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During 2015 open enrollment, 20,897 Alaskans selected qualified health plans (QHPs). About 24 percent of the residents who were eligible to use the marketplace signed up for QHPs for 2015. For 2014, only 15 percent of eligible residents enrolled in QHPs.
The stronger enrollment in 2015 came despite an average 31 percent increase in marketplace premiums for 2015. The 2015 average cost for a silver plan in Alaska is higher than in any other state.
The good news is that subsidies are tempering the higher cost for most enrollees. During the first two months of 2015 open enrollment, 89 percent of Alaskans purchasing plans qualified for premium subsidies.
Sign up to avoid 2015's higher penalties
While open enrollment is over, a special enrollment period (SEP) will give some consumers another chance to sign up for health insurance. The SEP runs from March 15 to April 30 and is available to consumers who learned they owe a tax penalty for not having health insurance in 2014. While these consumers will still be responsible for the 2014 penalty, they can avoid another penalty in 2015.
That's important, as the tax penalties increase next tax season. If you don't qualify for an exemption, you'll have to pay the higher of:
- 2% of annual household income, up to a maximum of the national average premium for a bronze plan, which is over $3,000.
- $325 per adult or $162.50 per child under 18. The maximum penalty per family using this method is $975.
Use this penalty calculator to see how much you may have to pay. Note: Native Americans and Alaska Natives are exempt from the penalty.
Other opportunities to enroll
If you missed open enrollment and don't qualify for the tax-related SEP, you can still sign up for coverage before the next open enrollment period if one of the following applies:
- You experience a qualifying event. You have 60 days from the date of the event to sign up for insurance.
- You are a Native American or Alaska Native. You can sign up for health insurance at any time during the year.
- You qualify for Medicaid. Medicaid accepts applications throughout the year.
Small businesses can enroll year-round
Alaskan businesses with 50 or fewer employees can now enroll online in the Small Business Health Options Program (SHOP). Employers set up an account, and then employees enroll in coverage online. Employers can work with an agent or broker if they want help with the process.
Small businesses can sign up on the SHOP at anytime and offer coverage to their employees throughout the year. Unlike the marketplace for individuals, there is no specific open enrollment period for small businesses.
Subsidies help consumers handle increases
Alaska insurance officials announced significant premium increases for 2015: 35 to 40 percent for policies sold by Premera Blue Cross and 22 to 29 percent for policies sold by Moda Health. According to the Commonwealth Fund, Alaska had the biggest average premium increase in the nation: 31 percent. The Commonwealth Fund also found that Alaska has the highest average cost for silver plans in 2015: $583, before subsidies.
Insurance Commissioner Lori Wing-Heier said the rate increases are justified. The state's small population and limited marketplace enrollment makes for a small risk pool. With high claim costs and few people to spread those costs across, insurance companies are raising rates to cover their expenses.
While the rate increases are certainly a burden for some, the impact will be muted for most consumers. Wing-Heier pointed out that people who qualify for subsidies — 88 percent of Alaskans using the exchange in 2014 — won't bear the brunt of those increases.
A study by the U.S. Department of Health and Human Services showed Alaskans who received subsidies in 2014 qualified on average for an 81 percent reduction in their premiums, resulting in an average monthly premium, after tax credits, of $94 in 2014. Forty-two percent of Alaska enrollees who received subsidies paid $50 or less per month in 2014.
2014 signups
Fewer than 13,000 Alaskans enrolled in private health insurance through the federal marketplace during the 2014 open enrollment period. Just Wyoming, the District of Columbia, North Dakota, and Hawaii had lower enrollment in private health plans. About 4,200 people qualified for Medicaid or the CHIP. Alaska's uninsured rate remains high at 16.1 percent.
Among Alaska residents selecting a QHP, 88 percent qualified for financial assistance, compared to 85 percent nationally. A report released in June by the U.S. Department of Health and Human Services showed the average monthly premium, after tax credits, for Alaska consumers was $94. Among states using the federal marketplace, the average was $82. Forty-two percent of Alaska enrollees pay $50 or less per month after subsidies.
Twenty-seven percent of Alaska residents selected a bronze plan (20 percent nationally), 65 percent selected a silver plan (65 percent nationally), 8 percent selected a gold plan (9 percent nationally), 0 percent selected a platinum plan (5 percent nationally) and 1 percent selected a catastrophic plan (2 percent nationally). Twenty-nine percent of Alaska enrollees were between the ages of 18 and 34.
Alaska's position on exchange, Medicaid expansion
Alaska is among the 26 states that opted to use the federal health insurance marketplace. Alaska refused all federal funding to evaluate and implement a health insurance marketplace, and it was one of the first states to announce it would leave responsibility for its marketplace in the hands of the federal government. While former Gov. Sean Parnell officially announced his decision in July 2012, he had previously made his opposition to the Affordable Care Act, which mandated the exchanges, well known.
Parnell also opposed expanding the Medicaid program. He rejected an expansion in late 2013, saying that Alaska wouldn't be able to afford the program if the federal government were ever to cut funding. (The federal government will pay 100 percent of Medicaid expansion through 2016 and 90 percent through 2020.) In January 2014, Democrats proposed legislation to expand Medicaid, and they included a provision to return to the current eligibility standards if federal funding drops below 90 percent. Even with that safeguard, the bill did not make it out of committee before the legislature adjourned.
However, the new governor — Bill Walker — vowed to reverse the course on Medicaid expansion. Walker took office Dec. 1, 2014, and announced his intention to authorize expansion within his first 90 days in office.
Valerie Davidson, Alaska's health department commissioner, appointed Chris Ashenbrenner to the new role of Medicaid expansion project director to help execute on Walker's pledge. Ashenbrenner, Davidson, and Walker need to convince the Republican-controlled legislature to help fund expansion costs and fix existing problems with the state's Medicaid systems. The recent drop in oil prices has created a huge budget problem in Alaska, so no funding requests are being warmly received, least of all one related to Obamacare.
In late February 2015, the House Finance Committee stripped Medicaid funding from Walker's budget and called for Walker to introduce legislation to expand Medicaid. The administration countered that Democratic legislators have introduced such a bill. In early March, Republican Sen. Pete Kelly announced he would introduce a bill to reform, but not expand, Medicaid. Kelly said the bill will include a managed care provision and health savings accounts.
If the state ultimately authorizes Medicaid expansion, about 30,000 Alaskans would qualify according to the Kaiser Family Foundation. The state would receive about $2.1 billion in federal funding through 2020 according to an Urban Institute study.
Alaska health insurance exchange links
State of Alaska Insurance Department: Affordable Care Act


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Grand Order of Mugunghwa
|This article needs additional citations for verification. (December 2012)
|Grand Order of Mugunghwa
Mugunghwa Daehunjang
|Graphical representation of the order insignia
|Awarded by South Korea
|Head of State of South Korea or its allies, as well as spouses of heads of state
|Awarded for
|Outstanding meritorious services in the interest of promoting the development and security of the Republic of Korea.
|President of South Korea
|Park Geun-hye
|13 August 1949
|Next (lower)
|Order of Merit for National Foundation
|Sash riband of the order
The Grand Order of Mugunghwa (Korean: 무궁화대훈장) is the highest order awarded by the government of the Republic of Korea. It can be awarded to the individuals who are serving or have previously served as the Head of State of the Republic of Korea and its allies, as well as to the spouse of a head of state. The order is presented for, "Outstanding meritorious services in the interest of promoting the development and security of the Republic of Korea." The order is traditionally awarded to the President of the Republic of Korea after their inauguration. Prior to his inauguration, President Roh Moo-Hyun declined the immediate award instead wishing to be invested with the order upon his departure from office. [1]
The Grand Order of Mugunghwa takes its name from the national flower of Korea, the Rose of Sharon. The Rose of Sharon is a cultivar native to the Korean peninsula and has great cultural significance in Korean history.[2]
The Grand Order of Mugunghwa consists of an insignia worn from a collar around the neck, a badge suspended from a sash, and a breast star. A lapel badge is also available.[1] The sash of the order is worn from the left shoulder to the right hip.[3] The Grand Order of Mugunghwa is made of gold and silver and includes ruby and amethyst gemstones. The decoration costs approximately 20 million won, or $19,000 U.S. dollars.[4]
The Grand Order of Mugunghwa is traditionally awarded to the incoming President of the Republic of Korea shortly after their inauguration. Before his inauguration, President Roh Moo-Hyun decided not to accept the award as is traditional, but to receive it from the incoming president upon his own departure from the office.[4] After their conviction for treason in 1996, the former presidents, Chun Doo-hwan and Roh Tae-Woo, were ordered to return the award along with other State decorations that they were awarded. While Chun agreed to return the awards, he and Roh have yet to actually return them.[citation needed]
List of notable recipients[edit]
- Sheikh Khalifa Bin Zayed, President of the United Arab Emirates[4]
- Alan Gabriel Ludwig Garcia Perez, former President of Peru[4]
- Abdullah II of Jordan, King of Jordan
- ^ a b "Awards and Decorations Act". Statutes of the Republic of Korea. Retrieved 10 November 2014.
- ^ Umoh, Kingsley (2014). Taekwondo Poomsae: The Fighting Scrolls: Guiding Philosophy and Basic Applications. Strategic Book Publishing & Rights Agency. pp. 233–234. ISBN 9781612048017.
- ^ http://home.earthlink.net/~dfifer/index.htm : Republic of Korea's Award System, click on "Methods of wear"
- ^ a b c d "Will Pres. Lee accept top gov`t honor before retiring?". The Dong-A Ilbo. 11 January 2013. Retrieved 10 November 2014.
External links[edit]
- "Image of the Grand Order of Mugunghwa". Government Award Information (in Korean). Ministry of Security and Public Administration.

