Update: Drug Susceptibility of Swine-Origin Influenza A (H1N1) = Viruses, April 2009

Since April 21, 2009, CDC has reported cases of respiratory = infection=20 with a swine-origin influenza A (H1N1) virus (S-OIV) that is being = spread=20 via human-to-human transmission (1). = As of=20 April 28, the total number of confirmed S-OIV cases in the United = States=20 was 64; these cases occurred in California (10 cases), Kansas = (two), New=20 York (45), Ohio (one), and Texas (six). The viruses contain a = unique=20 combination of gene segments that had not been reported previously = among=20 swine or human influenza viruses in the United States or elsewhere = (1).=20 Viruses from 13 (20%) of 64 patients have been tested for = resistance to=20 antiviral medications. To date, all tested viruses are resistant = to=20 amantadine and rimantadine but are susceptible to oseltamivir and=20 zanamivir. The purpose of this report is to provide detailed = information=20 on the drug susceptibility of the newly detected S-OIVs, which = will aid in=20 making recommendations for treatment and prophylaxis for swine = influenza A=20 (H1N1) infection. These data also will contribute to = antiviral-resistance=20 monitoring and diagnostic test development.

Adamantane susceptibility was assessed by conventional = sequencing or=20 pyrosequencing assay (2) with modifications (3), using viral RNA = extracted=20 from original clinical specimens and/or virus isolates. = Susceptibility of=20 virus isolates to the neuraminidase inhibitors (NAIs), including=20 oseltamivir and zanamivir and two investigative NAIs (peramivir = and=20 A-315675), was assessed by chemiluminescent neuraminidase = inhibition assay=20 using the NAStar Kit (Applied Biosystems, Foster City, California) = (4).=20 The generated IC50 values (i.e., = drug=20 concentration needed to inhibit 50% of neuraminidase enzyme = activity) of=20 test viruses were compared with those of sensitive seasonal = control=20 viruses. In addition, because H274Y is the most commonly detected = mutation=20 in oseltamivir-resistant viruses (4,5), a set of new primers for=20 pyrosequencing of the N1 gene was designed to monitor a residue of = the=20 neuraminidase protein at 274 (275 in N1 numbering) in viruses of = swine=20 origin (6,7) (Table=20 1).

All 13 specimens tested contained the S31N mutation in the M2 = protein,=20 which confers cross-resistance to the adamantane class of = anti-influenza=20 drugs (Table=20 2). In addition, a partial sequence deduced from the M2 = pyrograms=20 revealed changes characteristic for the M gene of S-OIVs. Existing = primers=20 used for the detection of adamantane resistance in seasonal = viruses do not=20 work with all tested S-OIVs. Optimized primers have been designed = and are=20 currently being validated. All 13 tested virus isolates exhibited = IC50 values characteristic of oseltamivir- = and=20 zanamivir-sensitive influenza viruses. A/Georgia/17/2006 (H1N1), = which is=20 a seasonal virus, was used as a control (Table=20 2). The IC50 for oseltamivir = ranged from=20 0.28 nM to 1.41 nM, whereas those for zanamivir ranged from 0.30 = nM to=20 1.34 nM. All tested viruses also were susceptible to peramivir and = A-315675. A subset of viruses (n =3D 2) tested in the fluorescent=20 neuraminidase inhibition assay showed IC50 = for oseltamivir and zanamivir ranging from 1.50 nM to 2.40 = nM,=20 similar to the sensitive control. Among the 36 specimens tested to = date=20 with pyrosequencing for the H274Y mutation in N1, none had = mutations at=20 residue 274.

Reported by: L Gubareva, PhD, M = Okomo-Adhiambo, PhD, V=20 Deyde, PhD, AM Fry, MD, TG Sheu, R Garten, PhD, C Smith, J Barnes, = A=20 Myrick, M Hillman, M Shaw, PhD, C Bridges, MD, A Klimov, PhD, N = Cox, PhD,=20 Influenza Div, National Center for Infectious and Respiratory = Diseases,=20 Coordinating Center for Infectious Diseases, CDC.

Editorial Note:

In the United States, two classes of antiviral drugs are = approved by=20 the Food and Drug Administration (FDA) for use in treating or = preventing=20 influenza virus infections: M2 ion channel blockers and NAIs. The = M2=20 blockers (adamantanes) are effective against influenza A viruses, = but not=20 influenza B viruses, which lack the M2 protein (8). However, use = of the M2=20 blockers has been associated with the rapid emergence of = drug-resistance=20 mutations of the M2 protein among human influenza A viruses of = H3N2=20 subtype, and in H1N1 subtype viruses circulating in certain = geographic=20 areas (2,3,9).=20 Adamantane resistance also has been detected in A (H5N1) viruses = in=20 Southeast Asia (10,11). In addition, adamantane resistance has = been=20 reported for swine viruses in Eurasia (12--14) but not in North = America.=20 This rapid increase in resistance has reduced the usefulness of = this class=20 of drugs for the management of influenza A infections, and since = 2005, CDC=20 has not recommended their use (15), although the emergence of = resistance=20 to oseltamivir in seasonal influenza viruses circulating during = the=20 2008--09 season led to changes in CDC recommendations.*

