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**HenryN** · June 23, 2008, 01:27 PM

Re: genbank anomalies

Originally posted by gsgs View Post

new paper, not yet available:

Anomalies in the Influenza Genome Database: New Biology or Lab Errors?
Michael, Krasnitz, Arnold Levine, Raul Rabadan,
Accepted J. of Virology.

(hopefully will shed some light on our previous discussion about
the conservations of regions, periods of slow evolution, "stasis",
recombinations, contamination)

404 Not Found

http://www.sns.ias.edu/~rabadan/?page_id=12

**HenryN** · June 23, 2008, 06:00 PM

Re: genbank anomalies

Originally posted by gsgs View Post

new paper, not yet available:

Anomalies in the Influenza Genome Database: New Biology or Lab Errors?
Michael, Krasnitz, Arnold Levine, Raul Rabadan,
Accepted J. of Virology.

(hopefully will shed some light on our previous discussion about
the conservations of regions, periods of slow evolution, "stasis",
recombinations, contamination)

Paper will be available on Wednesday.

**HenryN** · June 25, 2008, 09:56 AM

Re: genbank anomalies

JVI Accepts, published online ahead of print on 25 June 2008
<TABLE class=content_box_outer_table align=right><TBODY><TR><TD><TABLE class=content_box_inner_table><TBODY><TR><TD class=content_box_title_highlight colSpan=2>This Article</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Full Text (PDF) </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me when this article is cited </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me if a correction is posted </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>Services</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Similar articles in this journal </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me to new issues of the journal </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Download to citation manager </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Copyright Information </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Books from ASM Press </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>MicrobeWorld </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>Google Scholar</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Krasnitz, M. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Rabadan, R. </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>PubMed</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Krasnitz, M. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Rabadan, R. </TD></TR></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE><VARDEF id=TEXT>J. Virol. doi:10.1128/JVI.00101-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Anomalies in the Influenza Genome Database: New Biology or Lab Errors?

<NOBR>Michael Krasnitz<SUP>*</SUP>,</NOBR> <NOBR>Arnold J. Levine,</NOBR> and <NOBR>Raul Rabadan<SUP>*</SUP></NOBR>

Institute for Advanced Study, Einstein Dr., Princeton, NJ 08540, USA

<SUP>*</SUP> To whom correspondence should be addressed. Email: krasnitz@ias.edu<SCRIPT type=text/javascript></SCRIPT> . rabadan@ias.edu<SCRIPT type=text/javascript></SCRIPT> .

<TABLE cellSpacing=0 cellPadding=0 width="100%" bgColor=#e1e1e1><TBODY><TR><TD vAlign=center align=left width="5%" bgColor=#ffffff></TD><TH vAlign=center align=left width="95%"> Abstract</TH></TR></TBODY></TABLE>A search of the influenza genome database reveals anomalies<SUP> </SUP>associated with a non-negligible number of submitted sequences.<SUP> </SUP>There are many pairs of viral segments that are very close to<SUP> </SUP>each other in nucleotide sequence but relatively far apart in<SUP> </SUP>reported time of isolation, resulting in an abnormally low evolutionary<SUP> </SUP>rate. Also, some sequences show clear evidence of apparent homologous<SUP> </SUP>recombination, a process normally assumed to be extremely rare<SUP> </SUP>or nonexistent in this virus. These findings may point to surprising<SUP> </SUP>new biology, but are perhaps more readily explained by stock<SUP> </SUP>contamination or other errors in the sequencing laboratories.

**HenryN** · June 25, 2008, 09:58 AM

Re: genbank anomalies

J. Virol. doi:10.1128/JVI.00101-08
JVI Accepts, published online ahead of print on 25 June 2008
ABSTRACT

A search of the Influenza genome database reveals anomalies associated with a nonnegligible
number of submitted sequences. There are many pairs of viral segments that
are very close to each other in nucleotide sequence but relatively far apart in reported
time of isolation, resulting in an abnormally low evolutionary rate. Also, some sequences
show clear evidence of apparent homologous recombination, a process normally assumed
to be extremely rare or nonexistent in this virus. These findings may point to surprising
new biology, but are perhaps more readily explained by stock contamination or other
errors in the sequencing laboratories.

