1933 Sequences in 2004 Korean H1N1 Swine Isolates Raise Concern
submited by kickingbird at Dec, 22, 2004 14:45 PM from Recombinomics, Inc
The swine sequences release by GenBank this week would appear to be cause for concern. Six of the isolates were from swine in South Korea and they have both reassorted and recombined genes (between a common Korean avian, H9N2 sequences and genes virtually identical to WSN/33). This is of significant concern because WSN/33 is a neurotropic component of WS/33, the first human influenza virus isolated. WSN/33 was obtained from serial passages of WS/33 in mouse brains in 1940. It is lethal in mice and is H1N1 so it should also readily infect humans. The N in WSN has lost a glycosylation site and binds plasminogen to facilitate HA cleavage.
Two of the swine isolates are H1N1 and they have the same alteration which abolishes the glycosylation site. These two isolates, A/swine/Korea/S10/2004 and A/swine/Korea/S109/2004 have 7 WSN/33. Only PB2 is related to Korean H9N2 isolates. 4 other swine isolates are H9N2 and have 3-5 WSN/33 genes.
It would seem that swine shedding H1N1 virus from 1933 would pose a serious health threat. The 1933 virus has significant homology with the 1918 pandemic strain and was actually isolated for the study of neurotropic disease caused by the 1918 pandemic strain.
It seems that people born after 1933 would have limited immunity to the H1N1 virus isolated from the swine in South Korea .
The Korean sequences came out at the beginning of the week. Here is one of the H1N1s
Submitted (24-OCT-2004) Immunology Laboratory, College of
Veterinary Medicine, Chung Nam National University, Gung-Dong 220,
YuSeong-Gu, Dae Jeon, Chung Nam 305-764, The Republic of Korea
Veterinary Medicine, Chung Nam National University, Gung-Dong 220,
YuSeong-Gu, Dae Jeon, Chung Nam 305-764, The Republic of Korea
I was initially looking at the sequences for more recombination (which is present) and I was struck by the fact that the sequences were virtually identical to a 1933 isolate (showing that the virus does NOT change frequently via copying errors).
Since the human component was from a virus that is grown in various labs, I looked more closely at all 7 viruses from Korea, 6 of them contain the human (WSN/33) sequences, but in different combinations. However, the other component seems to be the same or similar in all (a H9N2 sequence closely related to avian sequences isolated in Korea, including one from 2003). Thus, the various isolates could be from the same two parents (WSN/33 which is H1N1 and an avian H9N2 from Korea), but with different combinations (2 have 7 WSN genes while the others have 3, 4 , or 5).
Because all have the same two parents, they could represent some sort of escape from a lab. However, it seems unlikely that they represent a planned experiment because WSN/33 is quite dangerous and combining it with H9N2 could unleash a pandemic strain that could infect birds, humans, and pigs.
Since two of the isolates are H1N1 (the other 4 are H9N2), they should have no problem infecting humans, and since there is no evidence for these viruses circulating in the wild, it would seem that most people would have limited immunity. Moreover, WSN was isolated in 1940 to study the neurotropic effects caused by the 1918 pandemic strain and I believe that WSN/33 is closely related to one of the parental strains that generated the 1918 pandemic strain (via recombination).
The bottom line however, is the fact that H1N1 from 1933 is in 2004 Korean pigs and who knows where else, and such a virus should be easily transmitted human to human (and the infected humans may have limited immunity because the virus is so old and seems to have been out of circulation in the general population - it may have been tucked away in South Korea fro the past 60-70 years).
For those looking for more background on WSN/33, here is a comment on a paper on the sequestering of plasminogen
As the likelihood of WSN/33 artifact diminishes, the possibility of bioterrorism and/or bioweapons reasearch increases.
The WSN/33 sequences are in several isolates, all of which are closely related to recent avian isolates from South Korea. Mixing human WSN/33 sequences with avian sequences would be a very dangerous experiment, as would putting the sequences into pigs. There is already quite a bit of recombination and reassortment.
It is much easier to imagine such combinations tracing to a military facility than a civilian one. It will be interesting to see how this sorts out, but is becoming increasingly deserving of serious investigation.
