to the forefront.  Preliminary review shows that A/Abakan/02/2009 demonstrates a mixture at HA amino acid position 246 coding for an alternate 246S, a polymorphism found once in the present pandemic with Auckland04, but more importantly found in sequences of H2N2 (Asian Flu Pandemic of 1957) and H2N8 (Asiatic Flu Pandemic of 1889-1890).  Abakan sits near a very large body of water (Krasnoyarskoye) and is somewhat centered north of Mongolia and east of Kazakhstan, geographically providing a mixing area for wild birds.

Of course, we would be remiss to fail mentioning that 225G is found in 3 of the 5 Vector isolates with 225E in the other 2.

This first reported instance (Novgorod01) of adjacent Receptor Binding Site amino acids on one sequence, 225E plus 226R, may equate to a direction change in ΣPF11 signalling a more persistent hyper-morphic behaviour in the reservoir.  At any rate, the recent reports from Russia demonstrate a geographically widespread incursion of polymorphism at amino acid 225, including 225E, 225N (Orenburg) and 225G, presumably on fatal cases. 

Will we also see 225S emerge in Russia or the surrounding states? 

Is ΣPF11 learning how to become a more deadly pandemic virus from previous pandemics of differing serotypes?  Is the ecosystem of Influenza reservoirs more promiscuous than our level of surveillance (granularity and geography) has led us to believe?

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Utah demonstrates Hypermorphic Behaviour at HA 225

A/Utah/42/2009 either demonstrates a profound series of lab errors or a population of sub-species that is essentially hypermorphic at 225.   The original sample was taken from a 28F on 2009-07-24 who has been confirmed as a fatality.

rrT is the representation of the tri-nucleotide (codon) that encodes the amino acid value(s) found in the mixed peaks of the sequencing trace at amino acid position 225 in Utah42.  "R" is a nucleotide Ambiguity Code that represents a Purine.  The Utah42 HA sequence carries the Purine Ambiguity Code at two adjacent nucleotide positions. 

Adjacent mixed peaks are more than rare within PF11.  We have not witnessed the event previously in any sequence that was not suggestive of lab error . . . until now.  Significant selective pressure would have credence as a driver for this type of multiple mixed peak scenario.  A genetic acquisition event of this type may be considered more probable at an antigenic position like 225 in the Receptor Binding Domain than at non-epitope. 

The Purine group includes Adenine (A) and Guanine (G).  4 permutations are demonstrated in the sample according to the output of the sequencing, not 4 possible values, but 4 actual differing values encoding at position 225.

Permutations of RRT Detected at Amino Acid 225 for Utah42

  • GAT=D
  • GGT=G
  • AAT=N
  • AGT=S

In this single original sample notated as Utah42, sub-species are detected that code for 225G, 225N, 225S and wt 225D.

Questions occur:

  • Hypermorphic
  • Special case indicating viral genetic acquisition direction
  • Anomaly

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Spain Introduces 2 November Sequences with Cross-Segment Pairing from Fatal Cases in the Ukraine

The HA and NA cross segment pairing of silent polymorphisms noted on the four fatal 225G cases from the Ukraine has now been tracked to 29 sequences around the globe on a cumulative study showing a quantifiable density in Spain.

  • HA:syn413K encoded from A1281G, AAa->AAg *
  • NA:syn407V encoded from T1221C, GTt->GTc *
* SNP Matches the 4 fatal flashfire cases

The most recent public database entries at GenBank are from Spain and were sampled in November 2009.
  • HA and NA CatNS7362 TamiFlu Resistant
  • HA and NA CatS1827
This background appears to be fertile ground and receptive to both major polymorphisms of interest today:

  • HA 225G correlated with deep lung damage and one "low reactor" vaccine antisera challenge result
  • NA 275Y conferring TamiFlu Resistance

The depth of this background continues to increase with 2 TamiFlu Resistant strains reported in 2 days.  The persistance of this cross segment correlation may signal a viral genetic acquisition response to widespread TamiFlu usage.  These silent polymorphisms are suggestive of an intermediate stage that is moving in the direction of antiviral resistance and continued Receptor Binding Domain revisions due to antiviral blanketing.

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225E in 5 of 9 HA from Spain during 4 Day Span in Mid-November

GenBank deposits on 2009-12-02 from Spain that were sampled from 2009-11-12 to 2009-11-16 demonstrate a significant percentage of 225E225E is now conserved in Spain.