TEXT

In the last few years, an extraordinary amount of Influenza genomic sequence has been
submitted to publicly available databases

1 . For instance, there are now over 3300 full
genome sets in the NCBI?s rapidly growing Influenza Virus Resource. To our knowledge,
no systematic attempt has been made to assess the quality of sequence data in this and
similar collections. Our observations show that a fraction of the sequences in the database
exhibit anomalous properties that point to either radically new biology, or, more likely, to
problems with the data. As a first example, we consider the rate of nucleotide substitution
in the Influenza A virus. This rate has been previously estimated at 0.001-0.007 per
nucleotide per year . Using the most conservative (lowest) estimate, we still find many
pairs of virus segments that are far closer to each other in nucleotide space than would

2

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randomly occur for a Poisson process with this evolutionary rate, given the difference in
time of isolation. Such sequences appear to be effectively ?frozen in time?. For instance,
the PB2 segments of the isolates A/duck/Taiwan/0526/1972(H6N1) and
A/chicken/Taiwan/G23/87(H6N1) differ in only 1 nucleotide position out of 2283 aligned
nucleotides, whereas the expected number of differences at 0.0015 substitutions per
nucleotide per year would be ~48 for 15 years. For a null Poisson process, this gives an
extremely low p-value of . Note also that 15 years is actually a lower bound on
the true evolutionary time between these two segments, since their latest common
ancestor is likely to predate both; this makes their virtual identity even more improbable.
To visualize this anomaly, consider the plot in fig.1.
6.6 10

20 ? /

One can see that the great majority of segment pairs lie on or above the dashed green line
representing a rough estimate of the expected number of nucleotide differences given the
distance in time between the isolates; most are above the line since the true evolutionary
time (the combined distance to the latest common ancestor) is generally greater than the
naive distance in years. However, a number of points lie significantly below the fit curve,
with corresponding extremely low p-values. These represent viruses that appear to be
?frozen in time?. We performed a systematic search for such ?frozen? sequences, the
results of which are listed in Appendix 1. We found about 60 isolates which show strong
evidence of anomalously low apparent evolutionary rate, with highly significant
Bonferroni corrected p-values. Most of these are viruses from avian and swine hosts,
many of them H5N1 isolates submitted from Asia in recent years. The phenomenon of
?frozen evolution? occurs at roughly the same rate in all Influenza segments, allowing for

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greater statistical power to detect it in the longer segments. Often, though not always,
multiple segments of the same isolate appear to have evolved at a very low rate with
respect to an ancestor virus. These results are insensitive to the exact rate estimates and
methods used to compute the p-values. Each of the anomalous sequences is so close to
some other sequence in the database compared to their distance in time that any model
would rule out the possibility of a random fluctuation; there is clearly something
extraordinary about these sequences. As discussed below, we omitted the human H1N1
viruses corresponding to the mysterious reemergence of H1N1 in 1977, as well as a few
other suspect isolates previously reported in the literature

3/7 .
Another anomaly present in the Influenza database relates to homologous recombination.
This mechanism is generally believed to be extremely rare or nonexistent in the Influenza
virus and in negative strand RNA viruses generally , and has never been observed
experimentally. However, we found many sequences in the database that show very
strong apparent evidence of homologous recombination. As a rough test for this, we
divided the nucleotide sequence of each segment into two equal halves. For each pair of
segments, we compared the number of nucleotide differences between them in the first
half (i.e. 5? in the positive strand) with the number of nucleotide differences in the second
half. The idea is that if two segments are nearly identical in one part of their sequence,
but very different in another part, this is strong evidence of homologous recombination,
with the divergent parts explained by a recombination event.

17,18

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In fig. 2, we plotted a sample comparison for pairs of PB2 segments of viruses isolated
from avian hosts. Most points cluster along the diagonal, as would be expected for
roughly uniform evolution along the segment with no homologous recombination.
However, there are some very significant outliers. For instance, the PB2 sequence of
A/shorebird/DE/236/2003(H11N9) differs from that of A/shorebird/DE/231/2003(H9N4)
by 6 nucleotides out of the first 1155, and by 80 out of the second 1155. Using a null
hypergeometric distribution, this gives an extremely low p-value of . As before,
these outliers are so extreme that possible corrections allowing for slightly non-uniform
evolution along the segment are irrelevant: no model would account for the difference
distribution of such a pair as the result of a random fluctuation. Beyond such extreme
outliers, a glance at fig.2 shows a larger scatter of points lying well off the diagonal
?cloud?, suggesting that many more sequences are potential apparent recombinants, with
less significant p-values.
1.6 10