"One of the unusual properties of WSN/33 is its ability to undergo HA cleavage activation in tissue culture without the addition of exogenous trypsin. As early as 1973, Lazarowitz, et al. (2) were able to explain this observation with the finding that WSN/33 had its HA cleaved by serum plasmin. They demonstrated that, in the absence of serum or in plasminogen-free serum, no HA cleavage occurred. Schulman and Palese (3) showed that the NA protein of WSN/33 was a necessary component for HA cleavage, and in 1993, Li et al. (4) found that the NA of WSN/33 lacked a crucial glycosylation site at residue 146 (residue 130 for WSN/33, which has an in-frame 48 base deletion). Restoration of this glycosylation site by reverse genetics yielded a mutant WSN/33 virus that could not undergo replication in tissue culture without exogenous trypsin. What the above papers lacked was a coherent model to synthesize these observations. The current study describes a model whereby plasminogen binds specifically to the NA of WSN/33. In so doing, the WSN/33 NA sequesters plasminogen on the cell surface so that it can be activated. Once activated, plasmin, also a serine protease, recognizes the single arginine motif at the cleavage site and cleaves HA0 into HA1 and HA2. "
The H1N1 isolates from the 2004 swine in Korea lack the same crucial glycosylation site as WSN/33 described above.
See also
"Neither the 1918 HA genes nor the NA genes possessed mutations that are known to increase tissue tropicity, which accounts for the virulence of other influenza strains such as A/WSN/33....."
Note also that the recent Nature paper showing the 1918 virulence was linked to the HA gene
used WSN/33 as the background virus and WSN/33 with HA from 1918 or without any new genes had similar pathogenicities in mice:
"The combination of Hsp and Nsp in an A/WSN/33 (WSN) (H1N1) genetic background was shown to be pathogenic in mice without prior adaptation of the proteins to growth in these hosts4, 5, usually a prerequisite for producing a lethal infection in mice with a human virus. However, WSN itself is both adapted to and highly pathogenic in mice, so it was not possible to assess the possible contribution that Hsp and Nsp had made to the pathogenicity of the recombinant virus."
Thus,a swine virus with 7 of 8 genes from WSN/33 is a serious situation. At a minimum, it would seem that a warning about possible virulent H1N1 infections would be in order.
To give a little better feel for the differences between the H1N1 swine sequences and the various relevant H1N1 sequences (WSN/33, WS/33, New Caledonia (isolate used in current human flu vaccine), I did a quick count. For H1 the number of amino acid differences between the two swine isolates, WSN/33, WS/33, and New Caledonia are 2, 5, 23, 60 (out of 290 amino acids). The numbers for N1 are 1, 2, 10, 45 out of 364 amino acids (the 16 additional amino acids in New Caledonia were not counted - the other 4 sequences all have a 16 amino acid deletion relative to the human N1 currently in circulation).
Thus, the two H1N1 swine isolates are very closely related to each other and WSN/33 (which was actually isolated in 1940, although it came from the 1933 mixture used to infect mouse brains in 1940). There are a fair number of differences between these three isolates and WS/33 and a very large number of differences between the swine isolates and the human H1 currently in circulation.
Thus, most people born after 1933 would have very little immunity to the two swine isolates, although both would readily grow in humans and be easily transmitted human to human because 7 of their 8 genes, including H1 and N1, are virtually identical to WSN/33.
The lab isolating the swine sequences has not knowingly had WSN/33 anywhere in the lab. The swine isolates were isolated in chicken eggs and the MDCK cells in the lab are PCR negative for WSN/33.
Further analysis of the H9N2 sequences indicates that there are at least 3 distinct H9N2 sequences that have recombined and/or reassorted with the WSN/33 sequences, Each set of WSN/33 sequences also has unique polymorphisms which follow well defined rules of influenza virus evolution and emergence.
The likelihood of these WSN/33 sequences being a lab artifact is very close to zero. The path from a lab to at least 6 swine on several farms in South Korea is still unclear, although all of the H9N2 sequences from WSN/33 positive isolates are closely related to each other, as well as recent H9N2 chicken and swine isolates from South Korea.