subset: commoncrawl


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No Chrome in Apollo!
Apr. 13, 2007 2 Comments Posted under: ActionScript, AIR, Development, Flex
There are lots of examples out there on the web of how to make you application window have no system chrome (the default system window close, minimise etc), but I was having problems getting it to work with my Flex project.
Most advice tells you to change some settings in the yourAppName-app.xml file:
<rootcontent systemchrome="none" transparent="true" visible="true">yourAppName.swf</rootcontent>
and then add some mx css:
background-color: "";
background-image: "";
padding: 0px;
However this wasn't removing the default Flex system chrome that you get when developing in MXML and ActionScript3.
I managed to find a comment on theflexblog which pointed out that you need to make sure you don't use the ApolloApplication mxml tag and instead just use the Application tag.
This took me a while to discover, especially with the Adobe live docs being very much dead… especially for Alpha Apollo. To make it worse all my code is behind the main mxml file in an AS class (Aral like) so I had to make sure my main class extended Application and not ApolloApplication…
So anyway, hope that helps someone out there!
This entry was posted on Friday, April 13th, 2007 at 6:46 pm and is filed under ActionScript, AIR, Development, Flex. You can leave a comment and follow any responses to this entry through the RSS 2.0 feed.


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Thai-style Sweet Sticky Wings
|Chicken drumstick
|10 Small (Very Small Ones Are Best)
|Spring onions
|2 (Scallions)
|2 Clove (10 gm)
|Chicken stock
|1 Tablespoon
|Seasoning sauce
|1 Tablespoon
|Oyster sauce
|1 Tablespoon
|2 Tablespoon
|Freshly ground black pepper
|1 Teaspoon
|Cooking oil
|2 Cup (32 tbs)
|1⁄2 Cup (8 tbs)
|Tempura flour
|1⁄2 Cup (8 tbs) (Enough To Coat Chicken)
1. Take your chicken drumsticks and slice either side of the bone to open up the meat and ensure quick cooking.
2. Mix the chicken together with the seasoning sauce and chicken stock powder and leave for 20-30 minutes or more, after has marinated you can coat with tempura flour.
3. Grind the black pepper, bash the garlic to a pulp and finely chop the spring onions, keep separate and leave for later.
4. Bring the oil to medium heat in a wok (or deep fryer)
5. Having coated the chicken in tempura flour lower it into the hot oil. Keep turning until you get a lovely light golden brown color, remove and leave to drain
6. To make the sauce, in a fresh wok heat the water to boiling, reduce to simmer and add the sugar, continue stirring until it reduces by about 50% it should now be a little sticky
7. Add the black pepper and garlic stirring in well, followed by the oyster sauce
8. When thoroughly mixed, chuck in the chicken and stir to give it a lovely sugary coating.
9. Lastly add the chopped spring onions and mix in well for a few more runs round the pan and it's done!
10. Serve with hot with some coriander (cilantro) to garnish!


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Big Time Rush With Victoria Justice: Summer Break Tour
* Additional fees apply. No coupon or promo codes necessary to enjoy the displayed discount price.
The last date listed for Big Time Rush & Victoria Justice was Sunday June 23, 2013 / 7:00pm.
Currently at Concord Pavilion
Idina Menzel: Tony-Winning Actress, Singer (Wicked, Frozen) in Concert
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Idina Menzel is a Tony Award-winning actress, singer and songwriter who's had a diverse career on… More
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Had a great time watching my daughter and her friend enjoy themselves. I almost felt like a nine year old again.
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Please tell me when they are coming again.. my daughters and wife were singing the whole show.. definately worth it. especially at goldstar prices.
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I received my tickets 48 hours before the event, as expected. I had no problem with the tickets, the seats were great for the price and the show was good as well. Overall good experience.
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This was such a fun concert.My 6 year old son had a blast!!! Goldstar hooked us up with great seats!!!


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Special Correspondent
To decide whether to field candidate or back independentArriving at a consensus would be ideal: Naqvi
BANGALORE: The National Democratic Alliance (NDA) leaders are meeting in Delhi on Wednesday to discuss the stand to be taken by them on the presidential election.
BJP vice-president Mukhtar Abbas Naqvi told a press conference here on Tuesday that the meeting would discuss various issues, including whether to field their own candidate or support an independent.
He said arriving at a consensus on the next president would be ideal. But the Congress was not interested in a consensus, and none of the Congress leaders had held consultations with the Opposition on the presidential election. Instead of striving for a consensus, the Congress was trying to dictate terms to the Opposition, he said.
"The Congress is searching for a Sonia-loyalist President. But the NDA wants a nation-loyalist President. This is the basic difference between the Congress and the Opposition parties on the presidential election," he said.
The run-up to the presidential election had exposed the real face of the Congress. In its effort to get the stage set for the election of a Sonia-loyalist candidate, the Congress was indulging in dirty politics, besides resorting to underhand agreements and compromises, Mr Naqui alleged. But he did not elaborate his charge.
When his attention was drawn to former Tamil Nadu Chief Minister Jayalalithaa's statement that the third front was ready to support the NDA candidate, he said the BJP was yet to decide on fielding a candidate.
Mr. Naqvi also maintained that the support extended by the Bahujan Samaj Party to the United Progressive Alliance candidate in the presidential election was not in keeping with the dignity of the top constitutional post.


