Reduced sensitivity of influenza A (H5N1) to oseltamivir.We tested the neuraminidase neuraminidase /neu·ra·min·i·dase/ (-ah-min´i-das) an enzyme of the surface coat of myxoviruses that destroys the neuraminic acid of the cell surface during attachment, thereby preventing hemagglutination. drug sensitivity of clade clade Cladus, subtype Genetics A branch of biological taxa or species that share features inherited from a common ancestor; a single phylogenetic group or line. See Inheritance, Species. 1 and clade 2 influenza A influenza A n. Influenza caused by infection with a strain of influenza virus type A. influenza A Infectious disease An avian virus, especially of ducks–which in China live near the pig reservoir and 'vector'; (H5N1). All viruses demonstrated similar sensitivity to zanamivir, but compared with the 2004 clade 1 viruses, the Cambodian 2005 viruses were 6-fold less sensitive and the Indonesian clade 2 viruses were up to 30-fold less sensitive to oseltamivir. ********** Two different strains of highly pathogenic avian influenza avian influenza: see influenza. A (H5N1) have been circulating since 2003. Clade 1 has been found in Vietnam, Thailand, Cambodia, Lao People's Democratic Republic, and Malaysia. Clade 2 subsequently emerged and spread from People's Republic People's Republic n. A political organization founded and controlled by a national Communist party. of China to Indonesia, Europe, and Africa in 2004-2005. Because of its systemic availability, oseltamivir is the drug of choice for treating infected persons (1). The Study We tested the drug sensitivity of neuraminidases (NAs) (2) from influenza (H5N1) from chickens, ducks, geese, and quail quail, common name for a variety of small game birds related to the partridge, pheasant, and more distantly to the grouse. There are three subfamilies in the quail family: the New World quails; the Old World quails and partridges; and the true pheasants and seafowls. (1) from 2004 from Vietnam and Malaysia (provided by N. Long, Regional Animal Health Centre, Ho Chi Minh City Ho Chi Minh City, formerly Saigon, city (1997 pop. 5,250,000), on the right bank of the Saigon River, a tributary of the Dong Nai, Vietnam. , Vietnam; and S. Hassan, Veterinary Research Institute, Ipoh, Malaysia), from 2004-05 from Cambodia (provided by S. San, National Animal Health Production and Investigation Center, Phnom Penh Phnom Penh (nŏm pĕn, pənŏm`) or Phnum Penh (pən  m`), city (1994 est. pop. , Cambodia), and from 2005 from Indonesia (provided by T. Usman). In
the absence of a validated cell culture assay, the 50% inhibitory
concentration measured in the NA enzyme inhibition Enzyme inhibitionThe prevention of an enzymic process as a result of the interaction of some substance with an enzyme so as to decrease the rate of the enzymic reaction. The substance causing such an effect is termed an inhibitor. assay is used as the benchmark for measuring drug sensitivity. (3) Despite their origins in different countries and different avian avian /avi·an/ (a´ve-an) of or pertaining to birds. a·vi·an adj. Of, relating to, or characteristic of birds. species, all clade 1 and clade 2 viruses had a similar sensitivity to zanamivir as the reference influenza (H1N1) NA, (Table 1, Figure, panel A). However, sensitivities of the NAs to oseltamivir (oseltamivir carboxylate carboxylate, n a carboxylic acid salt, ester, or ion. ) fell into 3 groups when compared to the NA of a reference human influenza (H1N1) strain (Table 1, Figure, panel B). The clade 1 isolates from 2004 were all more sensitive to oseltamivir than was the human influenza (H1N1) control, consistent with recent findings of Rameix-Welti et al. (4). However, the NAs of our 2005 Cambodian viruses showed a 6- to 7-fold decrease specifically in oseltamivir sensitivity in comparison to our 2004 Cambodian isolates. These 2005 isolates came from the same area as 1 of the more sensitive 2004 isolates (Kandal), which suggested that at least regional evolution had occurred. [FIGURE OMITTED] Of more concern was the third group. The NAs from all the clade 2 2005 Indonesian viruses demonstrated a 15to 