18 ? /

We performed a systematic search for ?recombinant? pairs. The sequences that show
strong apparent evidence of homologous recombination events are listed in Appendix 2.
With a very conservative p-value cutoff, we found more than 40 such isolates, again
mostly sequences from avian and swine hosts, with many recent H5N1 isolates from
Asia. There is a highly significant overlap between the sequences showing evidence of
apparent homologous recombination listed in Appendix 2, and the sequences showing
evidence of apparent ?frozen evolution? listed in Appendix 1. Similarly to the ?frozen?
viruses, isolates often have apparent evidence of homologous recombination in more than
one segment, and the overall incidence of recombinant pairs is consistent across the 8

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segments, allowing for greater statistical power to resolve such pairs in the longer
segments.

Discussion.

There have been some historically reported cases of ?frozen evolution? in
the Influenza virus. The most famous of these involves the reemergence in 1977 of H1N1
in the human population after an absence of 20 years. The viruses isolated in the former
USSR and China in 1977 were virtually identical in their nucleotide sequence to H1N1
viruses from 1949-1950. (We readily detected all previously known ?frozen viruses? in
our search, but omitted them from the list in Appendix 1). In this case, it is believed that
the term ?frozen? applies literally: these viruses were probably stored in a lab for 27
years, and then reintroduced into the population in a vaccination experiment gone wrong.
There are additional examples involving the common lab strains PR/34 and WSN that
appeared to reemerge unchanged in humans and camels in Mongolia in the 1980?s, and
pigs in South Korea in 2004, respectively. These cases are believed to be explained by
escaped vaccines in the former case, and stock contamination in the lab in the latter.

3,4
5

What can account for the many ?frozen? sequences reported here? One possibility is that
some interesting biological mechanism is at work. For example, it is possible that the
?frozen? viruses are mutating at a slower rate, perhaps because of a more faithful
polymerase. To examine this possibility, we searched for amino acid mutations in the
polymerase genes (and other genes) common to the ?frozen? viruses, but were unable to
find any such mutations (data not shown). Given the lack of error correction for RNA to
RNA polymerase, a mutation that dramatically reduces replication errors does not appear

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plausible. Another possibility is that these viruses have a much lower rate of replication,
perhaps because they persist without replicating within host cells, or even in the outside
environment; but there is no known latency mechanism for RNA viruses, and the very
long times (often decades) elapsed between isolation of nearly identical viruses make this
kind of mechanism seem somewhat unlikely . A recent article argues against the
likelihood of Influenza persistence in the outside environment, such as environmental
ice

19 . It is important to note that the notion of ?evolutionary stasis? which may or may
not hold for Influenza in certain hosts, is not relevant to these results: even viruses that
are ?static? at the amino acid level are expected to have normal rates of drift in
synonymous third codon nucleotide positions.

8

/16

We speculate that perhaps the most likely explanation for both anomalies reported here is
stock contamination in the sequencing laboratories (or wherever the viruses are stored). If
the virus stock containing virus A is contaminated with virus B, an experiment
supposedly sequencing A is actually sequencing B, thus resulting in apparent near
sequence identity between A and B; if A and B are separated by many years, this will
appear as an anomalously low evolutionary rate. If A and B are mixed in the stock, the
reverse transcriptase reaction used during sequencing could jump between an A and B
template, resulting in an apparent homologous recombinant. This possibility is consistent
with the fact that there is a very significant overlap between the sequences exhibiting
apparent slow evolution and those exhibiting apparent homologous recombination; this
overlap would be very difficult to explain on biological grounds, but is natural if stock
contamination has occurred. Also, there is a relative prevalence of old viruses (isolated

