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HOPEWELL - In the mid-1970s, Steve King and Steve Leonard were young disc jockeys working at WHAP, a 1,000 watt AM station based in Hopewell. Nearly 40 years later they have been reunited, along with former Virginia radio personality Bruce Wiseman, to la
HOPEWELL - In the mid-1970s, Steve King and Steve Leonard were young disc jockeys working at WHAP, a 1,000 watt AM station based in Hopewell. Nearly 40 years later they have been reunited, along with former Virginia radio personality Bruce Wiseman, to launch iKCG Retro Classics Radio.
The station officially launched Jan. 11 and broadcast live from the grand opening of the new USO Welcome Center at Fort Lee that same day.
King commented that, "1-11-11 just seemed like a great time to start this new venture."
iKCG is focused on serving the communities of Virginia's Tri-Cities with studios on S. 15th Ave. in Hopewell. The playlist will consist of songs from the '60s, '70s and '80s. King says he's hearkening back to the glory days of Top 40 radio. "Radio today has gotten very specialized and segmented," says King, who is the station's general manager. "Back then, you could play Steppenwolf and Elton John back to back; hard rock and pop together." The music will be a mix of the top-charting songs of those decades and songs from that era that are rarely heard anymore.
Though the iKCG sound will be inspired by the kind of radio King played all those years ago, it will have a very modern twist - iKCG will be exclusively available on the Internet at retroclassicsradio.com. The goal was to create a distinctly local radio station that could reach residents wherever they were, especially the Fort Lee community that is deployed all over the world. Listeners and fans can also interact with the station via Facebook and Twitter. iKCG - Retro Classics Radio is owned and operated by King Communications Group, LLC
King, who took the morning air shift, began his broadcasting career at Hopewell's WHAP while attending Hopewell High School. His radio career took him to stations in Harrisonburg, Richmond and Norfolk. Though he has primarily worked in television and video production for the past several years, he considers radio his first love and is happy to return.
Afternoons belong to Bruce Wiseman. He was known to listeners of Richmond's K95 as "The Bear." He also worked at other Richmond-area stations as well as the former WMXN Mix105.3 in Norfolk.
Steve "Mr. Beach" Leonard, who can be heard evenings also began his radio career at WHAP and has been a fixture on the radios of central Virginians since then. He is most famous for his popular Sunday Night Beach Party and is currently recruiting sponsors for its return on iKCG.


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Time: May 24, 2019 from 10am to 4pm
Location: Furtherfield Commons, Finsbury Park
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Would you like to imagine a Finsbury Park of the future? Join us for a free day-long workshop with Sci-Fi writer, Stephen Oram, and science experts to explore what the distant future holds for Finsbury Park.
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Runway item
Long jacket in crocodile-motif jacquard fabric
Just a few touches can give a classic garment a whole new look: Emporio Armani knows how to rework the details to keep designs fresh and modern. This jacket is given a new, fresh interpretation thanks to its special fabric. Long jacket in crocodile-motif jacquard fabric with matching-tone lacquered button and light wadding to pad the collar and cuffs.
Composition 100% Polyester
- Basic solid color
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- 1 button
- Long sleeves
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The model is 178 cm and is wearing size 40.
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The total you pay when you complete your order already includes all duties and taxes owed to local authorities. You will not need to pay any further fees when the package is delivered.Easy returnsYou have 30 days from delivery to follow our quick and easy return procedure. You can find more information in Returns and Refunds.
Product Code 2NG59T221281601


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The ultimate guide to one of Australia's most epic holiday islands
South East Queensland is known for its stunning beaches and charming coastal communities. Many opt for the glamour of Noosa or the hustle and bustle of the Gold Coast – not only do they boast magical beaches but shops, restaurants and direct access from Brisbane.
Enter North Stradbroke Island – a paradise that has beaches that rival the world's best and not one ounce of glitz.
