slow evolution in Chinese birds

[HenryN]

Re: low mutation rates in flu-viruses

There is another theory that could explain the phenomenom.

We know that viri held in deep freeze in artic and antarctic ice are still - sometimes - viable when released from deep freeze.

It is entirely possible that birds or other vectors have transported viruses to areas where subzero temperatures predominate, and that, with global warming and ice melts such viruses are released again into the environment.

Thus a virus from 1978 (or prior) could have become locked in ice, and then become released as warming has resulted in ice melts with a subsequent exposure to a viable host, restarting the cycle.

To test such a theory, where exactly were viral samples that matched earlier (unchanged) samples identified? Was it in a cold temperature area? Could it explain what has been seen in genetic sequence samples?

Just to add to the mix...

Please provide reference for viable virus isolated from ice.

[gsgs]

Re: low mutation rates in flu-viruses

minimum distance in 0/00 to groups :

19 times HK 1970s
059,077,092,097,047,393,025,057,   37.Ck/Germany/n/1949(H10N7)     
099,093,091,369,068,393,044,053,   38.fowl/Dobson/1927(H7N7)       
064,080,055,088,034,392,026,031,   40.Dk/UKR/1/1963(H3N8)          
040,081,071,082,040,071,031,025,   41.Ck/Scotland/1959(H5N1)       
046,082,066,093,074,123,054,050,   42.human/China/GD02/06(H5N1)    
042,076,079,090,074,114,058,051,   43.Bar-h Gs/Qinghai/62/05(H5N1) 
138,160,133,348,126,133,082,097,   44.Sw/1931(H1N1)                
134,151,129,343,114,129,068,059,   45.Brevig Mission/1/1918(H1N1)  
143,162,136,342,130,164,093,087,   46.Alaska/1935(H1N1)            
148,161,136,345,141,169,084,086,   47.Hickox/1940(H1N1)            
166,175,148,351,154,199,112,115,   48.Kansas/UR06-0068/2007(H1N1)  
150,074,144,081,146,069,093,087,   49.Japan/305/57(H2N2)           
153,085,145,062,154,096,096,104,   50.Russia/WO0200884/72(H3N2)    
158,117,158,148,169,149,111,124,   51.Texas/UR06-0356/2007(H3N2)   

6 times Hubei 1997
067,082,102,470,064,461,024,267,   37.Ck/Germany/n/1949(H10N7)     
103,100,101,466,072,466,048,058,   38.fowl/Dobson/1927(H7N7)       
064,084,066,473,061,397,024,035,   40.Dk/UKR/1/1963(H3N8)          
065,087,086,120,055,114,030,030,   41.Ck/Scotland/1959(H5N1)       
054,074,070,038,064,043,017,052,   42.human/China/GD02/06(H5N1)    
049,065,061,037,065,019,015,051,   43.Bar-h Gs/Qinghai/62/05(H5N1) 
140,159,138,350,130,154,082,096,   44.Sw/1931(H1N1)                
135,151,133,349,119,168,066,060,   45.Brevig Mission/1/1918(H1N1)  
147,162,140,362,134,187,093,091,   46.Alaska/1935(H1N1)            
153,158,141,364,142,197,086,087,   47.Hickox/1940(H1N1)            
170,175,152,370,153,213,115,117,   48.Kansas/UR06-0068/2007(H1N1)  
154,081,148,301,148,495,095,092,   49.Japan/305/57(H2N2)           
160,090,144,483,157,473,098,106,   50.Russia/WO0200884/72(H3N2)    
162,122,160,490,170,482,114,124,   51.Texas/UR06-0356/2007(H3N2)   

11 times Henan 2004
071,082,107,476,065,488,029,269,   37.Ck/Germany/n/1949(H10N7)     
105,103,102,466,074,491,052,053,   38.fowl/Dobson/1927(H7N7)       
064,086,066,480,064,398,029,036,   40.Dk/UKR/1/1963(H3N8)          
061,089,089,126,057,124,035,027,   41.Ck/Scotland/1959(H5N1)       
021,022,061,025,009,033,007,018,   42.human/China/GD02/06(H5N1)    
018,016,016,022,012,011,009,019,   43.Bar-h Gs/Qinghai/62/05(H5N1) 
146,160,139,352,133,168,087,096,   44.Sw/1931(H1N1)                
141,151,136,352,117,171,069,057,   45.Brevig Mission/1/1918(H1N1)  
152,162,143,367,133,190,096,090,   46.Alaska/1935(H1N1)            
157,162,139,363,140,199,089,085,   47.Hickox/1940(H1N1)            
171,176,151,370,151,216,116,109,   48.Kansas/UR06-0068/2007(H1N1)  
158,079,148,302,146,492,099,090,   49.Japan/305/57(H2N2)           
161,092,146,485,159,481,103,112,   50.Russia/WO0200884/72(H3N2)    
164,125,159,494,170,490,117,129,   51.Texas/UR06-0356/2007(H3N2)   