30-fold decrease in sensitivity specifically to oseltamivir compared with clade 1 viruses (Table 1, Figure, panel B). Govorkova et al. (5) also recently showed that the A/Turkey/15/2006 clade 2 virus was almost 60-fold less sensitive to oseltamivir in a plaque reduction assay than was the clade 1 A/Vietnam/1203/2004 virus. Both these results and those of Rameix-Welti et al. (4) contrast to those recently published by Hurt et al., who found no difference between the sensitivities of clade 1 and clade 2 isolates to oseltamivir in the enzyme assay Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Enzyme units Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of (6), although many of the isolates were the same as those tested here. The reason for the discrepancy is not known. The decrease in sensitivity is comparable to that conferred by the N294S recently detected in Egypt (7), known to be selected for by oseltamivir treatment (8). Although both drugs are based on the transition state analog of sialic acid sialic acid: see glycoprotein. , zanamivir has a single substitution of a guanidinium group at the 4' position on the sugar ring, whereas oseltamivir has an amino group amino group, in chemistry, functional group that consists of a nitrogen atom attached by single bonds to hydrogen atoms, alkyl groups, aryl groups, or a combination of these three. An organic compound that contains an amino group is called an amine. at the 4' position and, more importantly, a bulky hydrophobic hydrophobic /hy·dro·pho·bic/ (-fo´bik) 1. pertaining to hydrophobia (rabies). 2. not readily absorbing water, or being adversely affected by water. 3. pentyl pen·tyl n. See amyl. ether group replacing the glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. side chain at the 6' position. Reorientation Noun 1. reorientation - a fresh orientation; a changed set of attitudes and beliefs orientation - an integrated set of attitudes and beliefs 2. reorientation - the act of changing the direction in which something is oriented of E276 in the active site is required to create a hydrophobic pocket necessary to accommodate this pentyl ether group. Mutations that prevent this reorientation from occurring lead to high levels of specific oseltamivir resistance (H274Y, R292K). Decreased binding to oseltamivir can also be due to altered interactions with its 4-amino group (El 19V) (9). We therefore tested several viruses from each group for inhibition by a drug that shares the 4-amino substitution. The clade 1 and 2 viruses had similar sensitivity to 4-amino-Neu5Ac2en (Table 1, Figure, panel C), indicating the decreased binding to oseltamivir was specifically due to altered interactions around the 6-pentyl ether group, thus explaining why no altered binding to zanamivir was seen. Because of the potentially important implications of our findings for public health and stockpiling stock·pile n. A supply stored for future use, usually carefully accrued and maintained. tr.v. stock·piled, stock·pil·ing, stock·piles To accumulate and maintain a supply of for future use. strategies, and because of the apparent discrepancy with the results of Hurt et al. (6), we retested 2 viruses from each group against an independent source of oseltamivir carboxylate (provided by Biota, Notting Hill, Victoria Notting Hill is a suburb in Melbourne, Victoria, Australia, situated between Mount Waverley and Clayton North. Its Local Government Area is the City of Monash. History Its traditional centre is on Ferntree Gully Road. , Australia). We chose A/Indonesia/Wates/77/2005, which had a further slight decrease in sensitivity compared to other Indonesian isolates, as well as one that was in the same range as the remaining Indonesian isolates. Results shown in Table 2 confirm this decreased sensitivity of the clade 2 Indonesian isolates and a small further decrease in sensitivity of A/Indonesia/Wates/77/2005. Because we only had access to clade 2 isolates from Indonesia, we do not know whether this decreased sensitivity occurs only in the Indonesian clade 2 isolates or globally with all clade 2 isolates. Comparisons of the sequences in the