Read More at Culture Trip


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OPA Stands For : Online Publishers Association | Outcome | Overseas Performing Artist | Other Principle Arterial | Ohio Psychological Association | Oil Producers Association | Oklahoma Psychological Association | Oregon Pilots Association | Oregon Psychological Association
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Beth Davila Waldman
During a printmaking residency in Basel, Switzerland in August of 2018, I created my most abstract works inspired by the fragments of Plexiglas I have been using in collages since 2017. The resulting monotypes reference these Plexiglas forms with linocuts as well as use them directly as individual plates to represent a system in crisis and the potential navigation of something better. The fractured lines resulting from their extemporaneous shattering implicate displacement, disaster and disintegration. The isolated and intersecting forms highlight the singular nature of the impact that birthed each form and the potential of change with each suggestion of shift.
I am presenting a series of monotypes to exhibit from my 2018 "Devolution Series" both created at EditionBasel and Kala Institute and my "Intermittence Series" printed while in residency at Kala Institute in 2018-2019.
For more information about the artist, please visit her website.


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Let's Make Tequila Cocktails For Cinco de Mayo!
Cinco de Mayo is Friday and we've got some great tequila cocktails to celebrate the holiday. Pop in to the shop for tequila and other needs and then enjoy some deliciousness as you toast the unlikely victory of the Mexican army over the French at the Battle of Puebla.
Y'all Want This Party Started Right
Cinco de Mayo is definitely a time for tequila drinks and we think it's also a good time to serve up Insta-worthy cocktails. Go retro and start with a tequila sunrise. This simple drink of tequila, oj and grenadine is quick to make and yields impressive pictures.
How To Make A Tequila Sunrise
Serve this gorgeous cocktail in a collins or other tall, thin glass.
3 oz orange juice
Don't stir this drink, otherwise you will lose the distinct layers that make it so perfect for Instagram.
Pro tip: Boil water and allow to cool down a little before using it to make ice cubes. This is how you get gorgeous, clear cubes that look the best in drinks.
Y'all Want This Party Started Quickly
Let's face it: the quickest way to start the party is with shots. Keep it Instagram worthy by making these beautiful Slushy Margarita Shots (credit: Delish).
Concoct 4 Slushy Margarita Shots
6 oz tequila
Pro tip: Can't find agave? Don't worry. Just use honey!
Pace yourself to avoid brain freeze. If it happens anyway flatten your tongue against the roof of your mouth. This is the quickest cure.
Brunchworthy Bevvy
For a simple, gorgeous drink you can serve to everyone from your mother-in-law to your coworkers, consider these champagne margaritas from Delish. Serve in flutes.
Class It Up With Champagne Margaritas
½ cup fresh lime juice
1 cup silver tequila
½ cup orange liqueur
1 bottle champagne
Lime wedges and salt for rim
Pro tip: make the pitcher ahead of time but don't add champagne. Before serving put the champagne in serving ware and then pour the mixture over. This will mix the drink without killing the bubbles.
Get Spicy
This gorgeous cocktail, the Spicy Watermelon (credit: Cosmopolitan), is sure to get some major love on Instagram and you'll love its unique taste. Serve this puppy in a rocks glass and garnish it with a chili sliced lengthwise. If you really want to impress, make ice cubes each with a slice of chili. Don't forget to boil the water first and then let it cool for the clearest cubes possible!
Now That's A Spicy Watermelon
Fresno chili, sliced
Agave nectar
1 ½ oz tequila
½ oz lime juice
2 oz Tropicana Watermelon drink
Get Your Supplies!
Come on down to Jersey Wine & Spirits to stock up on tequila and other liquors to make these or any other delicious cocktails! And, be sure to tag us on Instagram and use the hashtag #cincodemayococktails so we can see your gorgeous drinks.


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UI Online:Certify for Continued Benefits Using UI Online
Welcome to UI online, your fast convenient and secure way to certify for continuing unemployment benefits and manage your claim. Today we'll walk you through one of the biggest features of UI online certifying for your continuing benefits. There are two ways to certify for continuing benefits, by UI online or UI online mobile.