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how to be generally annoying
In this world, we strive for our 15 minutes of fame, now you can effect people in every walk of life, an...
im 15 and love music.
║[o] ║
i am legend. i can fly and do seriously awesome magic tricks. i learnt some blackmagic from my sibling who is a bad witch. i can breathe underwater. how cool is that? i speak 12 languages. i am nine foot and three inches tall. i can bend a metal spoon just like that. i am a pirate too! just kidding. i'm a serial killer.
i love how i met your mother <3
i hate the fact that matt bomer is gay :(
god, i love BoysLikeGirls
Paul DiGiovanni is awesome. ilovehim
message me if you want me to read your stories. i know that those are awesome.
I fan back :)
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Want to upset the learning curve? Try some of these to see how it will effect your grade!
this story is the lamest story on the face of the planet earth. i wrote it when i was 12. haha its the s...
You never know when you're gonna meet someone And your whole wide world in a moment comes undone You'r...
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Track using Order ID
Login and do more!
- Track individual Orders
- View your entire Order history
- Cancel individual Orders
- Conveniently review products and sellers
True to the Tom Clancy's video games' tradition of innovation and excellence Tom Clancy's EndWar is an all-new breed of strategy game: a fusion of next-gen technology superior design and first-class production values.
2020 with resources growing scarce every nation must look after themselves. Tensions between the US Europe and Russia are near a breaking point. One spark will ignite the war to end all wars.
< ul>< li>All downloadable content packs from console versions included plus two exclusive missions maps.Cutting-edge technology.The "commander's-eye view" offers a view of the action from the perspective of any unit under control.The first strategy game to be entirely playable with voice command.Rock solid soldiers AI: they autonomously seek cover engage enemies and withdraw.Massive multiplayer endless replayability.The massively multiplayer "Risk-style" Theatre of War campaign regroups hundreds of thousands players in live action.Unique units customisation features (weapons armors gears) allow unlimited combinations in online and offline games.Your world in flames.40 real-life recognisable battlefields including Washington D.C. Paris and Moscow.Fully destructible dynamic environments.Hundreds of characters and vehicles on screen in real-time with a level of detail previously associated with first-person shooter.PC-specific features.PC-specific interface.More detailed graphics.Refined in-game camera.Revised "situation report" boosting a more tactical gameplay.Redesigned PC controls (keyboard mouse) using classic rea .< li>< ul>
Save More EXTRA 10% OFF* with Axis Bank Debit and Credit Cards Terms and Conditions
|Video Encoding
|Tom Clancy
|Multi-Player, Single-Player
|Xbox 360
|Tom Clancy's : ENDWAR
Please Note: All products sold on Flipkart are brand new and 100% genuine
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Sangram Jagtap
Oct 3, 2012
NICE!!This game is GREAT!
Feb 7, 2013
NEVER ENDING FUNGreat multiplayer game.Eagerly waiting for endwar2.but it seems they have put it on hold.anyone interested in real time strategy,then its a must have.control many uits simultaneously with great ease and fun.many means upto 12 units.great interface
Abhinav Krishna
Oct 10, 2012
Best Strategy game for Modern warFirst I must tell you that I am not a very big fan of the strategy games. But this game is really good. A friend recommended me and I bought it. But when I started playing I immediately started liking it.
Piyush Advani
Feb 9, 2014
Great Depiction Of WW3.Easily one of the best strategy games for any platform.Only drawback is that its very addictive!
Saravgya Tiwari
Nov 5, 2013
awesumm!!the game is awesumm.. specially for those who love RTT..
Ronith Maity
Apr 4, 2015
Ronith MAity123I have buy this PC GAME in 30th MARCH . this game is very good , interesting , enjoyeble. A realeastic WW3 experienced . you can act like a real general .it is really a money valuable PC GAME .
Muhammed Sajid Shaikh
Jan 28, 2014
don,t buyworst game so never look to buy it


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Innovative ROV Technology For Underwater Inspection
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Aquabotix Technology Corporation is based in southern New England, long-recognized as a hub for marine technology development. Our talented team of engineers and software developers innovate and integrate the latest technology for underwater inspection and exploration.
Aquabotix is the market leader in smart technology for ROVs. Our years of experience with underwater applications have advised a new breed of ROVs - the ROV/AUV hybrid. The HydroView and HydroView Professional are remotely controlled via a tether with added functionality of autopilot commands.
Our iPad and laptop applications are the most intuitive driving systems for underwater ROVs. From case to water in 3 minutes; from inexperienced to accomplished pilot in 3 hours; no formal training, lengthy instruction or special degrees are needed to profit from Aquabotix technology.
Aquabotix products are currently deployed in over 30 countries. All products are supported with our CEO guarantee, 1-year warranty and best-in-class customer support.
Made in the USA | Headquarters: Fall River, MA 02720 USA | Telephone: +1 508.676.1100 | FAX: +1 888.414.7363
© Copyright 2014 - Aquabotix Technology Corporation


text:
Within a sodium beam, atoms initially fly at speeds of some 1000 meters per second. Since each encounter with a photon can slow such an atom by three centimeters per second, every atom must absorb and reemit some 30,000 photons to come close to a standstill. Fortunately, this cyclic process repeats so rapidly that the atoms decelerate at rates of some 106 meters per second squared, or 100,000 times greater than the acceleration due to gravity. That suffices to bring them to a stop in as little as a millisecond, along a range of only 50 centimeters.
Similar techniques suffice to cool ions as well as atoms, and ions offer several advantages. Being electrically charged, it is possible to hold them for long periods by applying electric fields. Indeed, Hans Dehmelt, who has pursued his lengthy involvement with Priscilla the Positron, has similarly trapped a barium ion named Astrid. The opportunity to trap ions electrically, in turn, means that one can first trap them and then cool them, which can be easier than the cool-first, trap-later procedure with atoms. A third advantage is that ions that resist laser cooling can nevertheless reach low temperatures by storing them in a trap along with other ions that indeed respond to the laser. This technique, known as sympathetic laser cooling, treats the coolable ions as a type of ice, able to receive heat from the other ions and hence to slow their motions as well.
As an example of the measurements that then become possible, David Weinland and colleagues, at the National Institute of Standards and Technology, have carried out studies on a single positively charged