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before 1990), and viruses sequenced by laboratories in Asia and especially China, among
the anomalous sequences; it is tempting to speculate that such differences could reflect
differences in laboratory protocols. Along the same lines, nearly all anomalous sequences
come from avian and swine hosts; it seems natural to assume that viruses from human
hosts are generally handled with greater care (because of the potential public health
hazards resulting from their spread) and are thus less susceptible to stock contamination.
If stock contamination is indeed to blame for these anomalies, the results reported here
could represent just the tip of the iceberg. This is because we would detect the
contamination of viruses A and B only when A and B are sufficiently distant from each
other in time of isolation (resulting in a ?frozen? virus) and/or nucleotide sequence
(possibly resulting in a ?recombinant?). It is natural to assume, however, that most
contamination events in fact occur between viruses that are relatively close to each other
in both time and sequence, resulting in a reported sequence that is wrong, but not wrong
enough to be detectable by the present methods; this could perhaps account for the ?offdiagonal
clouds? seen in fig. 2. Thus, the present results suggest that an unknown, and
possibly quite nontrivial percentage of the data in the flu sequence database might be
compromised, and it is our hope that some steps will be taken by the Influenza
community to address the issue of quality control in the database. One simple, though
certainly insufficient, suggestion would be to regularly resequence the viruses; we would
predict that in most cases involving apparent ?recombinants?, a new sequencing assay
would result in a different sequence, since it seems unlikely that the reverse transcriptase
jumps would occur in the same positions as before. Aside from such detection steps,

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more should be done in the laboratories to prevent contamination from occurring in the
first place. If the rapid growth in the Influenza genome database can be accompanied by
addressing these apparent quality control issues, the Influenza research community will
truly be in the possession of an invaluable resource.

References and Notes

1. See, for instance:

http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html

http://www.flu.lanl.gov

http://influenza.genomics.org.cn

2. There are many references analyzing Influenza evolutionary rates in different
segments and different hosts. See, among others,
-

Parvin JD, Moscona A, Pan WT, Leider JM and Palese P. 1986.
Measurement of the mutation rates of animal viruses: influenza A virus
and poliovirus type 1. J. Virol. 59:377-83
- Fitch WM. 1996. The variety of human virus evolution. Mol. Phylogenet.
Evol. 5:247-58. Review.
- Fitch WM, Leiter JM, Li XQ, and Palese P. 1991. Positive Darwinian
evolution in human influenza A viruses. Proc. Natl. Acad. Sci. 88:4270-4.
- Gorman OT, Bean WJ and Webster RG. 1992. Evolutionary processes
in influenza viruses: divergence, rapid evolution and stasis. Curr. Top.
Microbiol. Immunol. 176:75-97

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-

Lindstrom S, Endo A, Sugita S, Pecoraro M, Hiromoto Y, Kamada M,
Takahashi T and Nerome K. 1998. Phylogenetic analyses of the matrix
and non-structural genes of equine influenza viruses. Arch. Virol.

143

:1585-98.
- Nelson MI, Simonsen L, Viboud C, Miller MA, Taylor J, George KS,
Griesemer SB, Ghedi E, Sengamalay NA, Spiro DJ, Volkov I, Grenfell
BT, Lipman DJ, Taubenberger JK and Homes EC. 2006. Stochastic
processes are key determinants of short-term evolution in influenza A
viruses. PLOS Pathog. 2:e125
- Chen R and Holmes EC. 2006. Avian influenza virus exhibits rapid
evolutionaty dynamics/ Mol. Biol. Evol. 23:2336-41.
3. Yamnikova SS, Mandler J, Bekh-Ochir ZH, Dachtzeren P, Ludwig S, Lvov
DK and Scholtissek C. 1993. A reassortant H1N1 influenza A virus caused fatal
epizootics among camels in Mongolia. Virology. 197:558-63.
4. Anschlan D, Ludwig S, Nymadawa P, Mendsaikhan J and Scholtissek C.

1996. Previous H1N1 influenza A viruses circulating in the Mongolian
population. Arch. Virol.

141:1553-69.
5. Seo SH, Kim JA, Jo SK. 2004. Reassorted swine H9N2 influenza virus in Korea.
Unpublished.

Enserink M.

2005. Infectious diseases. Experts dismiss pig flu scare as nonsense.
Science. 307:1392.

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6.

Bikour MH, Frost EH, Deslandes S, Talbot B and Elazhary Y. 1995.
Persistence of a 1930 swine influenza A (H1N1) virus in Quebec. J. Gen. Virol.

76

:2539-47.
7. Bikour MH, Frost EH, Deslandes S, Talbot B, Weber JM and Elazhary Y.