We will demonstrate how to certify for continuing benefits using UI online in order to certify for continuing benefits. Using UI online, you first need to register and create a UI online account. If you haven't created an account yet, view UI online overview and registration which will assist you in creating your UI online account.
After creating your account, login using your UI online username and password. After logging in, check the notifications section. When you have weeks that are ready to be certified, you'll see the certify for benefits button. Be sure to submit your certification by the specified date. Failure to certify timely may affect your eligibility for weeks if not. A message will display instead advising you when to check back.
To get started, select the certify for benefits button, next you'll see the certify for benefits page which provides information about the certification process as well as some useful resources for your reference. When you're ready select the certify for benefits button to begin certifying for your continuing benefits. Most customers certify for continuing benefits two weeks at a time. If you have multiple weeks that need to be certified, you'll start with the oldest week.
First at the top of this page, you'll notice a progress bar. This lets you know where you are in the certification process. In this example, we are currently certifying for continuing benefits for week 1. You will also notice that each certification question has the help icon. If you are unsure on how to answer any of the certification questions, select the help icon for more clarification.
You'll start with question 1 and there will be a total of 6 questions to answer. These are the same questions found on the paper. Continue completing claim form. Note if at any time while you are certifying for continuing benefits. If you decide to leave the page, you'll receive this warning message and you will have to restart the certification process again. However if you want to continue, select the stay on page button, once you have answered all the questions, select the next button to continue.
If further information is needed to help, process your certification. You'll be provided with clarification questions like the one shown on the screen in order to help the EDD determine if you meet the eligibility requirements. Let's briefly go over a few types of clarification questions.
You might see when you select YES for question 1 which asks where you too sick or injured to work. You'll be required to answer clarifying questions like the ones shown. If there is any question on whether you are eligible for payment or not, you may be contacted by a representative or a phone. Appointment will be scheduled and you'll be notified by mail.
Here's another example when you select yes for question 2 which asks if there was any reason other than sickness or injury that you could not have accepted full-time work. Each workday you'll be required to answer clarifying questions like the ones shown on this screen. When you answer yes to question three which asks if you look for work, you'll be presented with this optional screen that you can use to document your work.
Search efforts although you're not required to complete this screen. We encourage you to use this helpful tool since the information you enter here can be retrieved later at any time simply by accessing your certification record. If you do not wish to use this helpful tool, go ahead and select the next button to continue with the certification process.
When you select yes for question 4 which asks whether you refuse any work, you'll be required to answer clarifying questions like the one shown on this screen. When you select yes for question 5 which asks if you began attending any kind of school or training. You'll be required to answer clarifying questions like the one shown on this screen. When you select no for question 6 which asks if you work or earn any money.
Whether you're paid or not, you'll be required to answer this follow-up question regarding other types of income. Review the different types of income and select yes or no to indicate whether or not you've received any of the income listed.
In cases where you received a certain type of income that isn't listed and you're not sure if it needs to be reported, go ahead and select yes. After making your selection, select the next button to continue you. When you select yes as shown on the previous screen, you'll be presented with these additional questions and will need to provide a narrative description of the type of income you received.
Answering these questions will help the EDD determine whether or not the income you received is deductible from your weekly benefit payment. If the income you received isn't listed select not listed from the income type drop-down menu.
After entering the required information, select next to continue. We've completed week one. Let's start with week two. For week two, you'll follow the same steps as shown for week one except here. You'll notice two additional questions at the bottom.
You can request that federal income tax be withheld for both weeks or update your contact information if needed. After answering the questions, select next to continue. You've almost done once you answer all the certification questions.
It's time to review your answers for accuracy. Read the acknowledgement section below. This is important because submitting false information or withholding facts can result in a denial of benefits and penalties. If everything is correct, select the acknowledgment.
