Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

I have this in my NP-file :

NP:
p=0.0000000000000042323 1445 5480 l:1064 cut: 850 dif: 246= 151+ 95 A/Christchurch/14/2004(H3N2) A/quail/Shantou/1218/2003(H6N1)

but it involves H6N1.
I'll check later.

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

p=0.0000000000000042323 1445 5480 l:1064 cut: 850 dif: 246= 151+  95    >A/Christchurch/14/04(H3N2)     >A/Qa/Shantou/1218/03(H6N1)
p=0.0000089305815657764 1445  345 l:1496 cut: 300 dif:  51=  24+  27    >A/Christchurch/14/04(H3N2)     >A/HK/498/97(H3N2)
p=0.0000245520022496002 1445  481 l:1001 cut: 750 dif: 205= 130+  75    >A/Christchurch/14/04(H3N2)     >A/Dk/Shantou/2088/01(H9N2)
p=0.0000422254888616796 1445  334 l:1496 cut: 750 dif: 275= 108+ 167    >A/Christchurch/14/04(H3N2)     >A/Sw/Nebraska/209/98(H3N2)

Below is what is currently at Genbank, and I don't see any recombination

LOCUS CY002925 1565 bp ss-RNA linear VRL 15-SEP-2005
DEFINITION Influenza A virus (A/Christchurch/14/2004(H3N2)) segment 5,
complete sequence.
ACCESSION CY002925
VERSION CY002925.1 GI:75750237
KEYWORDS .
SOURCE Influenza A virus (A/Christchurch/14/2004(H3N2))
ORGANISM Influenza A virus (A/Christchurch/14/2004(H3N2))
Viruses; ssRNA negative-strand viruses; Orthomyxoviridae;
Influenzavirus A.
REFERENCE 1 (bases 1 to 1565)
AUTHORS Ghedin,E., Spiro,D., Sengamalay,N., Zaborsky,J., Feldblyum,T.,
Subbu,V., Sparenborg,J., Groveman,L., Halpin,R., Shumway,M.,
Sitz,J., Katzel,D., Koo,H., Salzberg,S.L., Jennings,L., Smit,M.,
Wells,V., Bao,Y., Bolotov,P., Dernovoy,D., Kiryutin,B., Lipman,D.J.
and Tatusova,T.
TITLE The NIAID Influenza Genome Sequencing Project
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1565)
CONSRTM The NIAID Influenza Genome Sequencing Consortium
TITLE Direct Submission
JOURNAL Submitted (15-SEP-2005) on behalf of TIGR/Canterbury Health
Laboratories, NZ/NCBI, National Center for Biotechnology
Information, NIH, Bethesda, MD 20894, USA
FEATURES Location/Qualifiers
source 1..1565
/organism="Influenza A virus
(A/Christchurch/14/2004(H3N2))"
/mol_type="genomic RNA"
/strain="A/Christchurch/14/2004"
/serotype="H3N2"
/isolation_source="gender:M; age:6y"
/specific_host="human"
/db_xref="taxon:345301"
/segment="5"
/lab_host="MDCK2 passage(s)"
/country="New Zealand: Christchurch"
/collection_date="08/07/2004"
gene 46..1542
/gene="NP"
CDS 46..1542
/gene="NP"
/codon_start=1
/product="nucleocapsid protein"
/protein_id="ABA26737.1"
/db_xref="GI:75750238"
/translation="MASQGTKRSYEQMETDGDRQNATEIRASVGKMIDGIGRFYIQMC
TELKLSDHEGRLIQNSLTIEKMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDG
KWMRELVLYDKEEIRRIWRQANNGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMD
PRMCSLMQGSTLPRRSGAAGAAVKGIGTMVMELIRMVKRGINDRNFWRGENGRKTRSA
YERMCNILKGKFQTAAQRAMVDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCL
PACAYGPAVSSGYDFEKEGYSLVGIDPFKLLQNSQIYSLIRPNENPAHKSQLVWMACH
SAAFEDLRLLSFIRGTKVSPRGKLSTRGVQIASNENMDNMGSSTLELRSGYWAIRTRS
GGNTNQQRASAGQTSVQPTFSVQRNLPFEKSTIMAAFTGNTEGRTSDMRAEIIRMMEG
AKPEEVSFRGRGVFELSDEKAANPIVPSFDMSNEGSYFFGDNAEEYDN"
ORIGIN
1 agcaaaagca gggttaataa tcactcactg agtgacatca aaatcatggc gtcccaaggc
61 accaaacggt cttatgaaca gatggaaact gatggggatc gccagaatgc aactgagatt
121 agggcatccg tcgggaagat gattgatgga attgggagat tctacatcca aatgtgcact
181 gaacttaaac tcagtgatca tgaagggcga ttaatccaga acagcttgac aatagagaaa
241 atggtgctct ctgcttttga tgaaagaagg aataaatacc tggaagaaca ccccagcgcg
301 gggaaagatc ccaagaaaac tgggggaccc atatacagga gagtagatgg aaaatggatg
361 agggaactcg tcctttatga caaagaagaa ataaggcgaa tctggcgcca agccaacaat
421 ggtgaggatg cgacagctgg tctaactcac ataatgatct ggcattccaa tttgaatgat
481 gcaacatacc agaggacaag agctcttgtt cgaactggaa tggatcccag aatgtgctct
541 ctgatgcagg gctcgactct ccctagaagg tccggagctg caggtgctgc agtcaaagga
601 atcgggacaa tggtgatgga actgatcaga atggtcaaac gggggatcaa cgatcgaaat
661 ttctggagag gtgagaatgg gcggaaaaca agaagtgctt atgagagaat gtgcaacatt
721 cttaaaggaa aatttcaaac agctgcacaa agagcaatgg tggatcaagt gagagaaagt
781 cggaacccag gaaatgctga gatcgaagat ctcatatttt tggcaagatc tgcattgata
841 ttgagagggt cagttgctca caaatcttgc ctacctgcct gtgcgtatgg acctgcagta
901 tccagtgggt acgacttcga aaaagaggga tattccttgg tgggaataga ccctttcaaa
961 ctacttcaaa atagccaaat atacagccta atcagaccta acgagaatcc agcacacaag
1021 agtcagctgg tgtggatggc atgccattct gctgcatttg aagatttaag attgttaagc
1081 ttcatcagag ggacaaaagt atctcctcgg gggaaactgt caactagagg ggtacaaatt
1141 gcttcaaatg agaacatgga taatatggga tcgagcactc ttgaactgag aagcgggtac
1201 tgggccataa ggaccaggag tggaggaaac actaatcaac agagggcctc cgcaggccaa
1261 accagtgtgc aacctacgtt ttctgtacaa agaaacctcc catttgaaaa gtcaaccatc
1321 atggcagcat tcactggaaa tacggaggga agaacttcag acatgagggc agaaatcata
1381 agaatgatgg aaggtgcaaa accagaagaa gtgtcattcc gggggagggg agttttcgag
1441 ctctcagacg agaaggcagc gaacccgatc gtgccctctt ttgatatgag taatgaagga
1501 tcttatttct tcggagacaa tgcagaagag tacgacaatt aaggaaaaat acccttgttt
1561 ctact