1995. Recent H3N2 swine influenza virus with haemagglutinin and nucleoprotein
genes similar to 1975 human strains. J. Gen. Virol.

76:697-703.
8. Urabe M, Tanaka T, Odagari T, Tashiro M and Tobita K. 1993. Persistence
of viral genes in a variant of MDBK cell after productive replication of a mutant
of influenza virus A/WSN. Arch. Virol. 128:97-110.
9. Urabe M, Tanaka T and Tobita K. 1993. MDBK cells which survived infection
with a mutant of influenza virus A/WSN and subsequently received many
passages contained viral M and NS genes in full length in the absence of virus
production. Arch. Virol. 130:457-62.
10. Tobita K, Tanaka T and Hayase Y. 1997. Spontaneous excretion of virus from
MDCK cells persistently infected with influenza virus A/PR/8/34. J. Gen. Virol.

78

:563-6.
11. Park CH, Matsuda K, Sunden Y, Ninomiya A, Takada A, Ito H, Kimura T,
Ochiai K, Kida H and Umemura T. 2003. Persistence of viral RNA segments in
the central nervous system of mice after recovery from acute influenza A virus
infection. Vet. Microbiol. 97:259-68.
12. Tobita K, Tanaka T and Hayase Y. 1997. Rescue of a viral gene from VERO
cells latently infected with influenza virus B/Lee/40. Virology. 236:130-6.

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13.

Marschall M, Helten A, Hechtfischer A, Zach A and Meier-Ewert H. 1998.
Persistent infection with an influenza C virus variant is dominantly established in
the presence of the parental wild-type virus. Virus. Res. 54:51-8.
14. Marschall M, Helten A, Hechtfischer A, Zach A, Banaschwski C, Hell W and
Meier-Ewert H. 1999. The ORF, regulated synthesis, and persistence-specific
variation of influenza C viral NS1 protein. Virology. 253:208-18.
15. Zach A, Marschall M and Meier-Ewert H. 1999. Influenza C virus persistence
depends on exceptional steps in viral RNA synthesis and transport. Arch. Virol.

144

:463-78.
16. Wang M and Webster RG. 1990. Lack of persistence of influenza virus genetic
information in ducks. Arch. Virol. 111:263-7.
17. Clare ER, Gould EA and Holmes EC. 2003. Phylogenetic analysis reveals a
low rate of homologous recombination in negative-sense RNA viruses. J. Gen.
Virol. 84:2691-703.
18. Boni MF, Zhou Y, Taubenberger JK and Holmes EC. 2008. Homologous
recombination is very rare or absent in human influenza A virus. J. Virol. Epub
ahead of print.
19. Worobey M. Phylogenetic evidence against evolutionary stasis and natural
abiotic reservoirs of influenza A virus. 2008. J. Virol. 82:3769-74.

Acknowledgements
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We thank Suzanne Christen and the whole group at the Simons Center for Systems
Biology for their comments and suggestions. This work was supported by the Simons
Foundation, the Leon Levy Foundation and the Martin and Helen Chooljian Foundation.
Raul Rabadan wishes to thank Fernando R. and Felicitas R. for their encouragement and
support.

Figure Legends

Fig. 1. Hamming distance vs. distance in years for PB2 segments from the avian
database. Each red plus represents a pair of different PB2 sequences of flu viruses
isolated from avian hosts. The x coordinate gives the difference in years between the
times of isolation of the two viruses, and the y coordinate the Hamming distance between
their sequences (number of nucleotide differences divided by the length of the segment).
The green dashed line represents a Jukes-Cantor fit for the expected Hamming distance.
An apparent slowly evolving pair is shown.
Fig. 2. Number of differences in nucleotides 1156-2310 vs. number of differences in
nucleotides 1-1355 for PB2 segments from the avian database. Each red plus represents a
pair of different PB2 viruses isolated from avian hosts. An apparent recombinant pair is
shown.

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**gsgs** · June 25, 2008, 10:42 AM

Re: genbank anomalies

> We speculate that perhaps the most likely explanation for both
> anomalies reported here is stock contamination in the
> sequencing laboratories (or wherever the viruses are stored).

many examples of slow evolution Hongkong 1975 - China 2004 here:

low mutation rates in flu-viruses - FluTrackers News and Information

http://www.flutrackers.com/forum/showthread.php?t=52214

that doesn't look like "freezing" ?!?