Check the box and enter your zip code then sign your electronic signature. That means you have read and understood the acknowledgement statement. If you need to make changes to any of your answers, simply select the previous button to go back to any of the previous screens.
When you are ready, select submit. Select submit finalizes and complete the certification process for these weeks. Congratulations! You have successfully submitted your certification to the EDD.
When you see this screen with your confirmation number, you should save this number for your reference or select the confirmation link to print a copy for your records. If you have additional weeks that are available to be certified, click the return to certify.
For benefits button, to repeat the process and keep in mind. You can only certify for two weeks at a time regardless of the number of weeks available. For example if you have four weeks available for certification, you will need to complete this process two times for added convenience.
You may access electronic copies of your submitted continue claim certifications any time by selecting claim history from the main menu. The claim history screen provides an overview of all the weeks you have certified for additional details for a specific week locate the desired week and select the certification link.
You can also view a copy of your certification confirmation detail screen which includes your answers and the clarifying information that you provided.
You may print a copy by selecting the print button or exit the screen by selecting the close window button. We hope you have found it helpful in certifying for your continuing unemployment benefits in a fast convenient and secure way online.
We invite you to visit our ETD website and view other instructional videos available to learn more about many different benefits and conveniences of using UI online.


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The Economic Principle of Political Liberalism
In his recent book The Community of Advantage (2018), Robert Sugden theorizes the Principle of Mutual Benefit. Nowithstandig Sugden's humean conventionalism, it emerges evidently the connection with John Rawls's contractualism, as recognized by the economist himself. Our paper inquires this intellectual relation. In particular, we try to understand the role that Sugden's Principle would occupy in Rawls's Political Liberalism, asking if it would be chosen by the contracting parties in the Original Position. The outcome integrates Rawls's and Sugden's systems, proposing a new economic principle for Political Liberalism. The broader aim is to soften the opposition between conventionalism and contractualism, which probably prevented to find a satisfactory idea of legitimacy for Liberal societies.
Keywords: mutual benefit - contractualism - liberalism - market ethics


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Have you identified your Lead Generation Target Market?
It would seem rather ridiculous to think that a business doesn't know its target market. Well, it turns out this is something that happens in reality, including you. Yes, you may know what kind of people they are, but do you really know them?
Email Writing Tips to a Better Lead Generation Campaign
Email marketing is already known within B2B circles as an effective means of getting high quality leads. But for some reason, companies struggle to realize the full potential of this platform in terms of getting prospects to engage or in other words, compel them to open an email and cultivate interest. We know for a fact though that content has a lot to do with these areas. Below, Really B2B's Libby Morgan lists some very effective tips for creating highly effective and engaging emails for a better B2B lead generation campaign. Know your audience Make use of buyer persona research to really get to know your target audience.
Lead Generation in Singapore: How Database Integration can Keep you in Profit
Photo Credit: Tom Raftery It's a data-driven world out there and B2B enterprises need to drift with the times. In the face of unpredictable market forces, it is important now for these companies to harness new technologies that can provide wide-ranging benefits to their lead generation and appointment setting campaigns.
Proper Demand Generation for Proper B2B Results
One of many challenges facing B2B companies today is increasing brand exposure. This comes as a wide variety of online lead generation tools are made available. Marketing expenditures in the industry are also set to increase relative to increasing competition. In demand generation, companies need to provide cost-efficient and effective strategies. The practice mainly involves broadcasting one's identity towards one's intended audience.
Productivity in B2B Lead Generation - Why Complacency could be a Bad Habit
Success in any undertaking is usually judged on paper, but sometimes, numbers and data aren't enough to paint the whole picture. Marketers are naturally inclined to rely on evaluative statistics to assess their progress, but there is a catch. There are things that statistics cannot measure, such as your marketing team's determination to reach your lead generation goals. You also cannot measure – with absolute certainty – the level of satisfaction of your clients.