032,011,016,080,013,120,013,014,   HK-Henan
010,003,016,020,003,029,005,008,   Hubei-Henan
034,011,017,072,014,095,008,012,   HK - Hubei

[gsgs]

Re: low mutation rates in flu-viruses

a good example is also
A/Guangxi/50/2005(H5N1) {gx50} which has only 49 differences
in 3 years to A/Henan/16/2004.
gx50 can be viewn as the best matching anchestor-virus to current
H5N1 in most segments.

These slow evolutions with few mutations are rare and not seen
in human flu AFAIK with the exception of 1977 H1N1.
In the Qinghai strain we have one (and only one) other such example :
the Hungary-Suffolk virus. It suggests an abiotic reservoir,
or some place inside the bodies, where the virus can survive for
some time without mutating/replication.
Could this be the key why the virus is so successful in
SE-Asia ? What could be the reservoir where the virus
survives without mutating ? Why is this obviously useful
feature so rare in influenza ?

[gsgs]

Re: low mutation rates in flu-viruses

strange low-mutation-rate in PB2:

                                           0000000000000000000000000000000000000000000011111111111111111111111111111111111111111111111111111111111111111112222222222222222
PB2                                        0001222223333333444445555666677788888999999900001112222223333334444444555555555566666677777777777888888999999990000000000111222
                                           4555557780001359047792289566612316788022348902494470247890112780566789113455678924445502234455667345678026778891233445799124128
                                           6576250953695943271752887403671805925014621080153736455706176164514321251947924361246972810605149921081599176982814694948842866
-codon-position----------------------------11                            1  1               1       1              11        1         1            1     2     2  1   1  
---Index-----------------------------------TCATGGATGTCAAGCTAACCTGTGAGGCCGTGTCCCCGTATGGACACACCTATATCAAGCACTGAGGGGTTGTCGCTCTGAGACGAGTCCACAGAGATTGTGGGTCCGCCCCCCATACCGCAGAGGG
A/Dk/Hongkong/7/1975(H3N2)                 ...............................................................................................................................
A/Dk/Nanchang/1681/1992(H3N8)              .......C.....A.CG.TGCA.AG....A..CT...A.G.......GTTCGAGC........A..A.A....A.......AG...A.T.GTG....C....AAC......T..GA..T....G...
A/Dk/Zhejiang/11/00(H5N1)                  ....A.............T....A........C.T....G.A......T....GC.......C..AAA...A......C..A......T.GT......C..........T..TT.....A......A
A/black Dk/Western AUS/4954/1983(H3N8)     ....AA..AA...AT.......CA.AATG.CA...TT.C.G.A...T.TT.....TGCA.GT..G..A..C.C.ATCT.A...AAG.C.T.T.AGA..C.......AAT.......GG.ATGA....
A/slaty-backed gull/Shandong/38/04(H5N1)   CTGC..G.A.TGG....GT....A.A.TT..A.T......A..GTG..T..........A.T.......C..........G..........T..G.G..ACA.A.T....T.....G.....A.AA.

omitting polymorphisms which only occur once:

                                           0000000000111111111111222
                                           2234667889122344677789001
                                           5837563162447767424640492
                                           2591438054755143180125944
-codon-position----------------------------        1 1              
Index--------------------------------------GGGCGCGTCACATCGGGCAATGAGG
A/Dk/HK/7/1975(H3N2)                       .........................
A/Dk/Nanchang/1681/1992(H3N8)              ..AT...CTGTGC.A.ATG..A...
A/Dk/Zhejiang/11/00(H5N1)                  A..T...C.G.GC.AAATG.C..A.
A/black Dk/Western AUS/4954/1983(H3N8)     AAA.ATA...T..T.A...GC.GAA
A/slaty-backed gull/Shandong/38/04(H5N1)   .A.TATA.T....T.....G.AG.A