text:
Genetic variability of West Nile virus in US blood donors, 2002-2005.
West Nile virus (WNV) is a small, enveloped, positive-strand RNA virus of the genus Flavivirus and a member of the Japanese encephalitis serocomplex. The plus-sense RNA genome is [approximately equal to] 11 kb and contains a single open reading frame flanked by 5' and 3' untranslated regions (UTRs). The encoded polyprotein is processed into 3 structural and 7 nonstructural (NS) proteins that are essential for viral replication, assembly, and release. WNV is maintained in nature by transmission between mosquitoes and birds but can also infect humans and other mammals (1) and reptiles (2). Most human infections are asymptomatic (70%-80%); symptomatic cases range from flulike illness to severe neurologic disease (>1% of cases) (3-5).
The first US outbreak of WNV occurred in 1999 in New York City; 68 human infections, mostly as meningoencephalitis, were confirmed, resulting in 7 deaths. Since 1999, WNV epidemics have reoccurred yearly with 23,975 reported human cases of disease and 962 deaths through 2006 (www.cdc.gov/ncidod/dvbid/westnile).
In 2002, the virus spread westward and the number of reported human cases increased dramatically. The North American epidemics of 2002 and 2003 represent the largest WNV outbreaks ever reported (6). Additional modes of WNV transmission were identified in 2002, including human-to-human transmissions from mother to child, organ transplantation, and blood transfusion (7-9). The quick spread of the virus raised questions regarding viral adaptation and prompted a detailed investigation of the genetic evolution of the virus.
The prototype WNV isolate, NY99-flamingo382-99 (WN-NY99; GenBank accession no. AF196835) was obtained from a flamingo infected during the 1999 outbreak in New York. This isolate belongs to lineage I and is most closely related to the Israel-98 goose isolate AF481864 (IS-98) (6,10). Phylogenetic comparisons of partial and complete nucleotide sequences from US isolates collected between the 1999 and 2000 epidemics with WN-NY99 isolate showed a high degree of genetic similarity with >99.8% nucleotide homology and >99.9% amino acid homology (11-14). A study of 22 different WNV isolates from 2001 and 2002 showed genetic variation of 0.35% (mean 0.18%) in the premembrane (preM) and envelope (env) genes compared with WN-NY99 (15).
Subsequently, 2 distinct genotypes were detected in strains obtained from Texas in 2002 (15). One new genetic variant widely spread over the United States diverged from the original WN-NY99 strain by several conserved nucleotide mutations and 1 aa substitution in the env protein. Recent studies have highlighted the emergence of a new WNV dominant genotype, named WN02, which has been increasingly prevalent in the United States since 2002 (16-19). Although 13 nt mutations became fixed in the new dominant genotype compared with the WN-NY99 prototype, the highest nucleotide sequence divergence of WNV strains isolated after 2002 is still in the range of 0.4%-0.5% (18,20). The reason for displacement of the WN-NY99 genotype by a new dominant genotype in North America is not clear, but could have been caused by differences in transmission efficiency of domestic mosquitoes that may offer a selective advantage for the newly emerged genotype (20,21).
Several studies on the genetic variation of WNV have been published (12,15,17,19,21,22). However, continuous monitoring of variability is needed because sensitivity of blood donor screening and diagnostic assays may be affected, producing a negative effect on public health. Genetic variability could also affect viral pathogenesis, development of vaccines, and development of efficacious therapeutic agents.
This study reports the genomic variation of WNV observed in clinical isolates obtained in the continental United States during 4 consecutive years (2002-2005). We observed an increase in the number of mutations in the full WNV genome from 0.18% in 2002 to 0.37% in 2005. It should be noted that 80% of the nucleotide changes in structural regions are transitions (T [left and right arrow] C) and 75% are silent mutations. Thus, WNV has continued to slowly diverge from precursor isolates as geographic distribution of the virus expanded.
Materials and Methods
Study Samples
This study included 30 plasma specimens (Table 1) obtained from blood donor units positive for WNV by nucleic acid tests used to screen blood donations under Food and Drug Administration (FDA)--approved nationwide clinical trials; the specimens were collected in 13 states in the continental United States. All samples were collected under Institutional Review Board-approved informed consent provided by each of the institutions performing donor screening.
Virus Isolation in Vero Cells
Vero cells were plated in T75 flasks and grown to 85% confluence in Eagle minimal essential medium (GIBCO BRL, Gaithersburg, MD, USA) containing 5% fetal bovine serum (Hyclone, Logan, UT, USA) and 10 [micro]g/mL of penicillin/streptomycin (GIBCO-BRL). For viral isolation, medium was removed, and 500 [micro]L of each plasma sample was added to individual flasks and the volume was adjusted to 5 mL with fresh medium. Cultures were incubated for 2 hours with the plasma, either at room temperature with gentle rocking or at 37[degrees]C with sporadic mixing. A total of 10 mL of fresh medium was then added and cultures were incubated at 37[degrees]C in an atmosphere of 5% C[O.sub.2]. Cultures were observed daily for cytopathic effect under phase microscopy. Supernatants were harvested when an extensive cytopathic effect was observed, centrifuged to remove cell debris, and frozen at -70[degrees]C until further analysis.
RNA Extraction, Reverse Transcription, and PCR
RNA extracts were obtained from 1-mL plasma samples by using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and extracts were resuspended in 20 [micro]L of water. RNA samples from viral passages were isolated from 140 [micro]L of culture supernatants by using the QiaAMP viral RNA extraction kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. RNA extracts were stored at -70[degrees]C until further analysis. Reverse transcription reactions were performed in a final volume of 20 [degrees]L that contained 10 [degrees]L of RNA and specific WNV reverse primers by using SuperScript Ill (Invitrogen) according to the manufacturer's instructions. Specific primers used for both PCR amplification and sequencing were designed according to available sequence information in GenBank and based on alignments of published WNV sequences. PCR amplification of overlapping fragments to cover the complete viral genome was performed by using 15 pairs of primers (Table 2). Partial sequences for the structural region were obtained by using primer sets 1, 2, and 3. cDNA specimens were amplified by using the Hi-Fidelity PCR system (Invitrogen) according to the manufacturer's instructions.
DNA Sequencing
PCR products were purified by agarose gel electrophoresis by using the MinElute Gel Extraction Kit (QIAGEN) according to the manufacturer's protocol. Both strands were subjected to direct sequencing by using amplifying primers (Table 2) and additional internal sequence primers. Sequencing reactions were performed by using the ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol and analysis by using the ABI Prism 3100 system (Applied Biosystems).
Sequence Analysis
Sequencing data were assembled and analyzed by using the Vector NTI Advance 10 software package (Invitrogen). Nucleotide and deduced amino acid sequences from each isolate were aligned by using the Align X program in Vector NTI and compared with prototype WN-NY99 and previously published sequences of isolates from different regions of the United States and other countries. Phylogenetic relationship studies were based on several methods of analysis (distance, parsimony, and likelihood algorithms) by using MEGA version 3.1 (www.megasoftware.com). All studied isolates were compared with each other, and phylogenetic trees were constructed by using the Kimura 2-parameter method that included transitions and transversions to show genetic relationships of isolates in this study with other WNV isolates in GenBank.
Because of limited volume of plasma specimens available, viruses were isolated from Vero cell cultures for sequence analysis. We assessed the potential of the viral isolation procedure to cause mutations in the viral genome by comparison of sequences of the structural region of 6 specimens and their respective isolates after 3 passages in Vero cells. There were no changes in the RNA sequences obtained in each original plasma sample and the RNA sequences obtained in each of the passages in culture.
We investigated the genetic variability of 30 WNV isolates collected from plasma specimens from nucleic acid amplification testing positive blood donors in 13 states during 2002-2005 (Table 1). Isolates were generated by cultivation in Vero cells. The fragment that encompasses the 5'-UTR, all structural genes, and part of NS1 (bp 1-2,685) from all 30 specimens were subjected to genomic sequencing. Eight of 30 isolates were fully sequenced.
The Appendix Table (online Appendix Table, available from www.cdc.gov/EID/content/14/3/436-appT.htm) compares conserved nucleotide mutations and deduced amino acid substitutions identified in structural regions of all 30 isolates with the WN-NY99 isolate (AF196835). Approximately 80% of the nucleotide changes in structural regions were transitions (T [left and right arrow] C) and 75% were silent mutations. All mutations in the preM and membrane (M) regions were silent, and 16 isolates shared the transition 660 C [right arrow] T. Several WNV strains from Europe and Asia, as well as Kunjin virus isolates, also had T at position 660, but both the prototype WN-NY99 and the IS-98 (AF481864) isolates contain 660 C. Twenty-nine of 30 isolates shared 2 conserved nucleotide mutations in the env gene: 1442 T C (Val 159 [right arrow] Ala) and 2466 C [right arrow] T that differentiates the new dominant genotype, WN02, from the preceding genotype WN99.
Construction of a phylogenetic tree by the maximum parsimony method (Figure 1) showed the degree of divergence of isolates from WN-NY99. The average nucleotide divergence for structural genes has increased from 0.18% in 2002 to 0.37% in 2005.
The env protein has several biologic roles, which include viral entry, virion assembly and release, agglutination of erythrocytes, and induction of B- and T-cell responses that are associated with protective immunity. Thus, this protein may be involved in WNV evolution. The phylogenetic tree shown in Figure 2 was constructed by maximum parsimony analysis with env gene sequences from US isolates from 1999-2006 in GenBank and sequences of human isolates in our study. The isolates clustered in 2 clades correlated with the parsimony-revealing mutation sites at positions 1442 and 2466.