Further analysis of the H9N2 sequences indicates that there are at least 3 distinct H9N2 sequences that have recombined and/or reassorted with the WSN/33 sequences, Each set of WSN/33 sequences also has unique polymorphisms which follow well defined rules of influenza virus evolution and emergence.
The likelihood of these WSN/33 sequences being a lab artifact is very close to zero. The path from a lab to at least 6 swine on several farms in South Korea is still unclear, although all of the H9N2 sequences from WSN/33 positive isolates are closely related to each other, as well as recent H9N2 chicken and swine isolates from South Korea.
A new H5N1 HA sequence from Macheng appeared at GenBank
The 2004 isolate has many of the polymorphisms that appeared in 2003 H5N1 isolates in Hong Kong.
New season, new sequences, new recombinations, new problems.
The significance of WSN/33 in 2004 Korean swine might be best demonstrated on page 70 of the January 2005 edition of Scientific American. The article on the "Mystery of 1918" begins on page 64 and gives some detail on the pandemic as well as efforts that lead to the sequences acquired from fixed tissue samples from 1918 victims.
Attempts are made to explain the 1918 data using reassortment, which of course ends in hand waving about unknown viruses emerging from unknown species, but the real story is seen on page 70 which has a "flu family tree". Just below the 1918 isolates is "Iowa 1930" which is A/swine/Iowa/15/30(H1N1), a classical H1N1 swine flu virus isolated in Iowa in 1930. Just above the 1918 isolates is "Wilson Smith N 1933", which is A/WSN/33(H1N1). The 1918 pandemic strains were formed by recombination between swine sequences like "Iowa 1930" and human sequences like "Wilson Smith N 1933".
Attempts are made to explain the 1918 data using reassortment, which of course ends in hand waving about unknown viruses emerging from unknown species, but the real story is seen on page 70 which has a "flu family tree". Just below the 1918 isolates is "Iowa 1930" which is A/swine/Iowa/15/30(H1N1), a classical H1N1 swine flu virus isolated in Iowa in 1930. Just above the 1918 isolates is "Wilson Smith N 1933", which is A/WSN/33(H1N1). The 1918 pandemic strains were formed by recombination between swine sequences like "Iowa 1930" and human sequences like "Wilson Smith N 1933".
The human sequences, Wilson Smith N 1933 are WSN/33, the sequences in Korean swine in 2004.
As the likelihood of WSN/33 artifact diminishes, the possibility of bioterrorism and/or bioweapons reasearch increases.
The WSN/33 sequences are in several isolates, all of which are closely related to recent avian isolates from South Korea. Mixing human WSN/33 sequences with avian sequences would be a very dangerous experiment, as would putting the sequences into pigs. There is already quite a bit of recombination and reassortment.
It is much easier to imagine such combinations tracing to a military facility than a civilian one. It will be interesting to see how this sorts out, but is becoming increasingly deserving of serious investigation.
Here is a chronology of events. There were 7 swine sequences deposited at GenBank on Oct 24 that were publicly available around the end of November.
I made my first Recombinomics´ What´s New post, Flu Bioterrorism? on Dec 4. I notified WHO on Dec 6 of my concerns that two of the isolates, A/swine/Korea/S10/2004(H1N1) and A/swine/Korea/S175/2004(H1N1), were H1N1, had 7 human genes from WSN/33 , and there was considerable recombination and reassortment among the six isolates with human genes.
I subsequently sent analysis, which included a number of reasons why the sequences appeared to be real and not artifacts. I initially contacted the Seos´ lab in Korea on Dec 7. I believe FAO began confirming sequences on Dec 13.
Interest in WSN/33 appeared to significantly increase in Japan on Dec 15 when the Japanese version of the wired.com story This Genome is Fowl appeared. This interest in Japan was quickly followed by interest in Korea and Geneva.
I made my first Recombinomics´ What´s New post, Flu Bioterrorism? on Dec 4. I notified WHO on Dec 6 of my concerns that two of the isolates, A/swine/Korea/S10/2004(H1N1) and A/swine/Korea/S175/2004(H1N1), were H1N1, had 7 human genes from WSN/33 , and there was considerable recombination and reassortment among the six isolates with human genes.