#differences in PB2                        1   2   3   4   5   6   7 
---------------------------------------------------------------------
  1 >A/Dk/HK/7/1975(H3N2)                  0, 43, 56, 94, 39, 98, 26
  2 >A/Dk/Nanchang/1681/92(H3N8)          43,  0, 89,115, 70,119, 45
  3 >A/black Dk/W.AUS/4954/83(H3N8)       56, 89,  0,111, 71,132, 68
  4 >A/Ck/Scotland/1959(H5N1)             94,115,111,  0,113,168,108
  5 >A/slaty-backed gull/SD/38/04(H5N1)   39, 70, 71,113,  0,125, 57
  6 >A/Bar-h Gs/Qinghai/62/05(H5N1)       98,119,132,168,125,  0, 78
  7 >A/Dk/Zhejiang/11/00(H5N1)            26, 45, 68,108, 57, 78,  0

the 168 differences in PB2 between the Qinghai virus and the 1959-virus
is already so low, that Qinghai might be a direct descendant of 1959.
But some of the other differences are even much lower, so that
we should assume either errors or slow evolution, which is unusual.
The HK/75 virus had already given some examples of slow evolution
above in this thread

But it's not just this one virus. Close to HK75 in PB2 are e.g.
A/Dk/HK/24/76(H4N2)
A/Dk/HK/562/79(H10N9)
A/Dk/HK/312/78(H5N3)

A/Dk/Nanchang/1681/92(H3N8)
A/black Dk/AUS/751/78(H3N8)

[gsgs]

Re: low mutation rates in flu-viruses

compare the differences in PB2 between these 4 viruses:

#differences                         1   2   3   4 
---------------------------------------------------
  1 >A/duck/England/1956(H11N6)      0  85 103 127 
  2 >A/Dk/HK/7/1975(H3N2)           85   0  26  56 
  3 >A/Dk/Zhejiang/11/00(H5N1)     103  26   0  34 
  4 >A/Dk/Guangxi/35/01(H5N1)      127  56  34   0

they are pretty much "in a line". No room for a lab-error,
the "error" can't know the required mutations.

This would all be not so surprising , if the date-differences
would match the mutations-differences But :
there are 25 years between 2) and 3), with
only 26 differences !!

[gsgs]

Re: low mutation rates in flu-viruses

now it seems that we can observe the same phenomenon
in North America. More and more sequences are being uploaded
and virus-segments with few differences but decades apart
appear.
I have not yet made a systematic list as above for
HK-Hubei-Henan but e.g.
A/pintail/Alaska/779/2005(H3N8)
seems to have some low-distance relatives from the 70s
or 80s in all segments.
While at the same time being close to other viruses from 2005.

segment 1 : 97.4% similarity for 14 years
segment 2 : 97.5% similarity for 12 years
segment 3 : 95.9% similarity for 26 years
segment 4 : 97.1% similarity for 27 years
segment 5 : 96.6% similarity for 24 years
segment 6 : 96.3% similarity for 28 years
segment 7 : 98.3% similarity for 27 years
segment 8 : 97.4% similarity for 26 years

these are different viruses for the segments, there
was apparantly much reassortment.

---------edit1----------
comparing 52 North American avian viruses from the 70s with
248 North American avian viruses from 2000 or later

1978-2005: 39,29,40,28,28,32,16,15

compare with Asia from post #1:

1977-2004: 29,11,17,81,13,99,14,19

minimum:

27 years: 29,11,17,28,13,32,14,15

(nucleotide-difference in 0/00)

This low-mutation evolution isn't seen in human influenza
and seems to be independent and rarer than "normal"
evolution in birds. But it contributes significantly,
so is the current H5N1 a result of this low mutation evolution
in most segments since the 70s as shown above.