 

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

The OBVIOUS HA sequences from Korea are below (recombination is between 575-1008 of submitted sequences)

A/Cheonnam/323/2002
A/Cheonnam/338/2002
A/Cheonnam/340/2002
A/Kyongbuk/320/2002
A/Daejeon/258/2002
A/Incheon/260/2002

 

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

maybe they didn't check HA

> in five different RNA segments

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

maybe they didn't check HA

> in five different RNA segments

No, they check all eight gene segments, but didn't find anything in HA.

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

TABLE 1. Results of recombination analysis of ten A/H3N2 influenza virus data sets.​

Segment Number
sequences
(distinct)
Alignment
length (nt)
Variable
Sites
3SEQ​

p-​

​

​

value
Number
?recombinant?
Number
?recombinant?
> 100 nt
Phylogenetic
signal for
recombination?​

PB2 1086 (912) 2347 898 10​

​

-3 24 1 Weak
PB1 878 (715) 2341 849 0.49 0 0 No
PA 1365 (1156) 2242 947 2.6 ? 10-6 28 0 No
HA 1365 (1154) 1772 918 1 0 0 No
413 (336) 1711 518 1 0 0 No
NP 1256 (938) 1570 620 2.9 ? 10-6 6 1 Weak
NA 1365 (1059) 1475 754 1.2 ? 10-10 240 0 No
413 (274) 1407 438 1 0 0 No
MP 1250 (682) 1028 366 0.04 1 0 No​

NS 630 (344) 906 318 1 0 0 No

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

what examples in Korea did they miss ?

they were searching for some "mosaic" structure.

Did they consider amino-acid sequences or nucleotides ?

Mosaic just means recombinant (sequences from two seperate sources). They looked at the nucleotide level.

I think they use a phylogentic tree to "confirm" the recombination, so they need the various pieces of genes to be 100 nt to create a tree to "prove" the recombination.

Of course most of teh recombination is between closely related sequences and the recombination is common, so its hard to fit their definition, which really only works for OBVIOUS recombination. i.e. the virus knows what it is doing, but the programmer doesn't.

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

16​

TABLE 2. Results of the recombination analysis of eight A/H1N1 influenza virus data sets.​

Segment Number
sequences
(distinct)
Alignment
length (nt)
Variable
Sites
3SEQ​

p-​

​

​

value
Number
?recombinant?
Number
?recombinant?
> 100 nt
Phylogenetic signal
for recombination?​

PB2 478 (421) 2346 871 1 0 0 No
PB1 478 (399) 2341 870 0.95 0 0 No
PA 478 (380) 2238 776 0.96 0 0 No
HA 482 (440) 1781 804 0.82 0 0 No
NP 478 (332) 1566 544 1 0 0 No
NA 481 (405) 1464 657 10​

​

-5 16 0 No
MP 478 (248) 1027 296 1 0 0 No​

NS 478 (297) 890 336 1 0 0 No

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

maybe they didn't check HA

> in five different RNA segments

I think the Korea sequences are about 1300 BP (out of 1700) so they didn't use them (although the recombination is in the center of the sequence).