5 of 9 have 225E

0 of 9 elicited 225G

Japan also adds one 225E sequence with NagasakiHA52 sampled on 2009-10-09.

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TamiFlu Resistant Sequence #14 from Spain in Late November Carries Cross Segment Pair of Markers from the Ukraine

HA and NA CatNS7362 is the most recent official entry into the TamiFlu Resistant list of 14 sequences.  This Spanish sequence sampled 2009-11-26 from a male in Catalonia also becomes the first recorded TamiFlu Resistant strain to participate in the cross segment pairing of HA syn413K and NA syn407V.

The NA of CatNS7362 is novel at the nucleotide level with no peer within ΣPF11.  The NA is an exact nucleotide match to A/Singapore/ON1156 (2009-07-14) and A/Catalonia/S1702 (2009-11-02) except for the SNP encoding the antiviral resistance.  The 7 polymorphisms found on the HA and the NA of this sample indicate a similar hyper-morphic behaviour that we saw with the Italian TamiFlu Resistant sequence, Pavia21.  Three of the polymorphisms are silent.  Inclusions are noted that previously appear on other sequences from Spain, Swine H1N1, Swine H1N2, Avian H1N1, Human H5N1, Avian H5N1 and Avian H6N1

NA Amino Acid Codings to 3 Rare Polymorphisms
  • syn149I
  • 275Y
  • syn407V
encoded from T447C, ATt->ATc 
Rare to ΣPF11 with 2 instances from Spain and 1 from Singapore.

encoded from T1221C, GTt->GTc 
Rare to ΣPF11 with 37 instances, yet common to Spain with 15 and the Ukraine with 9.
Progenitors may include:
H5N1 Human
H5N1 Avian

On CatNS7362 TamiFlu Resistance is indicated in typical PF11 fashion via a Single Nucleotide Polymorphism coding for 275Y on the Neuraminidase. The sequence displays the following NA Quadruple Combination:

106I, 248D, 275Y, 286S

106I is implied for CatNS7362 based on previous patterns as that portion of the sequence is truncated.

The following permutations are now represented on the fourteen PF11 anti-viral resistant sequences:

106V, 248N, 275Y, 286S = WA28, WA29, TX47
106I, 248N, 275Y, 286S = Osaka180
106I, 248D, 275Y, 286S = HK2369, Yamaguchi22, Denmark528, Hunan SWL3, Singapore57, Tokushima2, Iwate3, Quebec147365, Pavia21, CatNS7362

Until the 2009-08-21 deposit of the two Washington sequences, all 275Y TamiFlu-Resistant specimens on PF11 backgrounds were paired with 106I.  We continue to see only 3 of the 13 with 106V.  The addition of CatNS7362 heavily leverages position 248 toward Aspartate (D) with 10 specimens versus 4 with Asparagine (N). No TamiFlu Resistant specimen on file displays 286G as yet.

HA Amino Acid Codings to 2 Novel and 2 Rare Polymorphisms
  • 165N
  • 235A
  • 397D
  • syn413K
CatS1687 is the singular instance within ΣPF11.
Progenitors may include:
H1N1 Swine
H1N2 Swine
H6N1 Avian

Novel to ΣPF11.
Progenitors may include:
H1N2 Swine
H6N1 Avian
  Chukkar (first base SNP required)

Novel to ΣPF11.
Progenitors may include:
H6N1 Avian

encoded from A1281G, AAa->AAg 
Rare to ΣPF11 with 74 instances, yet common to Spain (12), the Ukraine (4), Japan (28) and Norway (9).
Progenitors may include:
H1N1 Avian
  red-winged tinamou/Argentina/MP1/2008

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Sweden Deposits 225G with Alternate Coding Matching and Pre-dating Norway3206-3 with NA Marker Homology

Sweden deposited a sequence today from mid summer that carries indicators worthy of study.

Matches include the rare alternate coding for 225G found in the Norway3206-3 fatality sampled 6 weeks after the Swedish case and the NA syn291V on that same Norwegian sample.  Six weeks appear to be enough time for these changes to travel across borders.  The HA sequences are identical, except for the 140S on Norway3206-3.

The two Scandinavian NA segments show a shared polymorphism at syn291V, a change that is found in 103 GenBank sequences including TamiFlu Resistant HK2369, Almati01, Tomsk01, Poland and with deep penetration through Italy and Catalonia.  The G873A coding for the syn291V is also present in CatNS2001 and CatNS2008.  The NA segment of Norway3206-3 is identical to the Swedish NA.