What could it be ? And why isn't it in human flu ?
(but some examples for swine too !)


some possible explanations:

* the virus could persist most of the time in the environment (water ?)
and only some of the time actively replicating in birds
* the virus could persist in some special organs/places inside
some special species of birds (or other animals ?)
* the virus has a method to replicate more reliably with fewer
mutation-rates in some species under certain circumstances
(as seen with influenza-B in humans)
* some gene-configurations are particularly stable and although
the mutation rate isn't lower, only few of them survive due
to selection (including selection for synonymous mutations)



I wish more bird-sequences from the 1970s from EurAsia

[gsgs]

Re: low mutation rates in flu-viruses

I want to add this for the evolution of the Alaska avian viruses
filename: swav18m.l6g

 18 >A/pintail/Alaska/779/2005(H3N8)            2314 2285 2203 1731 1530 1425  977  851   
  4:144,109, 99,447, 92,425, 69,273   A/fowl/Rostock/45/1934(H7N1)               2341 2341 2233 1741 1565 1393 1027  890
  5:141, 87, 90,494, 91,436, 64,276   A/Ck/Scotland/1959(H5N1)                   2341 2341 2233 1767 1565 1458 1027  890
  6:152,115, 91,498,116,434, 86,278   A/Bar-h Gs/Qinghai/62/05(H5N1)             2287 2298 2185 1698 1493 1361  982  831
  7:144,106, 73,489, 88,503, 65,282   A/tern/South Africa/1961(H5N3)             2341 2341 2233 1779 1565 1454 1027  890
  8:142,105, 61,491, 96,500, 57,275   A/duck/Germany/1215/1973(H2N3)             2341 2341 2233 1773 1565 1410 1027  890
  9:139, 96, 97,445, 95,483, 61,284   A/turkey/England/1963(H7N3)                2341 2341 2233 1738 1565 1369 1027  890
 10: 65, 89,115,435, 75,507, 36, 35   A/turkey/Oregon/1971(H7N3)                 2280 2280 2151 1691 1540 1419 1002  855
 11: 90,154,111,489, 64,504, 40, 50   A/duck/Memphis/546/1974(H11N9)             2341 2341  493 1760 1565 1460 1027  890
 12:142, 88, 78,493, 94,471, 75,281   A/duck/England/1956(H11N6)                 2341 2341 2233 1760 1565 1464 1027  890
 13:142, 86, 90,380, 90,481, 72,275   A/duck/Czechoslovakia/1956(H4N6)           2341 2341 2233 1738 1565 1426 1027  890
 14:140, 90,104,449, 86,516, 62,100   A/Ck/Germany/n/1949(H10N7)                 2341 2343 2233 1728 1565 1464 1027  890
 15:139, 94,105,451, 97,225, 78,281   A/quail/Italy/1117/1965(H10N8)             2341 2341 2233 1728 1565 1460 1027  890
 16:147, 83, 74,147, 93,216, 60,280   A/Dk/UKR/1/1963(H3N8)                      2341 2341 2233 1764 1565 1461 1027  890
 17:142,103, 65,152, 90,220, 65, 96   A/duck/Chabarovsk/1610/1972(H3N8)          2341 2341 2233 1765 1565 1460 1027  890
 19:104,163,141,213,138,218, 58,289   A/equine/New York/1/1983(H3N8)             2299 2302 2192 1723 1519 1425  992  850
 21:114,163,147,225,150,230, 64,289   A/swine/Chibi/01/2005/12/15(H3N8)          2341 2341 2233 1762 1565 1460 1027  889
 22:164,132,125,202,119,485, 88,283   A/swine/Hong Kong/5190/99(H3N2)            1285 1292 1535 1595 1459 1380  964  838
 23:155,125,164,206,172,487,103,274   A/Texas/UR06-0356/2007(H3N2)               2303 2297 2187 1701 1521 1428  988  848