This is a good example of the recombination not existing because the submitted sequences don't fit the program, so they don't exist!

It's like no H2H in Pakistan because the sample wasn't collected from the dead brother, and all of the other positives degraded so there is only one confirmed case.

However influenza really doesn't care about the sample integrity, the press releases, the peer reviewed papers, or the programming requirements for the obvious recombination.

This paper was an answer in search of a publication.

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

235​

REFERENCES​

236​

1. Aaskov, J., K. Buzacott, E. Field, K. Lowry, A. Berlioz-Arthaud, and E. C. Holmes.​

237​

2007. Multiple recombinant dengue type 1 viruses in an isolate from a dengue patient. J.​

238​

Gen. Virol. 88:3334-3340.​

239​

2. Aoki, F. Y., G. Boivin, and N. Roberts. 2007. Influenza virus susceptibility and​

240​

resistance to oseltamivir. Antiviral Therapy 12:603-616.​

241​

3. Bergmann, M., A. Garcia-Sastre, and P. Palese. 1992. Transfection-mediated​

242​

recombination of influenza A virus. J.Virol. 66:7576-7580.​

243​

4. Boni, M. F., D. Posada, and M. W. Feldman. 2007. An exact nonparametric method for​

244​

inferring mosaic structure in sequence triplets. Genetics 176:1035-1047.​

245​

5. Both, G. W., M. J. Sleigh, N. J. Cox, and A. P. Kendal. 1983. Antigenic drift in​

246​

influenza virus H3 hemagglutinin from 1968 to 1980: multiple evolutionary pathways and​

247​

sequential amino acid changes at key antigenic sites. J. Virol. 48:52-60.​

248​

6. Bush, R. M., W. M. Fitch, C. A. Bender, and N. J. Cox. 1999. Positive selection on the​

249​

H3 hemagglutinin gene of human influenza virus A. Mol. Biol. Evol. 16:1457-1465.​

250​

7. CDC. 2007. Deaths: Final Data for 2004. National Vital Statistics Reports 55.​

251​

8. Chare, E. R., E. A. Gould, and E. C. Holmes. 2003. Phylogenetic analysis reveals a​

252​

low rate of homologous recombination in negative-sense RNA viruses. J. Gen. Virol.​

253​

84:2691-2703.​

254​

9. Deyde, V. M., X. Xu, R. A. Bright, M. Shaw, C. B. Smith, Y. Zhang, Y. Shu, L. V.​

255​

Gubareva, N. J. Cox, and A. I. Klimov. 2007. Surveillance of resistance to​

256​

adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J.​

257​

Infect. Dis. 196:249-257.​

258​

10. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and​

259 high throughput. Nucleic Acids Res. 32:1792-1797.

11.​

​

Ferguson, N. M., A. P. Galvani, and R. M. Bush. 2003. Ecological 260 and immunological​

261​

determinants of influenza evolution. Nature 422:428-433.​

262​

12. Fitch, W. M., R. M. Bush, C. A. Bender, and N. J. Cox. 1997. Long term trends in the​

263​

evolution of H(3) HA1 human influenza type A. Proc. Nat. Acad. Sci. USA 94:7712-7718.​

264​

13. Gibbs, M. J., J. S. Armstrong, and A. J. Gibbs. 2001. Recombination in the​

265​

Hemagglutinin Gene of the 1918 "Spanish Flu". Science 293:1842.​

266​

14. Holmes, E. C., E. Ghedin, N. Miller, J. Taylor, Y. M. Bao, K. St George, B. T.​

267​

Grenfell, S. L. Salzberg, C. M. Fraser, and D. J. Lipman. 2005. Whole-genome​

268​

analysis of human influenza A virus reveals multiple persistent lineages and​

269​

reassortment among recent H3N2 viruses. PLoS Biol. 3:e300.​

270​

15. Kawaoka, Y., S. Krauss, and R. G. Webster. 1989. Avian-to-human transmission of the​

271​

PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J. Virol. 63:4603-​

272​

4608.​

273​

16. Khatchikian, D., M. Orlich, and R. Rott. 1989. Increased viral pathogenicity after​

274​

insertion of a 28 S ribosomal RNA sequence into the haemagglutinin gene of an​

275​

influenza virus. Nature 340:156-157.​

276​

17. Kilbourne, E. D. 1978. Molecular epidemiology ? influenza as archetype. Harvey​

277​

Lectures 73:225-258.​

278​

18. Nelson, M. I., L. Simonsen, C. Viboud, M. A. Miller, J. Taylor, K. S. George, S. B.​

279​

Griesemer, E. Ghedin, N. A. Sengamalay, D. J. Spiro, I. Volkov, B. T. Grenfell, D. J.​

280​

Lipman, J. K. Taubenberger, and E. C. Holmes. 2006. Stochastic processes are key​

281​

determinants of short-term evolution in influenza A virus. PLoS Pathog. 2:e125.​

282​

19. Niman, H. 2007. Swine Influenza A Evolution via Recombination ? Genetic Drift​

283​

Reservoir, Available from Nature Precedings.​

284 <http://hdl.handle.net/10101/npre.2007.385.1>.