Although preM, M, and env sequences show adequate phylogenetic representation, we also analyzed 8 complete genomes of WNV isolates for stronger evidence of evolutionary relationships between isolates and additional mutations that may have implications in phenotypic properties of these isolates. Nucleotide changes and deduced amino acid substitutions of complete genomic sequences from 8 isolates are shown in Tables 3 and 4, respectively. When compared with WN-NY99 sequences, these sequences showed an increased number of nucleotide mutations. FDA/Hu-02 isolated in 2002 showed 20 nt mutations plus 1 insertion at position 10497, and 5 mutations resulted in amino acid substitutions on the basis of deduced sequence of viral polyprotein. ARC 10-02 isolated in 2002 had 22 mutations, 3 of which resulted in amino acid substitutions. These 2 isolates showed [approximately equal to] 0.2% nucleotide divergence.
The 2003 isolate BSL5-03 showed 39 mutations (nucleotide divergence 0.35%), 7 of which were associated with predicted amino acid substitutions. These 3 isolates from 2002 and 2003 had 11 common mutations: 2 were in env (1442 T [right arrow] C resulting in Val 449 [right arrow] Ala and 2466 C T, a silent mutation); 8 were in the NS regions (4146 A [right arrow] G in NS2A; 2 C [right arrow] T transitions at positions 4803 and 6138 in NS3; 6996C [right arrow] T and 7015T [right arrow] C in NS4B; T C transitions at positions 7938 and 8811 and 9352 C [right arrow] T at NS5); and 1 in the 3-'UTR (10851 A [right arrow] G).
The 2004 isolate BSL5-04 had 42 mutations with 7 aa substitutions. The isolates from 2005 were as follows: GCTX1-05 had 56 mutations with 17 aa substitutions; GCTX2-05 had 41 mutations with 3 aa substitutions; BSL2-05 had 44 mutations with 10 aa substitutions plus a deletion of 14 nt (10480 to 10493) in the 3'-UTR; and BSL13-05 had 48 mutations with 8 aa substitutions. Isolates from 2004 and 2005 shared a nucleotide mutation 6721 G [right arrow] A, which resulted in amino acid substitution Ala2209Thr in the NS4A. Four isolates from 3 consecutive years (BSL5-03 from 2003, BSL5-04 from 2004, GCTX1 and BSL13-05 from 2005), shared an amino acid substitution (Lys2842Arg) in NS5. Three isolates from 2005 (GCTX 1-05, BSL2-05 and BSL 13-05) plus 1 isolate from 2004 (BSL5-04) also shared a silent mutation at position 8550 C [right arrow] T.
The overall nucleotide divergence from the WN-NY99 isolates from 2003, 2004, and 2005 showed a steady but small increase (BSL5-03, 035%; BSL5-04, 0.38%; BSL2-05, 0.39%; BSL 13-05, 0.43%; GCTX1-05, 0.5%; and GCTX2-05, 0.37%). These findings suggest relative stasis in WNV divergence. The 8 completely sequenced isolates shared conserved nucleotide mutations in the preM, M, env, NS2A, NS3, NS4B, and NS5 genes and the 3'-UTR (Table 4). The largest number of conserved mutations was in the NS3 and NS5 genes. No conserved mutations were observed in the core, M, NS1, and NS2B genes or the 5'-UTR.
Our study describes genetic variability observed among 30 clinical isolates of WNV from 13 states in the United States obtained during 2002-2005. Since the initial recognition of WNV in North America in 1999 (10), the virus has spread from the East to the West Coast and has become endemic. Genetic studies have shown that >50% of all WNV isolates in 2002 and >80% of all isolates in 2003 had a new genotype that emerged in the United States in 2001. The new dominant genotype (WN02) was characterized by 2 conserved nucleotide mutations in env (1442 T [right arrow] C and 2466 C [right arrow] T) and 1 deduced amino acid substitution (Val 159 [right arrow] Ala) in the env protein (16-18). Displacement of the initial genotype (WN99) by WN02 has been attributed to differences in efficiency of transmission of the virus by domestic mosquitoes (21). Genetic studies conducted during the early years of WNV activity in the United States identified a small number of mutations and showed no changes suggesting specific adaptations (6,12,15). After the genetic shift in 2001-2002, most nucleotide changes observed were silent transitions (T [left and right arrow] C and A [left and right arrow] G). Limited variability in structural genes observed by our group and others (15,16,18,20,22) suggests the absence of strong immune selective pressure, which led to limited evolution of WNV during 2002-2005. Data in this report show that WNV has continued to diverge from precursor isolates as geographic distribution of the virus expanded.
On the basis of nucleotide sequences, 6 aa substitutions were predicted in the core protein of 5 isolates. Two amino acid substitutions, Ser 11 [right arrow] Asn in isolate BSL 13-05 and Met 21 [right arrow] Thr in isolate FDA-Hu2002, were located within the core hydrophilic region outside ct helices. The other amino acid substitutions (Met 34 [right arrow] Val in isolate BSL06-04, Ala 52 [right arrow] Val in isolate FDA-Hu2002, Ala 77 Val in isolate ARC16-02, and Lys 79 [right arrow] Arg in isolate BSL10-05) were located within [alpha]l, [alpha]2, [alpha]3, and [alpha]4 helices, respectively (23) and were not expected to affect conformation, function, or antigenic properties of core protein.
Studies of WNV evolution have focused on the env protein because it plays a major role in immune response to infection and immunologic pressure could lead to its variation (12,15-21,24,25). A total of 29 of 30 isolates in this study contained 2 conserved nucleotide mutations in env (1442 T [right arrow] C and 2466 C [right arrow] T) that differentiate the dominant genotype from other genotypes and place these isolates in the WN02 clade relative to WN-NY99 (Figure 2). The mutation 2466 C [right arrow] T was a silent mutation. Mutation 1442 T [right arrow] C leads to the amino acid substitution Val 159 [right arrow] Ala, located in the variable region close to the glycosylation site. Glycosylation of env protein can influence virus infectivity and has been considered a potential determinant of virulence in a mouse model (26-28).
All 7 isolates from 2004 were from Arizona and had 2 common silent mutations in env: 1320 A [right arrow] G present in all isolates, and 1974 C [right arrow] T, present in 6 of 7 isolates. Mutation 1320 A [right arrow] G is also present in reported attenuated strains of WNV (AY532665, AY688948, M12294) (29) and was also reported in human isolate DQ164201 from Arizona in 2004 and in the red-tailed hawk isolate DQ164204 from Colorado in 2003 (18). Mutation 1974 C [right arrow] T, found in isolates from Arizona and Texas, was detected in 2000 in New York (AF404756), which indicated that this mutation occurred at least twice. A Kunjin virus isolate from 2003 (AY274505) and isolate EthAn4766 from Ethiopia (AY603654) also had that mutation.
Phylogenetic analysis of complete genomes of WNV isolates provided stronger evidence of evolutionary relationships between isolates. Eight fully sequenced isolates were compared by phylogenetic analysis with 39 published complete WNV genomes present in the United States during epidemics in 1999-2005 (Figure 3). The phylogenetic tree was constructed by maximum parsimony analyses and rooted by using WN-NY99 and IS-98; it clearly shows that these 2 isolates were the predecessors of other US WNV isolates. Analysis of nucleotide sequence alignment of complete WNV genomes indicated that most mutations that occur each year were not fixed. However, WNV has continued to diverge and the total number of fixed mutations and the overall nucleotide divergence have increased. Some mutations in US isolates may have appeared as a result of native adaptation and genetic drift of parental WNV isolates (18).
Within the WN02 genotype, a sequence from NY 2003 (DQ164189) formed 2 outgroups with moderately strong (96%) bootstrap support (Figure 3). Five of 8 isolates in outgroup 2a were from Arizona and had the 6721 G [right arrow] A mutation resulting in amino acid substitution Ala 209 [right arrow] Thr. This mutation was already present in the 2003 magpie isolate DQ164203 from Colorado and in the 2004 human isolate DQ164201 from Arizona (18). These isolates also shared silent mutation 8550 C [right arrow] T, which had been reported in the 2004 human isolate DQ164201 from Arizona (18).
Local concentration of closely related isolates in Arizona (Figures 2, 3) or in California (outgroup 1; Figure 3) may have been caused by introduction of 1 or few genetically similar viruses in the area with rapid spread to mosquitoes and local birds, which amplified the original genome. Human infections in that area would therefore result from 1 or a few local WNV-colonizing genotypes and reflect stochastic introduction of 1 or a few infected vectors, followed by rapid localized amplification (19). In contrast, a larger number of different viruses were introduced in other areas, as observed in Texas, which lead to a broader diversity of variants.
Several studies showed that mutations in conserved structures within the 3'-UTR did not affect WNV translation but could affect RNA replication and interaction between cellular protein eEF1A and WNV 3'-UTR, facilitating viral minus-strand synthesis (30-32). Isolate BSL2-05 from South Dakota had a 14-nt deletion (10480-10493) and isolate FDA/Hu-02 had 1-nt insertion at position 10497 in the 3'-UTR; the second isolate had larger plaques than WN-NY99. The recently published 2004 human isolate DQ431705 from North Dakota (19) had a 5-nt deletion (10434-10439) in the same 3'-UTR variable region. Previous studies had reported low genetic variation among WNV isolates in North America and emphasized a low frequency of nucleotide variants in the 3'-UTR. Subsequent observations showed that nucleotide changes in the 3'-UTR played a role in rapid spread of this virus in the New World because nucleotide changes in the 3'-UTR had co-evolved with amino acid changes and affected interactions of helicase and RNA polymerase with their RNA substrate (33). Mutations in 3'-UTR appear to play a role in production of small-plaque temperature-sensitive variants or mouse-attenuated phenotype variants (34).
Our study showed limited but continuous genetic variability with amino acid substitutions in WNV strains. We also found insertions and deletions in the 3'-UTR region that should be studied for assessment of their functional role. Phylogenetic studies of WNV isolates are needed to monitor microevolution of WNV that may affect pathogenic properties of the virus and to assess the effectiveness of available commercial donor screening and diagnostic assays for detection of variant strains. Study of genetic variability may also provide insights into the development of vaccines and therapeutic agents.