Two NAIs, oseltamivir (Tamiflu [Hoffman-La Roche, Ltd, Basel,=20 Switzerland]) and zanamivir (Relenza [GlaxoSmithKline, Stevenage, = United=20 Kingdom]) are FDA-approved drugs for use against type A and type B = influenza infections (16). The two drugs differ structurally, = resulting in=20 oseltamivir being orally bioavailable, whereas zanamivir is not = and must=20 be inhaled (17,18). A third NAI, peramivir (BioCryst, Inc., = Birmingham,=20 Alabama), is formulated for intravenous administration and is = undergoing=20 clinical trials, and a fourth, called A-315675 (Abbott = Laboratories,=20 Abbott Park, Illinois) has only been investigated in preclinical = studies.=20

Compared with M2 blockers, NAIs previously exhibited lower = frequency of=20 antiviral resistance during therapeutic use (16,19). However, = during the=20 2007--08 influenza season, emergence and transmission of=20 oseltamivir-resistant A (H1N1) viruses, with a H274Y mutation in = the=20 neuraminidase protein, was simultaneously detected in several = countries in=20 the Northern Hemisphere (4,20--22) and spread globally (7,9,23).= As=20 of April 2009, similar trends have been observed in the 2008--09 = influenza=20 season, with many countries reporting up to 100% oseltamivir = resistance in=20 A (H1N1) viruses. As a result, the World Health Organization = Global=20 Influenza Surveillance Network (GISN) and CDC have emphasized the = urgent=20 need for close monitoring of resistance to NAIs. Current interim = antiviral=20 recommendations for treatment and chemoprophylaxis of swine = influenza A=20 (H1N1) viruses include the use of either zanamivir or oseltamivir = and are=20 available at http://www.cdc.g= ov/swineflu/recommendations.htm.

References

CDC.=20 Swine influenza A (H1N1) infection in two children---southern=20 California, March--April 2009. MMWR 2009;58:400--2.
Bright RA, Medina MJ, Xu X, et al. Incidence of adamantane=20 resistance among influenza A (H3N2) viruses isolated worldwide = from 1994=20 to 2005: a cause for concern. Lancet 2005;366:1175--81.
Deyde VM, Xu X, Bright RA, et al. Surveillance of resistance = to=20 adamantanes among influenza A (H3N2) and A (H1N1) viruses = isolated=20 worldwide. J Infect Dis 2007;196:249--57.
Sheu TG, Deyde VM, Okomo-Adhiambo M, et al. Surveillance for = neuraminidase inhibitor resistance among human influenza A and B = viruses=20 circulating worldwide in 2004--2008. Antimicrob Agents Chemother = 2008;52:3284--92.
Lackenby A, Hungnes O, Dudman SG, et al. Emergence of = resistance to=20 oseltamivir among influenza A (H1N1) viruses in Europe. Euro = Surveill=20 2008;13(5).
Deyde VM, Okomo-Adhiambo M, Sheu TG, et al. Pyrosequencing = as a tool=20 to detect molecular markers of resistance to neuraminidase = inhibitors in=20 seasonal influenza A viruses. Antiviral Res 2009;81:16--24.
Lackenby A, Democratis J, Siqueira MM, Zambon MC. Rapid = quantitation=20 of neuraminidase inhibitor drug resistance in influenza virus=20 quasispecies. Antivir Ther 2008;13:809--20.
Hayden FG. Adamantadine and rimantadine---clinical aspects. = In: DD=20 Richman, ed. Antiviral drug resistance. San Francisco, CA: John = Wiley=20 and Sons Ltd.; 1996:59--77.
CDC.=20 Influenza activity---United States and worldwide, 2007--08 = season. MMWR=20 2008;57:692--7.
He G, Qiao J, Dong C, He C, Zhao L, Tian Y. = Amantadine-resistance=20 among H5N1 avian influenza viruses isolated in northern China. = Antiviral=20 Res 2008;77:72--6.
Cheung CL, Rayner JM, Smith GJ, et al. Distribution of=20 amantadine-resistant H5N1 avian influenza variants in Asia. J = Infect Dis=20 2006;193:1626--9.
Krumbholz A, Schmidtke M, Bergmann S, et al. High prevalence = of=20 amantadine resistance among circulating European porcine = influenza A=20 viruses. J Gen Virol 2009;90(Pt 4):900--8.
Schmidtke M, Zell R, Bauer K, et al. Amantadine resistance = among=20 porcine H1N1, H1N2, and H3N2 influenza A viruses isolated in = Germany=20 between 1981 and 2001. Intervirology 2006;49:286--93.
Gregory V, Lim W, Cameron K, et al. Infection of a child in = Hong=20 Kong by an influenza A H3N2 virus closely related to viruses = circulating=20 in European pigs. J Gen Virol 2001;82(Pt 6):1397--406.
Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane = resistance=20 among influenza A viruses isolated early during the 2005--2006 = influenza=20 season in the United States. JAMA 2006;295:891--4.
Moscona A. Neuraminidase inhibitors for influenza. N Engl J = Med=20 2005;353:1363--73.
Smith BJ, McKimm-Breshkin JL, McDonald M, Fernley RT, = Varghese JN,=20 Colman PM. Structural studies of the resistance of influenza = virus=20 neuramindase to inhibitors. J Med Chem 2002;45:2207--12.
Colman PM. Zanamivir: an influenza virus neuraminidase = inhibitor.=20 Expert Rev Anti Infect Ther 2005;3:191--9.
Monto AS, McKimm-Breschkin JL, Macken C, et al. Detection of = influenza viruses resistant to neuraminidase inhibitors in = global=20 surveillance during the first 3 years of their use. Antimicrob = Agents=20 Chemother 2006;50:2395--402.
Dharan NJ, Gubareva LV, Meyer JJ, et al. Infections with=20 oseltamivir-resistant influenza A (H1N1) virus in the United = States.=20 JAMA 2009;301:1034--41.
Hauge SH, Dudman S, Borgen K, Lackenby A, Hungnes O.=20 Oseltamivir-resistant influenza viruses A (H1N1), Norway, = 2007--08.=20 Emerg Infect Dis 2009;15:155--62.
Meijer A, Lackenby A, Hungnes O, et al. = Oseltamivir-resistant=20 influenza virus A (H1N1), Europe, 2007--08 season. Emerg Infect = Dis=20 2009;15:552--60.
Besselaar TG, Naidoo D, Buys A, et al. Widespread = oseltamivir=20 resistance in influenza A viruses (H1N1), South Africa. Emerg = Infect Dis=20 2008;14:1809--10.