20.​

​

Orlich, M., H. Gottwald, and R. Rott. 1994. Nonhomologous recombination 285 between​

286​

the hemagglutinin gene and the nucleoprotein gene of an influenza virus. Virology​

287​

204:462-5.​

288​

21. Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of DNA​

289​

substitution. Bioinformatics 14:817-818.​

290​

22. Scholtissek, C., W. Rohde, V. Von Hoyningen, and R. Rott. 1978. On the origin of the​

291​

human influenza virus subtypes H2N2 and H3N2. Virology 87:13-20.​

292​

23. Smith, D. J., A. S. Lapedes, J. C. de Jong, T. M. Bestebroer, G. F. Rimmelzwaan, A.​

293​

D. M. E. Osterhaus, and R. A. M. Fouchier. 2004. Mapping the antigenic and genetic​

294​

evolution of influenza virus. Science 305:371-376.​

295​

24. Strimmer, K., K. Forslund, B. Holland, and V. Moulton. 2003. A novel exploratory​

296​

method for visual recombination detection. Genome Biol. 4:R33.​

297​

25. Suarez, D. L., D. A. Senne, J. Banks, I. H. Brown, S. C. Essen, C. W. Lee, R. J.​

298​

Manvell, C. Mathieu-Benson, V. Moreno, and J. C. Pedersen. 2004. Recombination​

299​

resulting in virulence shift in avian influenza outbreak, Chile. Emerg. Infect. Dis. 10:693-​

300​

699.​

301​

26. Swofford, D. L. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other​

302​

methods). Version 4. Sinauer Associates, Sunderland, MA.​

303​

27. Taubenberger, J. K., and D. M. Morens. 2006. 1918 Influenza: the mother of all​

304​

pandemics. Emerg. Infect. Dis. 12:15-22.​

305​

28. Thompson, W. W., D. K. Shay, E. Weintraub, L. Brammer, N. Cox, L. J. Anderson,​

306​

and K. Fukuda. 2003. Mortality associated with influenza and respiratory syncytial virus​

307​

in the United States. JAMA 289:179-186.​

308​

29. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka. 1992.​

309 Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152-179.

30.​

​

Wittmann, T. J., R. Biek, A. Hassanin, P. Rouquet, P. Reed, P. Yaba, 310 X. Pourrut, L.​

311​

A. Real, J.-P. Gonzalez, and E. M. Leroy. 2007. Isolates of Zaire ebolavirus from wild​

312​

apes reveal genetic lineage of recombinants. Proc. Nat. Acad. Sci. USA 104:17123-​

313​

17127.​

314​

31. Worobey, M., A. Rambaut, O. G. Pybus, and D. L. Robertson. 2002. Questioning the​

315​

evidence for genetic recombination in the 1918 "Spanish Flu? Virus. Science 296:211-​

316​

211.​

317​

318

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

147​

RESULTS AND DISCUSSION​

148​

Two of the ten human influenza A/H3N2 virus data sets (PB2 and NP) analyzed here​

149​

contained sequences with statistically significant mosaic structure, as determined by 3SEQ, and​

150​

with putative recombinant sections that were each sufficiently long (>100 nt) that they could be​

151​

re-analyzed by phylogenetic recombination detection methods. Three of the remaining eight​

152​

A/H3N2 data sets (PA, NA, MP) and one of the A/H1N1 data sets (NA) also resulted in 3SEQ p153​

values that revealed a strong signal of mosaicism, but in all these cases the inferred breakpoints​

154​

were either close to the gene segment?s endpoints, or very close to each other, making it​

155​

impossible to infer a credible phylogeny. The remaining five A/H3N2 data sets (PB1, HA,​

156​

HA413, NS, NA413 ? the 413-suffix meaning that it is the HA or NA data set containing 413​

157​

sequences) and seven of the A/H1N1 data sets (PB2, PB1, PA, HA, NP, MP, NS) did not​

158​

contain any statistically significant mosaic signals that survived a Dunn-?id?k correction in​

159​

3SEQ. Recombination analysis results are summarized in Table 1 for A/H3N2 and Table 2 for​

160​

A/H1N1. The two putative recombinant data sets are discussed in more detail below.​

161​

The H3N2 PB2 data set assembled here contained 912 distinct sequences, one of which​

162​

? A/New York/11/2003 ? statistically supported a mosaic structure with both mosaic regions​

163​

longer than 100nt. The two most likely parental sequences, identified as A/Hong Kong/14/1974​

164​

(major parent) and A/New York/424/1999 (minor parent), revealed a strong mosaic signal​

165​

(corrected p = 0.013) in relation to A/New York/11/2003. However, while the phylogenies​

166​

inferred for the minor (positions 202?2189, Figure 1a) and major (positions 1?201 and 2190?​