 24:149, 88,153,161,157,488, 89,281   A/Russia/WO0200884/72(H3N2)                2341 2341 2233 1765 1565 1466 1027  890
 26:144, 83,151,483,145,486, 77,285   A/Index/1957(H2N2)                         2314 2317 2209 1730 1520 1447 1002  864
 29:140,164,147,484,151,426, 74,279   A/Index/1977(H1N1)                         2298 2300 2194 1731 1506 1426 1002  848
 34:148,175,157,495,156,431,103,287   A/Kansas/UR06-0068/2007(H1N1)              2283 2303 2197 1741 1506 1423  982  849
 36:119,153,141,477,122,420, 57,278   A/Index/early human/1935(H1N1)             2280 2274 2151 1701 1497 1410  982  838
 37:114,149,141,477,112,422, 51,275   A/Brevig Mission/1/1918(H1N1)              2280 2274 2151 1701 1497 1410  982  838
 38:127,158,140,484,120,422, 64,282   A/Sw/Index/early swine/1935(H1N1)          2280 2274 2151 1701 1497 1410  982  838
 43:139,167,161,480,152,431, 79,288   A/swine/Wisconsin/1/1957(H1N1)             2292 2298 2181 1730 1532 1418  995  855
 55: 87,122, 49,511,164,423, 85,294   A/Wisconsin/10/98(H1N1)                    1600 2341 1494 1064 1512 1458 1027  890
 57: 96,131, 66,467,164,429, 89,291   A/******/index/2009-02-01(H1N1)            2280 2274 2151 1701 1497 1410  982  863
 64: 87,128, 59,465,161,482, 95,295   A/Swine/Hong Kong/915/2004/10/14(H1N2)     2276 2266 2151 1701 1515 1428  984  861
 65: 97,125, 41,468,163,483, 72,293   A/Turkey/MO/24093/99(H1N2)                  787  706  729 1701  980 1434  360  846
 68: 78,123, 51,201,175,480, 80,291   A/Swine/Nebraska/209/1998(H3N2)            2335 2275 2151 1702 1565 1410  996  847
 69: 80,122, 46,200,163,481, 82,293   A/Sw/Index/triple-reassortant/1998(H3N2)   2341 2301 2202 1725 1565 1430 1012  870
 71: 81,123, 48,199,163,483, 80,293   A/mallard/SD/Sg-00125/2007/09/19(H3N2)     2334 2335 2226 1763 1557 1444 1017  880
 73: 66, 77, 68,479, 57,424, 33, 39   A/turkey/MO/21939/1987(H1N1)               2316 2294 2165 1748 1539 1418 1002  865
 74: 60, 56, 83,472, 58,426, 42, 33   A/swine/Saskatchewan/18789/02(H1N1)        2280 2274 2151 1701 1497 1410  982  838
 76:---,---,---,478,---,431,---,---   A/Anas crecca/Spain/1384/2007(H1N1)           3    3    3 1701    3 1410    3    3
 78:149,109, 79,480,100,430, 61,100   A/duck/Italy/69238/2007(H1N1)              2308 2308 2200 1743 1530 1422  989  887
 81:148,103,110,472, 96,432, 65,284   A/swine/Germany/2/1981(H1N1)=1578          2314 2313 2233 1778 1565 1412 1002  864
 89:163,129,134,468,119,439,106,311   A/Thailand/271/2005(H1N1)                  2301 2193 2120 1778 1541 1452  994  846
 90:141, 97, 50,481, 90,349, 69,280   A/shearwater/Australia/1/1973(H6N5)        2341 2341 2233 1744 1564 1454 1027  890
 91:142,114, 69,477,103,442, 62, 96   A/duck/Taiwan/0526/72(H6N1)                2341 2340 2233 1744 1563 1458 1027  890
 93: 93, 77,134,486, 69,483, 36, 36   A/Turkey/California/189/66(H9N2)           2257 2247 2140 1661 1505 1386  974  831
 94: 69, 72,132,486, 71,478, 38,280   A/turkey/Wisconsin/1/1966(H9N2)            2341 2341 2233 1742 1565 1467 1027  890
 95: 98,156,129,493, 69,435, 33,279   A/turkey/Ontario/6118/1968(H8N4)           2341 2341 2233 1744 1565 1462 1027  890