We thank Indira K. Hewlett, Deborah Taylor, and Caren Chancey for helpful discussion and review of the manuscript, and Valerie Winkelman for technical assistance.
This study was partially supported by the FY06-08 National Institute of Allergy and Infectious Diseases Trans National Institutes of Health/FDA Intramural Biodefense Program.
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Address for correspondence: Maria Rios, Laboratory of Molecular Virology, Division of Emerging Transfusion Transmitted Diseases, Food and Drug Administration, National Institutes of Health, Bldg 29, Rm 131, 8800 Rockville Pike, Bethesda, MD 20892, USA; email: maria.rios@fda. hhs.gov
Andriyan Grinev, * Sylvester Daniel, * Susan Stramer, ([dagger]) Susan Rossmann, ([double dagger]) Sally Caglioti, ([section]) and Maria Rids *
* Food and Drug Administration, Bethesda, Maryland, USA; ([dagger]) American Red Cross, Gaithersburg, Maryland, USA; ([double dagger]) Gulf Coast Regional Blood Center, Houston, Texas, USA; and ([section]) Blood System Laboratories, Tempe, Arizona, USA
Dr Grinev is a visiting associate in the Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland. His primary research interests include the molecular epidemiology and pathogenesis of flaviviruses and the evolution of WNV.
Table 1. Sequences of 30 West Nile virus isolates from the United States * No. passages in Vero GenBank Geographic cells accession Isolate ID Year location ([dagger]) no. FDA/HU-02# 2002# NY# 3# AY646354# ARC10-02# 2002# MI# 1# AY795965# ARC12-02 2002 OH 1 DQ666453 ARC13-02 2002 MI 1 DQ666454 ARC15-02 2002 MI 1 DQ666455 ARC 16-02 2002 IN 1 DQ666456 ARC 17-02 2002 GA 1 DQ666457 BSL5-03# 2003# UT# 1# D0005530# BSL-9-03 2003 TX 1 DQ666458 BSL56-03 2003 ND 1 DQ666459 BSL62-03 2003 SD 2 DQ666460 BSL114-03 2003 TX 2 DQ666461 RMS1-03 2003 MN 1 DQ666462 RMS2-03 2003 IN 1 DQ666463 RMS3-03 2003 IN 1 DQ666464 RMS4-03 2003 IA 1 DQ666465 BSL-1-04 2004 AZ 1 DQ666466 BSL-2-04 2004 AZ 1 DQ666467 BSL4-04 2004 AZ 2 D0666468 BSL5-04# 2004# AZ# 1# DQ666448# BSL6-04 2004 AZ 1 DQ666469 BSL7-04 2004 AZ 2 DQ666470 BSI-8-04 2004 AZ 2 DQ666471 GCTX1# 2005# TX# 1# DQ666449# GCTX2# 2005# TX# 1# DQ666450# BSL2-05# 2005# SD# 1# DQ666452# BSI-6-05 2005 AZ 1 DQ666472 BSI-9-05 2005 TX 1 DQ666473 BSL10-05 2005 LA 1 DQ666474 BSL13-05# 2005# AZ# 1# DQ666451# Note: Isolates have been completely sequenced are indicated with #. * Isolates in boldface have been completely sequenced. ([dagger]) Indicates passage of viral isolate from which RNA extracts were used to obtain the genomic sequence. Table 2. Primer sets used for PCR analyses of West Nile virus and sizes of overlapping amplicons * Set/location Amplicon Name Sequence (5' [less than size, kb or equal to] 3') 1/F1-R1300 1.3 F1 AGTAGTTCGCCTGTGTGAGCTGAC R1300 TTGGCGCATGTGTCAATGCT 2/F980-R2000 1.0 R980 CTTGGAATGAGCAACAGAGA R2000 GTTAGGTCGTTCAATGAAGC 3/F1690-R2685 1.0 R1690 GAGACGTTAATGGAGTTTGA R2370 CTTCACTGCTTCCCACATTTG 4/F2340-R3420 1.1 R2340 TTCGGAGGCATGTCCTGGAT R3420 CTGATCTCCATACCATACCAACA 5/F3330-R4120 0.8 R3330 GAGAGCTGCGGACACCGTGGACC R4120 CATAGCAGACTTGCTCCTTTCT 6/F4070-R4950 0.9 R4070 CTGTTGATGGTCGGAATAGG R4950 CCTGGTTTCGTCTGGACGTT 7/F4810-R5650 0.9 R4810 CGCCTGGACCCATACTGG R5650 CCATTCGTATCCAGAGTTCCA 8/F5510-R6430 0.9 F5510 AGCATTGCAGCAAGAGGTTA R6430 TAGTGCCTGGTGATCCGAGTACAC 9/F6290-R6770 0.5 F6290 CGACCGGAGGTGGTGCTTTGATGG F6770 CCTGGAACTTCAGCCATCCA 10/F6690-R7550 0.9 F6690 CCTCCTCATGCAGCGGAA R7550 GAGCTTGCTCCATTCTCCCA 11/F7420-R8260 0.9 R7420 CCACACCCATCATGCAGAA R8260 CGTTGGAGCAGCTCCATCTT 12/F8170-R9050 0.9 R8170 CATAGGACGATTCGGGTCCT R9050 CTCTTTCCCATCATGTTGTAAATGC 13/F8920-R9810 0.9 R8920 CAGCTTTGGGTGCCATGTT R9810 GAACCTGCTGCCAATCATACC 14/F9750-R10630 0.9 R9750 TCCTCAATGCTATGTCAAAGGT R10630 GGTCCTCCTTCCGAGACGGT 15/F10550-R11029 0.5 F10550 TGAGTAGACGGTGCTGCCTG R11029 GATCCTGTGTTCTCGCACCACCAG * Internal sequencing primers were also used and their sequences are available upon request. Table 3. Deduced amino acid substitutions in 8 completely sequenced West Nile virus isolates compared with isolate WN-NY99 * Nucleocapsid Isolate 11 21 52 294 298 342 WN-NY99 Ser Met Ala Leu Asn Asn FDA/HU-02 Thr Val ARC10-02 BSI-5-03 BSI-5-04 GCTX1-05 Ser GCTX2-05 BSI-2-05 Ser Ser BSL13-05 Asn Pro Residue no. Envelope glycoprotein Isolate 449 472 529 602 663 684 WN-NY99 Val Glu Leu Leu Ile Asn FDA/HU-02 Ala Ser ARC10-02 Ala BSI-5-03 Ala Val BSI-5-04 Ala GCTX1-05 Ala GCTX2-05 Ala BSI-2-05 Ala Gly Ile BSL13-05 Ala Phe NS1 Isolate 799 895 WN-NY99 Ile Leu FDA/HU-02 ARC10-02 BSI-5-03 Val BSI-5-04 GCTX1-05 GCTX2-05 Phe BSI-2-05 BSL13-05 Protein NS2A NS2B Residue no. 1169 1438 WN-NY99 Lys Ser FDA/HU-02 ARC10-02 BSI-5-03 Arg BSI-5-04 Asn GCTX1-05 GCTX2-05 BSI-2-05 BSL13-05 Protein NS3 Residue no. 1611 1688 1690 1702 1971 1991 WN-NY99 Val Met Arg Gly Pro Phe FDA/HU-02 ARC10-02 BSI-5-03 Ser BSI-5-04 GCTX1-05 Ala Thr Lys Asp GCTX2-05 BSI-2-05 Leu BSL13-05 Protein NS4A NS4B Residue no. 2188 2193 2209 2213 2261 2301 WN-NY99 Thr Phe Ala Val Leu Ser FDA/HU-02 Gly ARC10-02 Ala BSI-5-03 BSI-5-04 Ser Thr GCTX1-05 Leu Thr Met GCTX2-05 BSI-2-05 Thr BSL13-05 Ile NS4B NS5 Isolate 2488 2549 2755 2788 2820 WN-NY99 Ser Lys Gly Asn Ser FDA/HU-02 ARC10-02 BSI-5-03 BSI-5-04 Ser GCTX1-05 Arg Ser Leu GCTX2-05 BSI-2-05 Gly Arg BSL13-05 Gly NS5 Isolate 2826 2842 3056 3104 3132 WN-NY99 Glu Lys Pro Arg Arg FDA/HU-02 ARC10-02 Gly BSI-5-03 Arg BSI-5-04 Arg GCTX1-05 Arg Cys Ser GCTX2-05 Ser BSI-2-05 BSL13-05 Arg NS5 Isolate 3191 3238 3240 WN-NY99 Met Gin Pro Total FDA/HU-02 5 ARC10-02 3 BSI-5-03 6 BSI-5-04 Lys Gin 8 GCTX1-05 Thr 16 GCTX2-05 3 BSI-2-05 Gin 10 BSL13-05 Gin 8 * NS, nonstructural. Table 4. Nucleotide mutations conserved in fully sequenced West Nile virus isolates from 2002-2005 epidemics in the United States compared with isolate WN-NY99 Gene or region Isolate preM Env NS2A NS3 Nucleotide no. 660 1442 2466 4146 4803 WN-NY99 C T C A C FDA/HU-02 C T G T ARC 10 2002 C T G T BSL5 2003 T C T G T BSL5 2004 T C T G T GCTX1 2005 T C T G T GCTX2 2005 T C T G T BSL2 2005 T C T G T BSL13 2005 T C T G T Gene or region Isolate NS3 NS4A Nucleotide no. 6138 6238 6426 6721 WN-NY99 C C C G FDA/HU-02 T ARC 10 2002 T T BSL5 2003 T T BSL5 2004 T T T A GCTX1 2005 T T T A GCTX2 2005 T T T BSL2 2005 T T T A BSL13 2005 T T T A Gene or region Isolate NS48 NS5 Nucleotide no. 6996 7015 7938 WN-NY99 C T T FDA/HU-02 T C C ARC 10 2002 T C C BSL5 2003 T C C BSL5 2004 T C C GCTX1 2005 T C C GCTX2 2005 T C C BSL2 2005 T C C BSL13 2005 T C C Gene or region Isolate NS5 3'-UTR Nucleotide no. 8621 8811 9352 10851 WN-NY99 A T C A FDA/HU-02 C T G ARC 10 2002 C T G BSL5 2003 G C T G BSL5 2004 G C T G GCTX1 2005 G C T G GCTX2 2005 C T G BSL2 2005 C T G BSL13 2005 G C T G * preM, premembrane; Env, envelope; NS, nonstructural; UTR, untranslated region.
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From the Publisher"Edward Barbier's interpretation of the economic history of nations, seen through the lens of natural resource exploitation, is not only bold but brilliantly executed. The scholarship is immense and the analysis is acute. This is a terrific piece of work." - Sir Partha Dasgupta, University of Cambridge
"Ed Barbier's new book, Scarcity and Frontiers: How Economies have Evolved Through Natural Resource Exploitation, is vast in scope and remarkable in the breadth and depth of knowledge brought to bear on its central thesis—that the exploitation of natural resources and the expansion of frontiers in response to natural resource scarcity have been pivotal forces in the evolution of civilizations. Tracing long spans of pre-industrial history, Barbier demonstrates how expansion into unoccupied frontier areas provided the agricultural surpluses and natural resource raw materials needed to fuel economic growth in emerging civilizations. Particularly instructive are his comparisons between the fates of once powerful empires that failed to secure access to natural resource materials beyond their borders, and suffered economic stagnation and eventual decline as a consequence once their frontiers were closed, and the experience of western Europe, whose superior institutions of commerce and mastery of the seas enabled the exploitation of frontiers and natural resources globally and led to economic ascendancy during the age of exploration. Barbier's aim in writing this book is no less sweeping in scope, to help present day civilizations learn from these experiences and better understand how to cope with the coming age of ecological resource scarcity. He has produced an indispensable contribution in pursuit of that goal." - Robert T Deacon, University of California, Santa Barbara
"Wide-ranging in time (from 10,000 BC to the present day) and space (with case studies drawn from many parts of the world), this is a major and path-breaking analysis of the role of natural resources and the expansion of populations into new frontiers in shaping the evolution of economic and social changes. Based upon extremely wide reading and a detailed knowledge of the issues, this book adds to our understanding of past events while providing suggestions for handling today's resource and climate problems." - Stanley L Engerman, University of Rochester
"Ed Barbier has expanded his interest in problems of scarcity and marginalization in contemporary societies to a broadly based look at frontiers in a very long historical context, charting the interaction of economic growth and the resource frontier from the origins of settled agriculture to current concerns over the sustainability of expanding resource exploitation." - Knick Harley, Oxford University
"In his masterly and timely book, Ed Barbier rescues the resource endowment from its surprisingly passive role in development economics. He details the succession of natural resources which Bagehot's 'conquering swarm' of humanity has used to fuel its expansion. The pointers in the past from which a world of ecological scarcity may learn are fully revealed in this encyclopaedic study." - Eric L Jones, author of The European Miracle (2003) and Growth Recurring (2000)
"In this tour de force, Ed Barbier brings to bear the full weight of history on economics' age-old question about the significance of natural resource endowments for economic performance. Proceeding from the Paleolithic to the present, Barbier tests the ideas of natural-resource economics against the record of our species' efforts to make use of our environment. Economists, historians, and especially economic historians will learn from and admire this book for decades to come." - John R McNeill, University of Georgetown
"This book provides an ambitious re-interpretation of world economic history based on the notion of the frontier. Rather than a feature of US exceptionalism frontier expansion becomes central to explaining comparative historical development. This is a powerful and thought provoking work." - James Robinson, Harvard University