public databases show several mutations in the stalk region, which vary between clade 1 and clade 2 NAs, but 1 mutation in the globular globular resembling a globe. globular heart a spherical cardiac silhouette, usually greatly enlarged and lacking the detailed outline of the right and left atria and apex. Characteristic of pericardial effusion and cardiomyopathy. head, H252Y, varies between all clade 1 and clade 2 isolates. A further 3-aa variation occurs between most clade 1 and clade 2 NAs, S343P, E387G, and G459S (N2 numbering), including the Indonesian isolates studied here. An additional V338M variation is found in most Indonesian isolates, including the ones studied here (sequences submitted by N. Komadina). Whether this additional variation confers this decreased oseltamivir sensitivity remains to be elucidated. The A/Indonesia/ Wates/77/2005 NA has an additional unique I117V variation, which is adjacent to one of the catalytic arginines, R118 (which would be consistent with a further effect on drug sensitivity to oseltamivir), and a slight decrease in sensitivity to zanamivir (2.3 nmol/L vs mean of 1.4 nmol/L for other Indonesian isolates). Conclusions We have shown here that, compared with clade l isolates from 2004, some clade 1 Cambodian isolates and clade 2 Indonesian isolates from 2005 demonstrate reduced sensitivity to oseltamivir. Because none of the sequence variations in the public databases correlates with any mutation known to confer oseltamivir resistance, and none of the variations are in the active site (10), this suggests that the decrease in sensitivities may be due to drift mutations rather than from exposure to oseltamivir. Recent results show that human isolates can also demonstrate decreased sensitivity to oseltamivir and zanamivir with drift mutations in the NA remote from the active site (11). The difference in the amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. at position 252 may partially account for the altered binding between the clade 1 and clade 2 NAs as other researchers have suggested that the amino acid at position 252 can affect reorientation of the E276 (4,10). In clade 1 NAs, this is normally H252, but all clade 2 NAs have Y252. In the recently published N1 structure, although it is from a clade 1 influenza (HSNI HSNI Hypertonic Saline Nasal Irrigation ) NA, the authors had mutated the NA to Y252 (10), which is also found in human NAs. The E276 in this structure does not appear to have undergone full reorientation, which could contribute to the reduced oseltamivir binding found in to the Indonesian clade 2 isolates here. Testing the sensitivities of both NAs would provide valuable information on the role of H252. However, mutational analysis of the Indonesian and Cambodian isolates and structure determinations of the different NAs are necessary to fully understand the mechanisms of altered binding. The specific decrease in sensitivity to oseltamivir of both 2005 Cambodian clade 1 and especially the Indonesian clade 2 influenza (H5N1) isolates is disturbing, especially because they maintain their pathogenicity and transmissibility trans·mis·si·ble adj. That can be transmitted: transmissible signals. trans·mis in birds and are clearly pathogenic in humans, since Indonesia has the highest death rate from influenza (H5N1) infections of any country. This finding is in contrast to recent observations that mutations conferring zanamivir resistance in human strains have poor viability and are not genetically stable (12). Such a decrease in oseltamivir sensitivity could lead to suboptimal Suboptimal A solution is called suboptimal if a part of the solution has been optimized without regards to the overall objective. drug dosing in treating persons infected with these isolates, which is thought to facilitate Selection of viruses with a high level of resistance (13). Several groups have reported the emergence of resistant viruses in clade 1--infected influenza (H5N1) patients treated with oseltamivir and suggested that higher doses of oseltamivir may be needed (1,8). Because the clade 2 viruses studied here have a 15- to 30-fold decrease