-------edit----------

I'm eager to compare their lists with mine here:

http://www.setbb.com/fluwiki2/viewtopic.php?t=187&mforum=fluwiki2

http://www.setbb.com/fluwiki2/viewtopic.php?t=243&mforum=fluwiki2

(away from computer now until 10PM GMT)

**HenryN** · June 25, 2008, 11:52 AM

Re: genbank anomalies

I didn't see appendix 1 or 2, but the paper really doesn't address the compelling examples, such as the Canadian swine (which has sequences from isolates that have never been in the Wisconsin sequencing lab)

303 See Other

http://precedings.nature.com/documents/385/version/1

human HA sequences from Korea, which were confirmed by the CDC in Atlanta or lab in Japan,

http://www.recombinomics.com/phylo/H3N2_HA_Recombinants.html

or concurrent acquisition of G743A

303 See Other

http://precedings.nature.com/documents/459/version/4

**gsgs** · June 25, 2008, 04:46 PM

Re: genbank anomalies

I think, they are wrong.

Homologuous recombination and preservation do both happen in nature.
Although, maybe not in humans (except maybe 1977) and
probably rarely.

See here for a complete statistics from genbank 1.1.2008:

http://www.setbb.com/fluwiki2/viewtopic.php?t=387&mforum=fluwiki2

it's not just a few obvious examples but significantly more on
the "likely" and "probably" and "possibly" level too, so to
use popular words.

The best example for preservation is the recent Hungary/Suffolk virus,
see the picture in the Rostov-thread.
I mailed about this to DEFRA - no reply.
This was maybe not a natural preservation but
one which happened in poultry industry freezing.
However, it should be examined, they should comment.

**gsgs** · June 25, 2008, 04:51 PM

Re: genbank anomalies

the 1977 reoccurrance of human H1N1 is still being debated.

Here they doubt it could have been from a Chinese freezer:

Laver

http://virologyhistory.wustl.edu/Laver.htm

We did however make contact with the virologist Chu Chi Ming (his obituary is in Virology , 255, 1, 1999). And we visited Anshan in northern China where the first case of Russian Flu (H1N1) was isolated on May 4, 1977. We saw for ourselves how primitive Chinese labs had become during the dark days of the Cultural Revolution; no deep freezers, no equipment for storing live viruses for long periods of time. The usual explanation for the re-appearance in 1977 of a virus which was identical to one which was around in 1950 is that the Chinese were experimenting with a live H1N1 vaccine and the virus got away out into the general population. There has never been any evidence for this. I don't believe any live virus could have survived in a Chinese lab during the 27 years since 1950. Furthermore, for what it is worth, Chu Chi Ming was adamant that, in 1977, no work was being done in China with H1N1 viruses. So where this virus had been hiding, undetected and unchanged, for 27 years and what caused its re-emergence is a complete mystery.

**HenryN** · June 25, 2008, 05:51 PM

Re: genbank anomalies

Originally posted by gsgs View Post

I think, they are wrong.

Homologuous recombination and preservation do both happen in nature.
Although, maybe not in humans (except maybe 1977) and
probably rarely.

See here for a complete statistics from genbank 1.1.2008:

http://www.setbb.com/fluwiki2/viewtopic.php?t=387&mforum=fluwiki2

it's not just a few obvious examples but significantly more on
the "likely" and "probably" and "possibly" level too, so to
use popular words.

The best example for preservation is the recent Hungary/Suffolk virus,
see the picture in the Rostov-thread.
I mailed about this to DEFRA - no reply.
This was maybe not a natural preservation but
one which happened in poultry industry freezing.
However, it should be examined, they should comment.

I was waiting for appendix 1 and 2 to see what examples they came up with, but this paper is just a lot of hand waving. The take a couple of individual sequence pairs and same they are artifact base on zero data, and then say the additional examples are also artifact.

Since this paper does limit the database, they are stuck with MANY avian and swine examples, which they address with a few hadwaves citing lab error, but the human Korean sequences were CONFIRMED by two independent labs and the Canadian swine data would require dozens of selective errors, including sequences from isolates that have never been in the sequencing lab.

Paper is well into the hocus pocus category (but they do realize if the acknowledge ANY homologous rercombination, the house of cards callapses and the only two real explanations for the MANY examples are recombination or massive lab artifacts).