167​

2347, Figure 1b) segments revealed topological movement of the putative recombinant​

168​

sequence relative to the parental sequences, a general lack of phylogenetic resolution, reflected​

169​

in low levels of bootstrap support (particularly in the major segment), meant that there was​

170 insufficient signal to infer phylogenetic incongruence. Since support for phylogenetic

incongruence is necessarily made up of two components ? the phylogenetic​

​

171 relationship among​

172​

the parents and recombinant on the major and minor trees ? we call the signal ?weak? if one of​

173​

the components receives only low bootstrap support.​

174​

For the NP data set of A/H3N2 viruses, a single sequence ? A/Christchurch/14/2004 ?​

175​

supported a mosaic structure with both candidate recombinant regions longer than 100 nt. The​

176​

candidate parental sequences identified by 3SEQ were A/Beijing/1/1968 as the major parent​

177​

and A/New York/153/1999 as the minor parent (clonality among these three isolates is rejected​

178​

at corrected p = 0.032). The ML tree for the region 98-1454 is presented in Figure 2a while that​

179​

for regions 1-97 and 1455-1570 is shown in Figure 2b. In these phylogenies, the putative​

180​

recombinant sequence was clearly more closely related to a different parent in each sequence​

181​

region. The phylogenies also revealed sequence A/New York/381/2004 as a better candidate​

182​

for the minor parent than A/New York/153/1999; the mosaic signal when assuming A/New​

183​

York/381/2004 as the minor parent in the recombination event was still strong (corrected p =​

184​

0.052). However, as in the PB2 data set, the lack of bootstrap support in the phylogeny inferred​

185​

for the major segment indicates that there is in reality an insufficiently strong signal for​

186​

phylogenetic incongruence to conclude that homologous recombination has occurred.​

187​

For the two candidate recombinants A/New York/11/2003 (PB2) and​

188​

A/Christchurch/14/2004 (NP), it is also puzzling that the parental sequences were sampled 25​

189​

and 31 years apart, respectively. Hence, for one of these recombination events to have​

190​

occurred, a lineage of viruses closely related to an ?archaic? virus (either A/Hong Kong/14/1974​

191​

or A/Beijing/1/1968) must have circulated until at least 1999 and recombined with A/New​

192​

York/424/1999 or A/New York/153/1999. Given the rapid rate of influenza A virus mutation​

193​

through frequent RNA polymerase error, as well as the rapid lineage turnover driven by positive​

194​

selection on the major antigenic proteins (6, 11, 12, 23), this scenario seems extremely unlikely.​

195 Thus, laboratory error, such as template switching during amplification in a mixed or

contaminated sample, is a likely explanation of these apparent homologous​

​

196 recombination​

197​

events.​

198​

In sum, our study has revealed that no sequence of human influenza A virus contains a​

199​

clear signature of phylogenetic incongruence indicative of the action of homologous RNA​

200​

recombination. Given that more than 10,000 distinct sequences were analyzed, this constitutes​

201​

strong evidence that homologous recombination plays only a very minor role, if any, in the​

202​

evolution of human influenza A virus. More generally, the occurrence of phylogenetic​

203​

incongruence does not in itself constitute conclusive evidence for this process. Specifically,​

204​

because our analysis is necessarily based on viral consensus sequences rather than the myriad​

205​

individual viral molecules that characterize any infection, it is equally plausible that the​

206​

?recombinants? detected here in fact represent cases of mixed infection in individual hosts​

207​

followed by the amplification and sequencing of different viral molecules, thereby producing​

208​

laboratory-generated artificial recombinants. Hence, to demonstrate conclusively the​

209​

occurrence of homologous recombination in influenza A virus it will be necessary either to clone​

210​

(or plaque purify) and sequence multiple viral genomes from an individual host and demonstrate​

211​

the presence of the recombinant and both parental genotypes within the sample (1), or to show​

212​

that recombinant sequences form a distinct circulating lineage, with readily identifiable parents,​

213​

that is transmitted among multiple individuals in a population (30).​

214​

Finally, although there were 315 sequences in the data analyzed here that carried a​

215​

strong mosaic signal as identified by 3SEQ, it was impossible to verify the vast majority of these​

216​

as recombinants since the putative recombinant regions were too short to infer a credible​

217​

phylogenetic history. It is therefore possible that homologous recombination, should it occur in​

218​

influenza A virus, more commonly involves the transfer of very short sections of RNA, a process​

219​

that would be undetectable by the majority of other methods devised to detect recombination. If​

220​

homologous recombination of short segments is determined to be a relevant process in​

221 influenza A virus evolution, the basis of our more frequent observation of mosaicism in A/H3N2

viruses compared to A/H1N1 viruses will need to be investigated further. However,​

​

222 by far the​

223​

strongest signal in the influenza A virus sequence data analyzed here is that of strict clonality,​

224​

supporting most models of influenza virus evolution proposed to date.​

225
226​

ACKNOWLEDGEMENTS​

227​

The research undertaken in this study was funded in part by Resources for the Future (MFB),​

228​

NIH/NIGMS grant P50GM071508 (MFB), National Institutes of Health Grant GM28016 (MFB),​

229​

NIH grant number GM080533-01 (ECH), and the Intramural Research Program of the NIH, and​

230​

the NIAID (JKT). We thank John Zollweg and Linda Woodard at the Cornell University Center​

231​

for Advanced Computing for suggesting algorithmic improvements to 3SEQ, as well as two​

232 anonymous reviewers for helpful suggestions

 

[Malcolm]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

We need a translation of the post above into easily understood language.