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Exports - partners: Netherlands 14.6%, China 6.8%, Germany 6.8%, Italy 6.2%, Turkey 5.2%, Ukraine 5.2%, Belarus 4.7% (2012 est.)
Definition: This entry provides a rank ordering of trading partners starting with the most important; it sometimes includes the percent of total dollar value.
Source: CIA World Factbook - Unless otherwise noted, information in this page is accurate as of August 23, 2014See Also
© 2014 IndexMundi. All rights reserved.


text:
ASF's 31st Annual New York Gala Dinner and Auction
Details of the Atlantic Salmon Federation's Gala Dinner honoring Nathaniel P. Reed for his outstanding contributions to the protection of wild Atlantic salmon and the environment.
Where: The Plaza, Fifth Avenue and Central Park South
When: Wednesday, November 13th from 6pm
Schedule: 6:00 pm Cocktails and Silent Auction. 7:30 pm Dinner and Live Auction
Honoring: Nathaniel P. Reed for his outstanding contributions to the protection of wild Atlantic salmon and the environment.
The New York Dinner is the ASF's premier fund raising event. Proceeds from this event benefit the ASF, one of North America's oldest and most respected conservation organizations. Founded a half century ago, the ASF is a powerful conservations force with over 40,000 members.
An individual reservation for this event is $750 per person, which includes cocktails and dinner. All but $250 of the ticket cost is tax deductible.
Please click here to purchase tickets.
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Big Sky cross country championships Saturday at Bozeman
Women racing at 10 a.m., men at 11 a.m. at Bridger Creek Golf Course
The 2010-11 cross country and track seasons mark Courtney Babcock's third year at Montana. She was hired in July 2009 after serving in the same role on an interim basis during the 2008-09 season.
If Babcock's athletes had a successful season in 2008-09, they had a breakout campaign in 2009-10.
The women's and men's cross country teams both finished third at the 2009 Big Sky Conference championships after being picked for fourth- and fifth-place finishes by the league's coaches prior to the season.
The women notched their fourth top-three finish of the last five years, while the men recorded their first outright top-three finish since 1990.
Katrina Drennen (third), Lynn Reynolds (fifth) and Kesslee Payne (seventh) had All-Big Sky Conference finishes. Drennen and Payne became Montana's first pair of all-conference finishers at the same meet since 2000.
Drennen's 17th-place performance at the NCAA Mountain Regional would allow her to advance to the NCAA championships, Montana's first representative at the national meet since 2006.
That trend continued - especially for the women - at the 2010 Big Sky indoor championships.
The UM women's team's top five individual-event finishers all came from Babcock's distance crew, highlighted by Drennen's first-place finish in the 3,000 meters. She became Montana's first women's indoor distance champion since 2001.
Brooke Andrus and Kara DeWalt finished second in the 800 meters and mile, respectively, and Drennen and Emily Eickholt placed third in the 5,000 and 800 meters, respectively.
Montana's other champion at the indoor meet was its distance medley relay team, led by Andrus, DeWalt and Payne.
The same athletes came through at the Big Sky outdoor championships as well.
Drennen was runner-up in both the 1,500 and 5,000 meters, Andrus took third in the 800 meters and Payne pulled off the single-day performance of the meet, placing third in the 1,500 meters, fourth in the 800 meters and fifth in the 5,000 meters within a three-hour window.
On the men's side, Reynolds finished fourth in the steeplechase.
During the season Drennen would break Montana's 11-year-old 1,500-meter record and compete in that event at the NCAA West Regional. Payne, who also ran a 1,500 meters faster than the previous school record, joined Drennen at regionals in the 1,500 meters and also ran the 800.
Drennen's career-best and improved-school-record time of 4:19.36 at regionals earned her a spot at the NCAA championships, giving her a cross country-outdoor track single-season national championships double.
A native of Chatham, Ontario, Babcock is a 1996 graduate of Michigan. While competing for the Wolverines from 1992-96, she was a one-time NCAA champion (distance medley relay, 1994 NCAA indoor championships), an eight-time All-American in indoor and outdoor distance events, a member of four Big Ten championship teams and a three-time Academic All-Big Ten selection.
She was inducted into the Michigan Women's Track and Field Hall of Fame in 2004. (Note: Babcock's father, Larry, was inducted into the Michigan Hockey Hall of Fame in 1970. He played center for the Wolverines for three seasons [1960-61 to 1962-63], captaining the team his senior year, and was a Montreal Canadians draft pick.)
Babcock moved to Missoula in 1997 to train and compete with the Mountain West Track Club.
Between 2001 and 2003, Babcock won four titles at the annual Canadian Championships, winning two in the 5,000 meters and two in the 10,000 meters.
She had her most successful season of racing in 2003, posting Canadian national records in the 5,000 meters at the IAAF World Championships in Paris, France, and the 10,000 meters at the Cardinal Invitational in Palo Alto, Calif.
Babcock's 5,000-meter time of 14:54.98 placed her eighth at Worlds; her 10,000-meter time of 31:44.76 broke a 15-year-old national record.
Babcock followed her World Championships performance with a 4:01.99 1,500 meters a week later in Brussels, Belgium, the third-fastest 1,500-meter time in Canadian history.
Babcock competed for Canada in both the 1,500 and 5,000 meters at the 2004 Summer Olympics in Athens, Greece.
She and her husband, Miles Key, have two sons, Ridley and Hudson.
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Small boat cooking
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Small boat cooking
A few things we learned cruising on our 30-footer (she had a 2-burner Origo alcohol stove):
1) Magna Propane Grill on the stern rail!!!!!!! We cooked almost every dinner there.
2) Before you leave home for the weekend, "prep" some meals. I would chop up veggies for coleslaw or salad, put them in a ziplock bag, and make dressing in a jar to mix on board. I''d also put meat or chicken in ziplock bags with marinade and FREEZE them at home. I''d pre-chop anything messy I might need like onions or garlic. I would pre-make potato salad or pasta salad at home. Then when it was time to go sailing I would put it all in one of those soft-sided coolers, get a block of ice on the way to the boat, and load it all into the icebox (if you don''t have an icebox maybe bring along a real, hard-sided cooler and lash it below?). The meat/chicken would thaw slowly and be ready for grilling the next day (or that day if not kept right next to the ice). It made mealitimes super easy and we were still able to have really good, gourmet meals aboard. I would grill extra meat the first night and then for lunch the next day, chop it up and serve with the pasta salad leftovers. Yum. I rarely used the stove below, just for coffee/hot water in the morning and occasionally for rice or pasta or pancakes. Oh, and for heating wash-up water. You could do all this on a Magma Propane Grill, though: it has a holder for pots and pans.
3) Get an insulated pump-top thermos. We fill it with hot water each morning and then have it available for tea, cocoa, or "cuppa-soups" all day, as well as washing up.
4) Zip-lock bags rule on board. For leftovers, etc.
Have fun!
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All our fish at Eataly NYC is responsibly sourced and comes from right around the corner at Eataly's Seafood Counter. Every day, we offer a Whole Fish "al forno", the chef's choice roasted to perfection and a Whole Fish "alla piastra", expertly pan seared market fish. You can always indulge in a daily selection of oysters, as well as unbelievably fresh crudo offerings often seasoned with no more than Extra Virgin Olive Oil and sea salt. Visit our Seating Point located near our Bookstore and Fresh Pasta Department to get a counter seat or table today.
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pc shuts off automatica
lly after only 1-2 minutes
computer shuts off after only a few minutes.
Re: pc shuts off automatica
lly after only 1-2 minutes
Try this procedure:
Unplug the PC and open it up. Clean out all the dust. Carefully remove and replace all the cables going to the motherboard one at a time. Do the same for the memory dimms and the video and sound cards if you have any. You might want to buy a can of compressed air to blow the dust out of the CPU heat sink. Plug your PC back in and give it a go.
HP DV9700, t9300, Nvidia 8600, 4GB, Crucial C300 128GB SSD
HP ENVY 17t Touch,i7-5500u,16GB, 256 GB SSD (on loan)
HP Photosmart 6520
HP Touchpad, HP Chromebook 11
Custom i7-4790k,Z-97, 16GB 2400 mhz, Vertex 3 SSD, Plextor M.2 SSD, Samsung EVO SSD, Corsair HX650, GTX 760TI
Windows 7/8 UEFI/Legacy mode, MBR/GPT
Re: pc shuts off automatica
lly after only 1-2 minutes
You may also check if some of the CPU heatsink screw holder has not broken (it may exceptionally happen on heatsinks with plastic holder) loosing heatsink from CPU. If CPU has insufficient contact with heatsink it overheats in a minute and thermal protection shuts computer down.
I work for HP but my posts and replies are my own.
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Make it easier for other people to find solutions by marking a Reply 'Accept as Solution' if it solves your problem.