Only this profiled private Swedish sequence from mid summer, CatNS2001 and CatNS2008 from early August and Norway3206-3 from September carry this variant Glycine coding at amino acid 225 that is seen as T717A, GGt->GGa.  Neither of the cross segment pair, HA syn413K or NA syn407V, appears in the recent Scandinavian samples. 

The Swedish sequence is a nucleotide match with Norway2674 and Norway2690 except for the RBD polymorphism 225E on the Norwegian sequences.  Dialing back the resolution by 1 nucleotide, we find that the Swedish sequence is a 1724/1726 match with 2 more Norway 225E cases, Norway3059 and Norway4023.  Delta is indicated after link to sequence.

2009-07-27  Norway2674  Δ 225E
2009-07-29  Norway2690  Δ 225E
2009-08-11  Norway3059  Δ 225E + 1 nucleotide
2009-10-14  Norway4023  Δ 225E + 1 nucleotide

If we return to the tighter resolution again, we find that the Swedish sequence is a exact nucleotide match except for the two nucleotides coding for 225D on 6 additional sequences from Sweden (1),  Germany (1) and Russia (4).  Delta is indicated after link to sequence.

2009-__-__ Bayern66         Δ 225D
2009-05-21 MoscowIIV01    Δ 225D
2009-05-30 Stockholm33    Δ 225D Traveler from US
2009-06-09 MoscowIIV04    Δ 225D
2009-06-20 MoscowIIV05    Δ 225D
2009-09-22 Russia01          Δ 225D

This particular background is far from isolated.  The homology between these 225D, 225E and 225G sequences raises a very basic question. 

Does some mechanism in the protocol from sample to sequence potentially vary the outcome or block 225G materialisation on the resultant sequence?  We know that lungs are being destroyed across extensive geography, yet few 225G sequences are being produced?  Backing studies lead us to believe that 225G bearing samples will grow well in a receptor environment rich in α2-3-linked sialic acid

MDCK cell lines specialised to overexpress α2-6-linked sialic acid at the expense of α2-3-linkedSA are being employed in Neuraminadase Inhibitor sensitivity testing, demonstrating that these canine cell lines are adaptable in linkedSA expression.  Exactly how sensitive is the culture environment that is being used as a predecessor to sequencing?  How consistent are the protocols and materials between labs?

Does a possibility exist that current MDCK cell lines being used are underexpressing α2-3-linked sialic acid in a manner consistent with the low numbers of resultant 225G sequences that have been published?  Are samples that are originating with 225G not growing well due to a limited ratio of matched receptors?  We specifically enquire here on the cell lines, but obviously all aspects of the growth environment must be inspected for inhibitors to 225G materialisation if the presenting sample sequences are to be accurately elicited. 

Similar rare codings across 4 geographically dispersed sequences for Glycine at HA residue 225 and an NA syn291V occurring in temporal sequence from Sweden in late summer, to Catalonia in early August, and onward to Norway on September 1st beg for any explanation other than "spontaneous" mutation within a pandemic reservoir, ΣPF11, that is actively demonstrating an ability to share data. 

Is 225G transmitting?  Is 225G transmitting in 2 variant forms?  Is 225G now confirmed as Hydra?

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NA 106V and 248D Pairing Reaches US and Russia

A/Chita/01, sampled from human lung in Russia with no identifying host gender or age, becomes the 5th distinct sequence on file for the previously discussed 4th permutation of the NA Dual Combination at amino acid positions 106 and 248.  No HA was published for this sample, but the sample location points to fatality and 225G is suspect.

This PF11 sequence demonstrates 106V and 248D and is distinct from the four previous isolates with this characteristic pair:

2009-04 A/Toronto/3184
2009-04 A/Auckland/4
2009-07 A/Toronto/R8564
2009-05 A/Wisconsin/629-D00487

Chita01 is most similar (1417/1419) to Toronto3184, yet all 5 sequences with this pairing are distinct.  2 silent polymorphisms are found on Chita01, coding for syn257R and syn315G.

The NA Dual Combination of 106V and 248D is now found 5 times within ΣPF11:

2009-04 Canada and New Zealand
2009-05 Wisconsin
2009-07 Canada
2009-xx Russia

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