It would be interesting to have a discussion of the paper's stated rarity of recombination as opposed to the reality of recombination's daily occurrence.

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

Influenza A viruses are a major cause of respiratory disease in humans,​

​

43 responsible for​

44​

36,000 annual deaths in United States alone (7, 28) and occasional widespread pandemics​

45​

associated with much higher levels of mortality and morbidity (27). The viral genome is​

46​

comprised of eight negative-strand RNA segments, with a combined length of ~13.6 kb, that can​

47​

evolve through a variety of mechanisms. Most notably, the lack of a proof-reading mechanism​

48​

during RNA replication results in a high frequency of point mutations which, when combined​

49​

with large population sizes and short generation times, gives influenza A virus the ability to​

50​

generate quickly both antigenic variants that can escape host immunity ? a process termed​

51​

antigenic drift (5, 29) ? as well as genotypes that provide resistance to anti-viral agents such as​

52​

the adamantanes (9) and neuraminidase inhibitors (2). In addition to generating genetic​

53​

diversity by rapid mutation, when multiple viruses co-infect a single cell the eight segments of​

54​

the influenza virus genome can reassort and yield progeny virions with a novel combination of​

55​

segments ? a process termed antigenic shift. Such reassortment is well documented among​

56​

those viral strains that differ in their host species, such as humans and birds. Reassortment of​

57​

this type, involving the acquisition from avian hosts of new polymerase PB1, hemagglutinin​

58​

(HA), and/or neuraminidase (NA) segments to which there was no prior human immunity,​

59​

played a major role in the genesis of the human influenza pandemics of 1957 and 1968 (15, 22).​

60​

More recently, intra-subtype reassortment has also been shown to occur frequently among co61​

circulating human H3N2 influenza A viruses (14, 18), which may also impact ongoing antigenic​

62​

evolution (14). In addition to reassortment among RNA segments, intragenic recombination​

63​

between different RNA segments, commonly referred to as non-homologous recombination (3,​

64​

20, 25), as well as intragenic recombination between viral RNA and exogenous RNA (16) have​

65​

been observed and may possibly play a role in determining pathogenicity (25).​

66​

More controversial, however, is the occurrence of homologous recombination in​

67​

influenza viruses, most likely involving copy-choice (template-switching) replication of RNA​

68 molecules that co-infect a single cell. Although bioinformatic evidence for homologous

recombination has been suggested (13, 19), these results remain unsubstantiated,​

​

69 with​

70​

extensive lineage-specific rate variation a likely source of a false-positive signal for at least​

71​

some putative recombination events (24, 31). Indeed, because the genomic RNA generated​

72​

during replication is rapidly packaged with ribonucleoprotein, which will act to prevent the​

73​

occurrence of template-switching that is central to copy-choice replication, homologous RNA​

74​

recombination is thought to occur rarely, if at all, in both influenza viruses (17), and negative75​

strand RNA viruses in general (8). In particular, a comprehensive phylogenetic analysis of​

76​

recombination in negative-sense RNA viruses found only sporadic evidence for recombination,​

77​

and not among influenza viruses (8), although the process was recently demonstrated in Zaire​

78​

ebolavirus, an unsegmented negative-sense single-stranded RNA virus (30). If proven to occur,​

79​

homologous recombination would facilitate two evolutionary processes in influenza virus: the​

80​

purging of deleterious mutations and the rapid generation of novel genotypes, potentially​

81​

including new antigenic and drug-resistant variants.​

82​

To assess whether homologous recombination has played a role in shaping the genetic​

83​

diversity of human influenza A virus we compiled a data set of 13,852 sequences representing​

84​

all eight RNA segments of isolates of A/H1N1 and A/H3N2 subtypes. Using an exhaustive​

85​

search method (4), we statistically assessed the possibility of every potential two-breakpoint​

86​

homologous recombination event, considering each sequence as a possible recombinant and​

87​

searching over all possible parents and all possible breakpoints. In our data set, this translated​

88​

to considering over seven billion sequence triplets, where two of the sequences in each triplet​

89​

are posited to have recombined to form the third sequence in the triplet. For those sequences​

90​

identified by this method to contain putative recombinant sections longer than 100 nucleotides​

91​

(nt), we used more stringent phylogenetic methods to further verify that they contained an​

92​

evolutionary signal (i.e. phylogenetic incongruence) compatible with the action of homologous​

93 recombination.