in sensitivity compared to the clade 1 viruses, this suggests the standard dosing of oseltamivir may be even less effective in treating clade 2 influenza (H5N1) A-infected patients. Many laboratories are developing rapid PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) sequencing methods for detecting the known mutation (H274Y) that confers high-level resistance in influenza (H5N1). However, we have shown here the importance of phenotypic phe·no·type n. 1. a. The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences. b. testing of isolates in an enzyme assay rather than just genotypic genotypic emanating from or pertaining to genotype. genotypic selection selection of breeding stock on the basis of known inherited characteristics. screening (14). Because the clade 2 virus is now spread through parts of Europe and Africa, continued global collaboration and phenotypic testing of drug sensitivity of circulating highly pathogenic avian isolates for NA inhibitor sensitivity are critical. This knowledge is essential for developing appropriate management strategies for pandemic pandemic /pan·dem·ic/ (pan-dem´ik) 1. a widespread epidemic of a disease. 2. widely epidemic. pan·dem·ic adj. Epidemic over a wide geographic area. n. planning. No altered sensitivity to zanamivir occurred, which further supports the hypothesis of minimalist min·i·mal·ist n. 1. One who advocates a moderate or conservative approach, action, or policy, as in a political or governmental organization. 2. A practitioner of minimalism. adj. 1. drug design (15) and of maintaining the inhibitor as close as possible to the natural substrate to minimize the emergence of resistance. Our results suggest that zanamivir may also play an important role in pandemic stockpiles. Parts of this work were funded by the Australian Department of Agriculture, Fisheries fisheries. From earliest times and in practically all countries, fisheries have been of industrial and commercial importance. In the large N Atlantic fishing grounds off Newfoundland and Labrador, for example, European and North American fishing fleets have long and Forestry; and a National Health and Medical Research Council The National Health and Medical Research Council (NHMRC) is Australia's peak funding body for medical research, with a budget of nearly A$500M a year . The Council was established to develop and maintain health standards and is responsible for implementing the of Australia grant no. 414400. References (1.) De Clercq E, Neyts J. Avian influenza A (H5N1) infection: targets and strategies for chemotherapeutic intervention. Trends Pharmacol Sci. 2007; 28: 280-5. (2.) McKimm-Breschkin JL, Blick TJ, Sahasrabudhe A, Tiong T, Marshall D, Hart G J, et al. Generation and characterization of variants of NWS/G70C influenza virus influenza virus n. Any of three viruses of the genus Influenzavirus designated type A, type B, and type C, that cause influenza and influenzalike infections. after in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment. in vi·tro adj. In an artificial environment outside a living organism. passage in 4-aminoNeu5Ac2en and 4-guanidino-Neu5Ac2en. Antimicrob Agents Chemother. 1996; 40: 40-6. (3.) Wetherall NT, Trivedi T, Zeller J, Hodges-Savola C, McKimm-Breschkin JL, Zambon M, et al. evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network. J Clin Microbiol. 2003 ;41: 742-50. (4.) Rameix-Welti MA, Agou F, Buchy P, Mardy S Mardy can refer to:
(5.) Govorkova EA, Ilyushina NA, Boltz DA, Douglas A, Yilmaz N, Webster RG. Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus. Antimicrob Agents Chemother. 2007; 51: 1414-24. (6.) Hurt AC, Selleck P, Komadina N, Shaw R, Brown L, Barr IG. Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes. Antiviral antiviral /an·ti·vi·ral/ (-vi´ral) destroying viruses or suppressing their replication, or an agent that so acts. an·ti·vi·ral adj. Res. 2007; 73: 228-31. (7.) World Health Organization. Tamiflu resistance found in Egypt patients. Press release 2007 Jan 22. [cited 2007 Feb 2]. Available from http://www.emro.who.int/csr/media/pdf/ai_press_22_01_07.pdf (8.) Le QM, Kiso M, Someya K, Sakai YT, Nguyen TH, Nguyen KH, et al. Avian flu avian flu: see influenza. : isolation of drug-resistant H5N1 virus. Nature. 