**Oracle** · June 25, 2008, 06:20 PM

Re: genbank anomalies

New antigenic variants of influenza A(H1N1) virus detected in the USSR in 1979. [Article in Russian] Iakhno MA, Kendal A, Zakstel'skaia LIa, Molibog EV, Shenderovich SF. Vopr Virusol. 1981 Mar-Apr;(2):136-41.

Studies of influenza A (H1N1) viruses isolated in the spring of 1979 in the USSR showed all the 73 strains to belong to influenza A (H1N1) virus but to be heterogeneous. Apart from the strains identical with the reference A/USSR/90/77 and A/Brazil/11/78 as well as intermediate ones, 14 strains were identified and found to be new drift variants. A composite analysis of representative strains of this group (A/USSR/50/79 and A/USSR/61/79) by HI test with diagnostic rat and ferret sera as well as monoclonal antibody and by immunoadsorption method confirmed their individual natures and showed them not to be identical completely to any one of the previously known drift variants of the A (H1N1) subtype. The neuraminidase of the new strains also acquired some changes in the antigenic composition and resistance to detergents destroying disulfide bonds indicating certain alterations in this subunit as well. Besides, a comparison of the drift variants of influenza A (H1N1) virus variants isolated in 1947-1957 and in 1977-1979 showed the drift of the new A (H1N1) strain to occur in a different way than in those isolated in 1947-1957.

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Reversion in the natural variability of influenza A virus. [Article in Russian]
Iakhno MA, Isachenko VA, Molibog EV, Iamnikova SS, Vorkunova GK. Vopr Virusol. 1978 Mar-Apr;(2):146-51.

In mid-November, 1977, local outbreaks of acute respiratory diseases (ARD) in institutionalized communities began to be recorded in a number of geographical zones of the USSR, and by the end of the month a general increase in the incidence was observed in some areas of the country. The epidemic outbreaks extended gradually and were characterized by moderate development involving mainly young subjects. The strains causing the epidemic had no antigenic relationship with reference A (H3N2) virus and the H1 test were neutralized with antisera to influenza A virus with the antigenic formula H1N1 to 1 1/4 titer. Their neuraminidase was inhibited by the antiserum to the recombinants containing neuraminidase of the subtype N1, especially A/New Jersey 8/76. These results permit to classify the strains causing the epidemic outbreaks in November--December, 1977, as influenza A virus with the antigenic formula H1N1.

What is important about A/New Jersey 8/76?

The great Swine Flu Debacle of 1976, with exactly one death, at Fort Dix, NJ. This is the isolate used for manufacture of influenza vaccine.

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Previous H1N1 influenza A viruses circulating in the Mongolian population.
Anchlan D, Ludwig S, Nymadawa P, Mendsaikhan J, Scholtissek C. Arch Virol. 1996;141(8):1553-69.
Four influenza A viruses of the subtype H1N1, isolated from Mongolian patients in Ulaanbaatar between 1985 and 1991, were analysed by sequencing of various RNA segments. The isolate from 1985 was found to be highly related in all genes sequenced to strains isolated from camels in the same region and at about the same time. These camel isolates were presumably derived from a UV-light inactivated reassortant vaccine (PR8 x USSR/77) prepared in Leningrad in 1978 and used in the Mongolian population at that time [19]. The human isolate from 1988 was also found to be a derivative of a reassortant between PR8 and USSR/77; in contrast to the 1985 isolate, however, it contained an HA closely related to PR8. One of the Mongolian isolates from 1991 (111/91) was in all genes sequenced closely related to PR8, while the other isolate from 1991 (162/91) was closely related to H1N1 strains isolated around 1986 in other parts of the world. About 12% of 235 convalescent sera collected in various parts of Mongolia contained antibodies against PR8, while none of German control sera contained such antibodies. The mutational and evolutionary rates of the Mongolian strains seem to be significantly lower when compared to the rates of human influenza A strains isolated in other parts of the world. This might indicate that these rates depend to a certain extent on the population density. Thus, viruses from remote areas might keep the potential to reappear in the human population after several years to cause a pandemic as it had happened in 1977.