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

MATERIALS​

​

​

95 AND METHODS​

96​

Sequence data. Nucleotide sequences of human influenza A virus were obtained from​

97​

the Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/Database/select.cgi)​

98​

and aligned using MUSCLE (10). Sixteen sets of sequences, two for each RNA segment, were​

99​

obtained by downloading all of the full-length subtype A/H3N2 and subtype A/H1N1 sequences​

100​

generated through the NIH/NIAID Influenza Genome Sequencing Project. In addition, two​

101​

previously published data sets comprising 413 HA and NA segments of human A/H3N2 viruses​

102​

were also included in the analysis (18). After removing duplicate sequences that were identical​

103​

at the nucleotide level, a final subset of 10,492 sequences was analyzed.​

104​

Recombination analysis. As an initial screen for possible recombination, each of the​

105​

18 data sets was first analyzed using the 3SEQ program (4). 3SEQ tests all possible two106​

breakpoint recombination events for each triplet of sequences in the data set, assigns a​

​

p-value​

107​

(rejecting clonality) to each sequence triplet, and infers breakpoints. Breakpoint pairs are found​

108​

using a parsimony criterion, with the most likely breakpoint positions being those that minimize​

109​

the number of mutations between the putative recombinant sequence and a two-breakpoint​

110​

mosaic of the parental sequences. Breakpoint pairs are reported as ranges of nucleotide sites​

111​

since there are multiple pairs of breakpoints that can satisfy this parsimony criterion. 3SEQ​

112​

reports a p-value by calculating the exact probability that this type of recombination signal would​

113​

be observed under the null hypothesis of clonal (non-recombinant) evolution. Finally, all p114​

values are corrected with a Dunn-?id?k correction for the large number of triplets tested. If a​

115​

particular sequence triplet had a corrected p < 0.05, and if the inferred breakpoints guaranteed​

116​

that the shortest possible recombinant segment was longer than 100nt (which we deemed​

117​

suitable for phylogenetic analysis), a secondary phylogenetic analysis of the data was used as​

118​

an independent verification of putative homologous recombination identified among these​

119 sequence triplets. Given that 3SEQ is one of the most powerful methods for detecting

recombination (4) and is the only method available that can scan hundreds​

​

120 of sequences at a​

121​

time and identify the candidate recombinants with breakpoints and p-values, it is an appropriate​

122​

method for detecting recombination in large data sets of influenza A virus. However, although​

123​

simulations show that 3SEQ is generally robust to false-positive results (4), lineage-specific rate​

124​

variation can generate apparent recombinants that triplet methods (like 3SEQ) detect as real​

125​

recombinants.​

126​

To minimize the possibility of false-positive results, we performed a secondary​

127​

phylogenetic analysis of recombination in our data sets of influenza A virus. For each putative​

128​

recombinant (or set of recombinants with the same breakpoints), the entire data set alignment​

129​

was divided at the breakpoint positions established by 3SEQ. If two recombination breakpoints​

130​

were found in a single sequence, the sequence region between the breakpoints is denoted the​

131​

?minor? region, generated by the minor parent, and the remainder referred to as the ?major?​

132​

region, generated by the major parent. Because of the very large size of the data sets in this​

133​

study, initial neighbor-joining (NJ) phylogenetic trees were inferred using the PAUP* package​

134​

(26) on either side of the putative breakpoints. If evidence for phylogenetic incongruence was​

135​

apparent due to a change in topological position of specific sequences, a more detailed analysis​

136​

using maximum likelihood (ML) phylogenetic trees was undertaken. In this case​

137​

phylogenetically representative sequences, along with those closely related to the putative​

138​

recombinants, were selected from the data sets to comprise a final data set of 30-40 sequences​

139​

on which rigorous phylogenetic analyses could be undertaken using the breakpoints determined​

140​

using 3SEQ. For these analyses, the best-fit model of nucleotide substitution was determined​

141​

using MODELTEST (21) (details available from the authors on request) and phylogenetic trees​

142​

were inferred under this model using the ML method available in PAUP* (26), employing TBR​

143​

branch-swapping in each case. Finally, to assess the degree of support for the differing​

144​

phylogenetic positions of each putative recombinant, a bootstrap re-sampling analysis was​

145 undertaken using 1000 replicate NJ trees inferred under the best-fit substitution model.

 

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

search over all pairs of sequences and all (double) breakpoints whether
the relative number of differences within the breakpoints differs from
the relative number of differences outside the part enclosed by the breakpoints.

No triples needed, just pairs.
55 million pairs, not 192 billion triplets (how do they get 7 billion ?)

took me and Frenchie 2 days per segment of 5000 sequences for single breakpoint
in 50 nucletide intervals.
(not yet speed optimized)

quite some recombinations in H5N1,H9N2 few in H3N2,H1N1.
several obvious sequence-errors

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

We need a translation of the post above into easily understood language.

It would be interesting to have a discussion of the paper's stated rarity of recombination as opposed to the reality of recombination's daily occurrence.