2005; 437: 1108. (9.) McKimm-Breschkin JL. Management of influenza virus infections with neuraminidase inhibitors: detection, incidence, and implications of drug resistance. Treat Respir Med. 2005; 4: 107-16. (10.) Russell RJ, Haire LF, Stevens DJ, Collins PJ, Lin YP, Blackburn GM, et al. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature. 2006; 443: 45-9. (11.) Monto AS, McKimm-Breschkin JL, Macken C, Hampson AW, Hay A, Klimov A, et al. Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use. Antimicrob Agents Chemother. 2006; 50: 2395-402. (12.) Zurcher T, Yates PJ, Daly J, Sahasrabudhe A, Waiters M, Dash L, et al. Mutations conferring zanamivir resistance in human influenza virus N2 neuraminidases compromise virus fitness and are not stably maintained in vitro. J Antimicrob Chemother. 2006; 58: 723-32. (13.) Kiso M, Mitamum K, Sakai-Tagawa Y, Shiraishi K, Kawakami C, Kimura K, et al. Resistant influenza A viruses in children treated with oseltamivir: descriptive study. Lancet. 2004; 364: 759-65. (14.) Smith GJ, Fan XH, Wang J, Li KS, Qin K, Zhang JX, et al. Emergence and predominance of an H5N1 influenza variant in China. Proc Natl Acad Sci U S A. 2006; 103: 16936-41. (15.) Varghese JN, Smith PW, Sollis SL, Blick TJ, Sahasrabudhe A, McKimm-Breschkin JL, et al. Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase. Structure. 1998; 6: 735-46. Address for correspondence: Jennifer L. McKimm-Breschkin, CSIRO CSIRO Commonwealth Scientific & Industrial Research Organization (Australia) Molecular and Health Technologies, 343 Royal Parade, Parkville, Victoria Parkville is an inner city suburb north of Melbourne, Victoria, bordered by North Melbourne to the south-west, Carlton and Carlton North to the south and east, Brunswick to the north, and Flemington to the west. It includes the postcodes 3052 and 3010 (University). 3052, Australia; email: jennifer.mckimm-breschkin@csiro.au Jennifer L. McKimm-Breschkin,* Paul W. Selleck, ([dagger]) Tri Bhakti bhakti (bŭk`tē) [Skt.,=devotion], theistic devotion in Hinduism. Bhakti cults seem to have existed from the earliest times, but they gained strength in the first millennium A.D. Usman, ([double dagger double dagger n. A reference mark (  ) used in printing and writing. Also called diesis.Noun 1. ]) and Michael A. Johnson ([dagger]) * CSIRO Molecular and Health Technologies, Parkville, Victoria, Australia; ([dagger]) CSIRO Livestock Industries, Geelong, Victoria This article is about the Victorian city; the name may also refer to City of Geelong or Geelong city centre. Geelong is the second largest city in the state of Victoria, Australia and is the largest regional centre in the state. , Australia; and ([double dagger]) Disease Investigation Centre Region IV, Yogyakarta, Indonesia (1) Isolates tested were the following: A/Mississippi/3/2001 wt, A/Mississippi/3/2001 H274Y; A/quail/Kelantan/6309/2004, A/ chicken/Kelantan/5858/2004 , A/quail/Vietnam/007B/2004,A/duck/ Vietnam/007A/2004,A/chicken/Vietnam/0024/2004, A/chicken/ Vietnam/0023/2004 A/chicken/Vietnam/0018/2004, A/chicken/ Vietnam/0015/2004, A/chicken/Vietnam/0010/2004, A/chicken/ Vietnam/008/2004; A/chicken/Cambodia/Siem Reap/77B/2004, A/chicken/Cambodia/Siem Reap/76/2004, A/chicken/Cambodia/ Takeo/45/2004, A/chicken/Cambodia/Kandal/23/2004, A/chicken/ Cambodia/Pong Peay/1B/2004, A/chicken/Cambodia/Pong Peay/ 1A/2004, A/gooselCambodialKandall2005, A/chicken/Cambodia/ Kendal/3/2005, A/chicken/Cambodia/Kandal/2/2005, A/chicken/ Cambodia/Kendal/1/2005, A/chicken/lndonesialWates/130/2005, A/chickenl/ndonesia/Wates/126/2005, A/chicken/Indonesia/Wates/ 83/2005,A/chicken/lndonesialWates/80/2005,A/chicken/lndonesial Wates/77/2005, A/chicken/lndonesia/Wates/1/2005. Dr McKimm-Breschkin is a chief research scientist in the CSIRO Division of Molecular and Health Technologies in Australia. Her institute was involved in the development of zanamivir. Her research interests focus on understanding mechanisms of influenza resistance to the neuraminidase inhibitors.