I subsequently sent analysis, which included a number of reasons why the sequences appeared to be real and not artifacts. I initially contacted the Seos´ lab in Korea on Dec 7. I believe FAO began confirming sequences on Dec 13.
Interest in WSN/33 appeared to significantly increase in Japan on Dec 15 when the Japanese version of the wired.com story This Genome is Fowl appeared. This interest in Japan was quickly followed by interest in Korea and Geneva.
WSN/33 Diversity in Korean Swine
Further analysis of the WSN/33 isolates in Korean swine has indicated that the human sequences are probably widespread. In addition to the two H1N1 isolates, there are 4 H9N2 isolates with WSN/33 sequences.
However, each of the H9N2 swine isolates is unique. One, A/swine/Korea/S109/2004 (S109), has WSN/33 sequences in all six internal genes, although the 3´ end of PB2 is avian. Another, A.swine/Korea/S83/2004 (S83) has WSN/33 sequences in only 3 internal genes. However, the two isolates with WSN/33 in 4 genes, A/swine/Korea/S81/2004 (S81) and A/swine/Korea/S190/2004 (S190), have very distinct NA genes.
There are at least two distinct avian H9N2 strains with WSN/33 sequences. Both are related to each other and earlier Korean H9N2 isolates. One is represented in the H9N2 swine isolate without WSN/3 sequences A/swine/Korea/S452/2004 (S452), which is very similar to the chicken isolate from last year, A/chicken/Korea/S1/2003 (S1). The other group may be more like the two chicken sequences on deposit from this year, A/chicken/Korea/S20/2004 (S20) and A/chicken/Korea/S22/2004 (S22).
The 4 swine isolates with WSN/33 sequences have at least 3 distinct NA genes. S81 has an NA like last year´s chicken sequence, S83 is like this years chicken sequences, and S109 and S190 have an NA that is a recombinant between the two chicken sequences.
Thus, all 4 H9N2 swine isolates with WSN/33 sequences are unique, indicating that there is considerable diversity in the swine WSN/33 isolates and that the infections are quite widespread and quite real.
However, each of the H9N2 swine isolates is unique. One, A/swine/Korea/S109/2004 (S109), has WSN/33 sequences in all six internal genes, although the 3´ end of PB2 is avian. Another, A.swine/Korea/S83/2004 (S83) has WSN/33 sequences in only 3 internal genes. However, the two isolates with WSN/33 in 4 genes, A/swine/Korea/S81/2004 (S81) and A/swine/Korea/S190/2004 (S190), have very distinct NA genes.
There are at least two distinct avian H9N2 strains with WSN/33 sequences. Both are related to each other and earlier Korean H9N2 isolates. One is represented in the H9N2 swine isolate without WSN/3 sequences A/swine/Korea/S452/2004 (S452), which is very similar to the chicken isolate from last year, A/chicken/Korea/S1/2003 (S1). The other group may be more like the two chicken sequences on deposit from this year, A/chicken/Korea/S20/2004 (S20) and A/chicken/Korea/S22/2004 (S22).
The 4 swine isolates with WSN/33 sequences have at least 3 distinct NA genes. S81 has an NA like last year´s chicken sequence, S83 is like this years chicken sequences, and S109 and S190 have an NA that is a recombinant between the two chicken sequences.
Thus, all 4 H9N2 swine isolates with WSN/33 sequences are unique, indicating that there is considerable diversity in the swine WSN/33 isolates and that the infections are quite widespread and quite real.
- USCDC: Avian Influenza A(H5N1) U.S. Situation Update and CDC Activities 18 hours ago
- USCDC: Urgent field correction notice 1 days ago
- Joint FAO/WHO/WOAH preliminary assessment of recent influenza A(H5N1) viruses 2 days ago
- Primers and probes for H5 and N1 qRT-PCR detection [CNIC] 3 days ago
- USDA: Actions to Protect Livestock Health From Highly Pathogenic H5N1 Avian Influenza 4 days ago
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