Actually, the paper says it is hard to find recombination that the author's like and can verify with phyogenetic trees (>100 nt). The paper acknowledges smaller regions of recombination and ignores the possibilty that the smaller regions became smaller because of additional recombination. In addition, the paper limits analysis to a human dataset. Thus, recombination with swine or birds is not analyzed. Similarly, the fact that some obvious examples are missing suggests that the human dataset was also limited, or the program just misses the obvious (like the 2002 South Korean HA sequences which have human sequences in circulation a decade earlier).

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

Actually, the paper says it is hard to find recombination that the author's like and can verify with phyogenetic trees (>100 nt). The paper acknowledges smaller regions of recombination and ignores the possibilty that the smaller regions became smaller because of additional recombination. In addition, the paper limits analysis to a human dataset. Thus, recombination with swine or birds is not analyzed. Similarly, the fact that some obvious examples are missing suggests that the human dataset was also limited, or the program just misses the obvious (like the 2002 South Korean HA sequences which have human sequences in circulation a decade earlier).

The analysis only looks at full sequences, so sequences like the south Korean HA sequences with obvious recombination are excluded.

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

We need a translation of the post above into easily understood language.

It would be interesting to have a discussion of the paper's stated rarity of recombination as opposed to the reality of recombination's daily occurrence.

I am still going through the problems withn this paper, but here are a few general considerations.

First and foremost is the fact that most scientists shy away from publishing negative data because they know that negatives can be created by MANY factors that can creep into the experimental design that lead to false negatives, and this paper is a case in point.

The paper has put a LARGE number of restrictions on the data, which takes a VERY biased database and biases it further.

The limitations include a requirement for a full human sequence for inclusion in the database. This elimnates many sequences from China, Korea, and southeast Asia, where much of the diversity and recombination originates. Similarly, birds and pigs are excluded, which represent signifiant influenza genetic reservoirs. Data analysis imposes further limitations. Short stretches of recombination are not detailed because they do not lend themselves to phylogenetic analysis.

As a result, the analysis fails to find some glaring examples in human influenza, and fails to address glaring examples in swine (other than a quick handwave to suggests positives examples are lab error due to contamination).

Some of the above can be seen in the glaring South Korean H3N2 isolates from 2002. The HA sequences are around 1650 base pairs in length, but it looks like all HA sequences less than 1700 were excluded from this analysis.

The South Korean sequences switch from a contemporary 2002 H3 sequence at about position 575 and through approximately position 1000 switch over to a human H3 sequence from a decade earlier. However, most of the sequneces from a decade earlier are only about 1000 base pairs, so they would also be exlcuded from the database used in the paper.

Consequently, the analysis would miss the recombinants (there are six) and the parents (many, but only 1000 BP).

This series alone would invalidate the major conclusion of the paper. The same general recombination was OBVIOUS in six isolates, which formed two subgroups. The smaller subgroups eliminates the possibility of a 1990 H3 contaminant creating a recombinant during amplification. Moreover, the sequences show that the earlier sequences can be maintained for a decade, which was the same type of result seen in the swine paper in Nature Precedings, which is referenced by this paper, but charaterized as "controversial" (because it has real data conclusively demonstrating influenza homologous recombination in multiple genes in multiple swine isolates).

 

[gsgs]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

The analysis only looks at full sequences, so sequences like the south Korean HA sequences with obvious recombination are excluded.

they should still have found it.
E.g.:

se7a43.10
1112 : >A/Denmark/18-2/03(H3N2)
1118 : >A/Cheonnam/323/02(H3N2)
--------------------------------------------------------....
............................................................
............................................................
............................................................
............................................................
............................o...............................
............................................................
............................................................
............................................................
............................................................
..............................oo.o........o............o...o
.......o.........................................o.........o
.........o...oo....o.......................................o
............................................................
......................o...................oo................
....o..........................o..o.........................
.......o..........................o.......................o.
...............................o...........o................
............................................................
............................................................
.........................o..................................
............................................................
............................................................
............................................................
...................................................o........
............................................................
............................................................
............................................................
.............................--------------

Full sequences.
I think the problem is that they require triples, both parents

 

[HenryN]

Re: Homologous Recombination is Very Rare or Absent in Human Influenza A Virus

they should still have found it.
E.g.:

se7a43.10
1112 : >A/Denmark/18-2/03(H3N2)
1118 : >A/Cheonnam/323/02(H3N2)
--------------------------------------------------------....
............................................................
............................................................
............................................................
............................................................
............................o...............................
............................................................
............................................................
............................................................
............................................................
..............................oo.o........o............o...o
.......o.........................................o.........o
.........o...oo....o.......................................o
............................................................
......................o...................oo................
....o..........................o..o.........................
.......o..........................o.......................o.
...............................o...........o................
............................................................
............................................................
.........................o..................................
............................................................
............................................................
............................................................
...................................................o........
............................................................
............................................................
............................................................
.............................--------------

Full sequences.
I think the problem is that they require triples, both parents

No, the Korean sequence is about 1650 BP, so it was excluded. The other parent is from a decade earlier, like A/Seoul/45/91. It will match the 0's in the above figure, but it just goes to position 1000.

Your figure shows obvious diversion in the middle of the gene, but the sequences from the early 90's show where the middle sequence originated.