text: The New Yorker Blog August 7, 2009 Finger Painting: American Wing By The New Yorker Jorge Colombo sketches the interior of a museum, using Brushes, an application for the iPhone. The New Yorker offers a signature blend of news, culture, and the arts. It has been published since February 21, 1925. Bio All work &


text: Georgia outside linebacker Jordan Jenkins said freshman outside linebacker Lorenzo Carter mixed it up with junior offensive tackle John Theus in practice this week after a shove by Theus at the end of the play. 'ÄúHim and Theus got into a little bout the other day. We broke it up before things got a little too hectic. I got a feeling he'Äôs going to get into it with somebody again real soon.'Äù 'ĶHow does a player get through seven straight days of practices so far? 'ÄúIf you don'Äôt get in that cold tub, I don'Äôt know how you'Äôre going to make it,'Äù Jenkins said of his 10 to 15 minutes after each practice. Georgia practiced under the hottest conditions so far this preseason Thursday afternoon with the temperature at 96 degrees early in the workout. …Jenkins said Davis who has played both safety and cornerback under Pruitt, has looked like a scholarship player and has earned the right to be awarded one. 'ÄúHe'Äôs working hard,'Äù Jenkins said. 'ÄúIt'Äôs up to big daddy Richt.'Äù


text: 218 Dogwood Ln Dogwood Landing, Wilmington, NC 28403 Status: Off Market MLS#: 487827 Contact Email this listing Share on Pinterest Share on Google+ Share on Facebook Share on Twitter Calculate Mortgage Print Flyer Get Directions Request a Showing Off Market 218 Dogwood Ln Wilmington, NC 28403 is no longer on the market or has been removed from the MLS. Similar to 218 Dogwood Ln : Ask an Agent About 218 Dogwood Ln * Required fields Send Your Question × Monthly Payment Estimator Price: Down Payment: % Loan Amount: Loan Type: 30 Year Fixed 15 Year Fixed 5/1 ARM Interest Rate: % Est. Tax & Insurance: % ($/mo.) Est. Monthly Payment: $478 per month × Email a Listing 218 Dogwood Ln, Wilmington NC, 28403 From: Name: Email: To: Name: Email: Subject: Message: Take a look at this property I found on WilmingtonNCHomeFinder.com: http://www.wilmingtonnchomefinder.com/homes/218-Dogwood-Ln/Wilmington/NC/28403/29204327/ Send a copy of this message to me Send Email