TABLE 1.=20 Sequences of swine-origin influenza A (H1N1) primers for=20 pyrosequencing targeted NA codon 274
Primer=20	Primer=20 sequence (5' to 3')
Forward primer=20 (Uni-sw-N1-B-F780)	GGG GAA GAT TGT = YAA ATC AGT=20 YGA
Reverse primer=20 (Uni-sw-N1-B-R1273-biot)	CWA CCC AGA ARC = AAG GYC TTA=20 TG
Sequencing primer = (Uni-sw-N1-B-F804seq)	GYT GAA TGC MCC = TAA=20 TT

TABLE 2. Drug=20 susceptibility of human influenza A (H1N1) viruses of swine=20 origin
CDC identification no.	Strain designation	Date specimen collected	Adamantane susceptibility	M2 mutation	NAI* susceptibility (IC50, nM)=E2=80=A0
Oseltamivir	Zanamivir	Peramivir	A-315675
2009712047	A/California/04/2009	04/01/09	Resistant	S31N	1.37	1.34	0.13	0.66
2009712097	A/California/05/2009	03/30/09	Resistant	S31N	1.41	1.30	0.15	1.78
2009712110	A/California/06/2009	04/16/09	Resistant	S31N	0.28	0.49	0.08	0.11
2009712111	A/California/07/2009	04/09/09	Resistant	S31N	0.56	0.31	0.10	0.18
2009712113	A/California/08/2009	04/09/09	Resistant	S31N	0.73	0.93	0.09	0.19
2009712175	A/Texas/04/2009	04/14/09	Resistant	S31N	0.64	0.62	---	---
2009712177	A/Texas/05/2009	04/15/09	Resistant	S31N	0.54	0.44	0.10	0.35
2009712190	A/Mexico/4482/2009	04/14/09	Resistant	S31N	0.39	0.51	0.06	0.63
2009712191	A/Mexico/4486/2009	04/14/09	Resistant	S31N	0.42	0.50	0.12	0.39
2009712192	A/Mexico/4108/2009	04/03/09	Resistant	S31N	0.39	0.56	0.12	0.50
2009712389	A/Mexico/4516/2009	04/03/09	Resistant	S31N	1.01	0.86	0.26	1.94
2009712390	A/Mexico/4603/2009	04/14/09	Resistant	S31N	0.34	0.35	0.07	1.03
2009712391	A/Mexico/4604/2009	04/14/09	Resistant	S31N	0.44	0.30	0.07	0.68
Control = (seasonal)	A/Georgia/17/2006	---	Sensitive	S31	0.61	0.56	0.16	0.67
Control = (seasonal)	A/Georgia/20/2006=C2=A7	---	Sensitive	S31	200.73	0.80	13.87	1.59
* Neuraminidase = inhibitor. =E2=80=A0=20 Drug concentration needed to inhibit 50% of neuraminidase = enzyme=20 activity (determined by chemiluminescent NAI assay). =C2=A7=20 Oseltamivir resistant, zanamivir=20 sensitive.

* Available at http://www.cdc.gov/fea= tures/dsfluview2009.

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