Table 1. Mean I[C.sub.50]s of NA sensitivities in MUNANA *-based
enzyme inhibition assay for influenza (H5N1) isolates from each region
compared with a human influenza (H1N1) control and known resistant
H274Y isolate ([dagger])
Zanamivir, Oseltamivir,
([double dagger]) ([double dagger])
([section]) mean ([section]) mean
I[C.sub.50], I[C.sub.50],
Isolate nmol/L nmol/L
Subtype H1N1
A/Mississippi/3/2001 [2] 1.18 (0.24) 2.16 (0.31)
([paragraph]) wt
A/Mississippi/3/2001 H274Y 1.41 (0.26) 475.1 (344)
[4] ([paragraph])
Clade 1 subtype H5N1 2004
Malaysia 2004 [2] 1.21 (0.13) 0.47 (0.07)
Vietnam 2004 [8] 1.40 (0.44) 0.55 (0.26)
Cambodia 2004 [6] 1.96 (0.56) 0.41 (0.24)
Clade 1 subtype H5N1 2005
Cambodia 2005 [4] 1.53 (0.4) 2.88 (0.58)
Clade 2 subtype H5N1
Indonesia 2005 [6] 1.42 (0.63) 11.45 (4.32)
4-Amino-Neu5Ac2en,
([double dagger]
([section]) mean
Isolate IC50, [micro] mol/L
Subtype H1N1
A/Mississippi/3/2001 [2] 1.12 ([dagger])
([paragraph]) wt
A/Mississippi/3/2001 H274Y 1.31 ([dagger])
[4] ([paragraph])
Clade 1 subtype H5N1 2004
Malaysia 2004 [2] 2.82 (0.77)
Vietnam 2004 [8] 2.47 (0.42)
Cambodia 2004 [6] 2.59 (0.15)
Clade 1 subtype H5N1 2005
Cambodia 2005 [4] 2.56 (0.53)
Clade 2 subtype H5N1
Indonesia 2005 [6] 2.00 (0.77)
* Sigma, Saint Louis, MO, USA.
([dagger]) I[C.sub.50], drug concentration for 50% inhibition of
enzyme activity; NA, neuraminidase; wt, wild type.
([double dagger]) Drugs provided by GlaxoSmithKline (Stevenage,
Hertfordshire, UK).
([section]) Nos. in brackets indicate the nos. of isolates tested
against zanamivir and oseltamivir in the same assay. Nineteen
influenza (H5N1) isolates were retested in an independent assay
against oseltamivir and 4-amino-Neu5Ac2en. Means for oseltamivir are
the results of the 2 independent experiments. I[C.sub.50]s for each
isolate were calculated by using the Sigmaplot nonlinear curve-fitting
function (Systat Software Inc., London, UK). Values in parentheses
are standard deviations for the means of the individual IC50s for the
isolates in each group.
([paragraph]) Several plaques were tested from the A/Mississippi/3/2001
against zanamivir and oseltamivir, but only 1 plaque of each was
tested against 4-amino- Neu5Ac2en.
Table 2. Mean I[C.sub.50]s of isolates from each group against
alternative source of oseltamivir * ([dagger])
Oseltamivir,
Virus I[C.sub.50],
nmol/L
A/Mississippi/3/2001 wt 3.2 (2.1)
A/Chicken/Vietnam/008/2004 0.7 (0.4)
A/Chicken/Cambodia/Kandal/23/2004 0.4 (0.2)
A/Goose/Cambodia/Kandal/2005 4.2 (0.5)
A/Chicken/Cambodia/Kandal/3/2005 4.8 (2.5)
A/Chicken/Indonesia/Wates/77/2005 25.6 (2.5)
A/Chicken/Indonesia/Wates/126/2005 14.7 (2.3)
* I[C.sub.50], drug concentration for 50% inhibition of enzyme
activity; wt, wild type.
([dagger]) Isolates were tested in duplicate in 2 separate assays.
I[C.sub.50]s and standard errors (in parentheses) were calculated by
using the nonlinear curve-fitting function in Sigmaplot (Systat
Software Inc., London, UK).
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