[Vibrant62]

Re: low mutation rates in flu-viruses

Question - we know influenza virus can remain viable for an indefinite time in ice, and we are in a period of global warming and ice melts. Is it possible that viral reservoirs are being freed from ice during melts at the same time as possible hosts are around for wider dissemination, re-introducing older viral forms back into the mix?

[gsgs]

Re: low mutation rates in flu-viruses

we have no evidence for this. flu has never been isolated from ice
in nature, AFAIK.
When you say: "indefinite time" then there should also be older
viruses around, 100 years or such, but that has never happened before,
AFAIK.
Also the samples from 1918 in permafrost were only small fragments,
hard to reconstruct. And they had to search a lot until they found
a body with some fragments.

maybe frozen droppings or frozen eggs or frozen bird-cadavers ?
Maybe some birds find and eat these frozen eggs, cadavers.

OK for Alaska, but in Hong Kong ??


We have a lot new bird sequences from USA and Alaska recently
shovelers,pintails,mallards, sandpiper

maybe I can make a more complete analysis when ******
stays calm


--------------------------------------

http://alaska.usgs.gov/science/biology/avian_influenza/pdfs/Pearce_et_al_Evol_Apps_2009.pdf

[gsgs]

Re: slow evolution in Chinese birds

The available epidemiological data indicate that H9N2 influenza viruses were not detectable in chickens in southern China from 1975 to 1985, for extensive surveillance in live poultry markets in Hong Kong during this period detected the H9 subtype only in ducks (10). The first reported isolates of H9N2 in Asia from domestic poultry were in 1992 and 1994 (11), and by 1997 reports from Korea and Europe indicated widespread H9N2 influenza virus activity (12, 13, 18).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC17788/?tool=pubmed

[gsgs]

Re: slow evolution in Chinese birds

http://www.cdc.gov/eid/content/16/4/728.htm

ahh, not a single word about the _strange_ preservation of virus-RNA over ~8 years
with only minimal mutations, if any.
Swine flu in mallards is very unusual and such preservation is also unusual and unexplained.
Waterborne transmission is assumed to be prevalent in mallards, but survival of flu in water
over 8 years is not documented.
Are these multiple waterborne transmissions over several generations but with viruses
spending most of their time in water without mutating ?
Are the viruses surviving for years without mutation in some organ
or in the environment ?
We did see similar things in H5N1 viruses isolated from egg-shells in Vietnam 2005.


----------------------------------------------
Inasmuch as we identified a swine lineage virus in waterfowl, we first investigated laboratory contamination by using trace back and history of swine virus isolations during the time the surveillance samples were processed. No H3N2 subtype were isolated from swine sources in the Minnesota Veterinary Diagnostic Laboratory during this period. Furthermore, phylogenetic analysis of all HA segment sequences from isolates obtained in that 4-month period confirmed no contamination. We then investigated whether an ecologic niche existed for potential exposure of waterfowl to pigs. We identified a swine herd near the wildlife refuge area where the waterfowl sampling occurred. Pigs were housed outdoors, and the owner of this swine herd reported that geese and ducks inhabit the water ponds/stock dams/slough area to which the pigs had access
...
Respiratory secretions from the pigs possibly could have spread to birds through aerosols or droplets. It is also likely that swine and waterfowl shared common water sources, which contained feces from influenza-infected waterfowl or respiratory secretions from influenza-infected swine. This mode of influenza virus transmission from birds to pigs has been documented (6–9). Indeed, a waterborne source for transmission is most likely because influenza A virus can persist in water for several months depending on environmental factors such as pH, temperature, and salinity (10). Finally, because the swine herd in this area was housed outdoors in open pens, direct interaction with waterfowl was possible.
--------------------------------------------------

--------edit-------
mixed populations in these mallards:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007105

[mixin]

Re: slow evolution in Chinese birds

ahh, not a single word about the _strange_ preservation of virus-RNA over ~8 years with only minimal mutations, if any.

Can the virus survive freezing and thawing multiple times?

[gsgs]

Re: slow evolution in Chinese birds

it has not been demonstrated. I assume the virus wouldn't notice
freezing at all.
You won't find these extreme preservations, even in Alaska.
Once there was a paper about flu in Sibirean ice, but
it was probably just a lab-error, contamination.


(the preservation is on the nucleotide-level, not just amino-acids,
and it's in swine-viruses, distant from the bird-index)

[mixin]

Re: slow evolution in Chinese birds

I found this New Scientist article from Nov, 2006 discussing flu viruses frozen in Siberian lakes.

Can flu viruses survive winter in frozen lakes?

Stoye says that whether or not the viruses are infectious depends largely on how the virus was frozen. Viruses frozen in water are likely to be inactivated by the water's relatively low pH. "But if the virus was in droppings, which presumably is how it was deposited, there seems to be no reason why it should not freeze and survive at low temperatures."

He adds that viruses are more likely to survive in a frozen state if they freeze and thaw only once, as the freeze-thaw process kills at least 90% of virus each time.

http://www.newscientist.com/article/dn10676-can-flu-viruses-survive-winter-in-frozen-lakes.html

[gsgs]

Re: slow evolution in Chinese birds

here some more links and a graph

http://www.bioone.org/doi/abs/10.1637/8760-033109-Reg.1