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Persistence of the genes of epidemic influenza viruses (H1N1) in natural populations. [Article in Russian] L'vov DK, Iamnikova SS, Shemiakin IG, Agafonova LV, Miasnikova IA. Vopr Virusol. 1982 Jul-Aug;27(4):401-5.

A comparative study of antigenic determinants and genome of the analogues of epidemic H1N1 variants isolated from various animal species and from man in the USSR and Mongolia in 1978--1980 was carried out. The analysis of the antigenic determinants of hemagglutinins of the viruses isolated in this period from man, domestic and wild birds and animals performed with a set of monoclonal antibodies to the epidemic reference A/Brazil/11/78 virus revealed their close similarity in the structure of the antigenic sites. Study of neuraminidase of these viruses with a set of monoclonal antibodies to the A/Denver/1/57 strain showed their relationship with neuraminidase of the viruses isolated in 1957 and revealed its trend for changes in individual antigenic determinants. The analysis of genomes of the viruses isolated in Mongolia from man and animals by the methods of RNA polyacrylamide gel electrophoresis and RNA--RNA hybridization showed them to be close to each other and to differ from the epidemic A/USSR/90/77 strain. At the same time, the Mongolian isolates showed differences in the electrophoretic mobility of the genome fragments coding for hemagglutinin and neuraminidase synthesis.

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I don't think this is an issue of 'lab freezer escapee'. Probably more like an improperly prepared (UV-inactivated) vaccine that went awry.

Now, the key issue is this: how long do you think you could store an influenza virus isolate, circa early 1930s and have it remain viable, so as to remain free of indication of drift, using 1930s-1950s era technology?

Thirty years?

BS!

**HenryN** · June 26, 2008, 01:58 AM

Re: genbank anomalies

>>One simple, though certainly insufficient, suggestion would be to regularly resequence the viruses; we would predict that in most cases involving apparent “recombinants”, a new sequencing assay would result in a different sequence, since it seems unlikely that the reverse transcriptase jumps would occur in the same positions as before<<

The above suggestion for identifying sequence artifacts in fact was done in the Korean HA sequences from 2002 and the re-sequenced sequences exactly matched the earlier sequences, which were obvious recombinants.

http://www.recombinomics.com/News/06010801/H3N2_Recombination_Confirmed.html

**gsgs** · June 26, 2008, 02:12 AM

Re: genbank anomalies

the polymerase jumped from a 1991-virus to a 2002-virus ?

**gsgs** · June 26, 2008, 02:28 AM

Re: genbank anomalies

Oracle,

A/USSR/50/79 and A/USSR/61/79 are not available. It's not clear, how much the new
strains drifted, I assume they were still descendants of the 1977 virus.

the 1976 virus is swinish and quite different from the 1977 virus.

Mongolia 1991 was not a natural event, but a vaccine.

USA more likely than China had the technology to store viruses for 30 years in 1950.
And they probably did some vaccine experiments in 1976, maybe in swine ...

**HenryN** · June 26, 2008, 02:30 AM

Re: genbank anomalies

Originally posted by gsgs View Post

the polymerase jumped from a 1991-virus to a 2002-virus ?

The authors of the paper assume that the recombinant sequence is created because the sample has a contaminating sequence (that escaped from the freezer). They maintain that when the sequence is amplified via PCR, the polymerase jumps from the new sequence to the "contaminating sequence" generating the recombinant "artifact". They suggest re-sequencing, which they predict will creat a new recombinant based on different cross over points.

However, the samples that were originally sequenced in South Korea, were re-sequenced by the CDC and Japan, several years later. These re-sequences were in the recent Science paper, and the independent resequencing produced the EXACT sequences deposited by South Korea's CDC, showing that the original sequence was NOT and artifact, and the six recombinant H3N2 HA sequences were in fact quite real.

I have e-mailed the three authors to let them know that their paper has some MAJOR problems, and will be doing several commentaries and posting the analysis in Nature Precedings.

This paper is much like the earlier J Virol paper, which also attributed the OBVIOUS homologous recombination to hundreds of lab errors.

Both papers are excellent examples of authors with preconceived notions that prevent them from believing there own analysis.

The flat earth society is alive and well and both papers should be taught in courses on the scientific method which includes examples of resistance to paradigm shifts.

Both of these J Virol papers are well into the "emporer has no clothes" category and signal the final throws of "random mutations".

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