How to Develop a Fully-Functioning Content Marketing StrategyB2B Lead Generation Company in Malaysia
A marketing team hires a good writer, puts up a blog, produces a constant flow of relevant articles, and proliferates the content throughout a web of subsites and social media networks. Sounds like a good strategy, right? The thing is, a B2B lead generation campaign that relies on content marketing is not as easy as it sounds. You have to have a system that works. Meaning, it has to have a clearly defined set of goals and a definite measure of success. According to Ann Smarty, founder of My Blog Guest, co-founder of Viral Content Buzz and Brand and Community manager at Internet Marketing Ninjas, these are the ways to create a content marketing strategy that works:
Prevent your Lead Generation Campaign from Capsizing by Knowing these Five Warning Signs
If there's an appropriate metaphor for your lead generation campaign, it's a cargo vessel. And like any ship that crosses seas and oceans, we just can't expect the journey to be a relaxing boat ride on the pond for our precious cache of prospects. Elements of failure abound, and B2B marketers are always at their toes drawing up contingency plans in anticipation of a collision. And it takes a good sense of perceiving critical cracks to make sure things abide by your campaign objectives. Here are five warning signs your campaign could be unfit to traverse turbulent waters.
Marketing Activities you should Outsource to Professionals
Some things are better left to experts, especially when the fate of a business is on the line. This is certainly true in the world of marketing. No one is really an expert of everything; each person can only specialize in a handful of fields. When we desperately try to be a "Jack of all trades", we end up not reaching the maximum potential of our efforts. That's why we need to understand that there are marketing channels that need external help for them to be truly effective.
Software Company Transforms Marketing Activities after Using Callbox
The Client The Client is a leading supplier of shipping software and IT solutions. Based in Singapore, it has more than 100 employees serving over 200 clients worldwide. Its IT solutions are being used by well-known customers around the world, mostly shipping companies, liner agents, short sea carriers, NVOCCs, terminals, and depots.
How to Improve your B2B Lead Generation Telemarketing for Better Sales
In recent years, telemarketing has seen a boom in usage despite pessimistic notions that it is being phased out by social media and email. Within the B2B industry, the practice is very much alive, particularly for lead generation purposes. However, not all of these businesses are doing it right.


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Ty Pennington, the Emmy Award winning star of the shows "Extreme Makeover: Home Edition" and "Trading Spaces," was on hand at Riverstone Books in McCandless on June 7 to talk about and sign copies of his new book "Life to the Extreme."
The humorous book focuses on difficulties Pennington encountered after being diagnosed with Attention Deficit Hyperactivity Disorder while in college and how he managed a successful career while dealing with the condition.
To support the new book, publisher Harper Collins is sponsoring a $25,000 Kitchen Makeover sweepstakes that includes a personal design consultation and advice from Pennington.
Copyright ©2019— Trib Total Media, LLC (TribLIVE.com)


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Finishing touch
in Shooting, Small Sided Games
Use this session to improve the passing technique of your players so they can set up strikers with solid goalscoring opportunities close to goal. MORE
Coaching these soccer shooting drills will enhance your players' ability to beat the goalkeeper from many different positions. Shooting is a core soccer skill for EVERY player and we have some great ways to coach it. We look at shooting from dead ball situations, shooting while running, shooting first time, drive shots and how to make the ball bend, curl and dip. We have great soccer shooting drills and games to help you coach the skills to all your players.
in Shooting, Small Sided Games
Use this session to improve the passing technique of your players so they can set up strikers with solid goalscoring opportunities close to goal. MORE
in Shooting, Small Sided Games
The training session is an excellent workout if you want to get your strikers on their toes and ready to fire shots at the goal. This is a strengthening and reaction exercise that gives players a good workout to sharpen up scoring instincts. MORE
This is good for attackers working the ball through an organised defence, and it has lots of chances for the attack to catch opponents on the counter. It's a good session for tactics and techniques. MORE
in Shooting, Small Sided Games
This session gets teams to link up play from back to front, with movement and passing options in each zone and plenty of scoring opportunities for strikers. MORE
in Shooting, Small Sided Games
In this session, strikers are encouraged to hit a shot at the first opportunity from wherever they are in the area. MORE
This is an intense and creative session that has players taking advantage of any space they can get. Because the pitch is small it also encourages shooting, so individual skill and team work are vital to success. MORE
in Ball Control and Footwork, Shooting
It takes a skilful player to hit a shot on the volley first touch, but if your players have practised this session, they will have a great chance of taking the opposition by surprise. MORE
Your attackers will have a better chance of beating the opposition goalkeeper if they strike the ball first time. A goalkeeper or defence is less likely to be ready to deal with such a shot and this could result in more goals for your team. MORE
in Email newsletters, Shooting
Tournaments are all about scoring goals – from the Women's World Cup to your local tournament on the village green. Score more with my sessions MORE
To score goals, players need to have the correct techniques and ability to use all parts of either foot. This session will have your players practising a variety of shooting techniques. MORE
Great session for movement and combination play in the final third. Also has 1v1 against the attacker and shots on goal. MORE
in Shooting, Small Sided Games
Shooting from midfield is a great way to get your team to catch the opposition out. Use this session to get players lining up shots. Players can be scared to shoot from distance but this session will make sure they start doing it in matches. MORE
Have a bit of fun with this Easter-themed session that also has the serious goal of improving your players' first-time shooting skills. In the final game, if you can put all your eggs (balls) in the basket (goal) before the opposition, you are the Easter champion. MORE
in Email newsletters, Shooting
Every one of your players will love scoring goals – even the goalkeeper. It is an important part of your team strategy that you do not rely on just one player to score for your team during the season. MORE
If a striker can shake off defenders in tight areas and create room to take a shot, your team is bound to score more goals. Here's how you can teach that technique. MORE
in Arrival activities, Fun soccer games, Shooting
This fun, shooting activity gives all players the opportunity to shoot in a fast paced competitive game. MORE
In this series we will show you how to get the ball into positions where your strikers can score more. This week we focus on being creative in front of goal MORE
To increase your team's chances of scoring goals, you can increase the number of players in your attack. This session will help improve decision-making abilities in your strikers for overload situations and the net result will be more goals. MORE
in Shooting, Soccer drills and skills
Use this fun training game to ensure your players are both quick-thinking and quick into the box for a shooting chance. Players must think quickly so they can be first to the ball and shoot. It's a great game for concentration, balance and movement. MORE
in Email newsletters, Shooting
Miroslav Klose is the all time leading scorer for the German national team. Now retired he is giving his skills to the 17-year-olds of Bayern Munich. Use the sessions this week to help you coach strikers to be like Klose MORE
In this series we will show you how to get the ball into positions where your strikers can score more. This week we focus on how to recognise a first-time shooting opportunity MORE
This session allows players to work on first-time shooting in a competitive game-like situation. It works on technique of shooting and tactical positioning so your players make themselves open to shoot MORE
in Email newsletters, Shooting
If you want to see your forwards scoring like they're on fire, get them to model their game on Boca Juniors 00striker Carlos Tevez, who plays with passion and scores with regularity MORE
Use this fast-scoring game to teach your players how to latch onto goalscoring opportunities. This session is all about scoring before your opponents and knocking them out. MORE
"I took a free trial and I was blown away by the drills; it made my training sessions so much easier. "
Coach Gardiner
"As soon as the boys became used to the new way I was training the team went through the roof and went on a undefeated run of 9-10 games"
Coach Wylie
"Great ideas for fun ways of coaching kids."
Coach Brelsford
"A fresh and valuable source of ideas from which I create practices which are specific to my teams."
Coach DiCarlo
"Soccer Coach Weekly is simply excellent."
Coach Belcher


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No more Notre Dame
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On the evening of April 15 in Paris, alarms were sounding, indicating the Notre Dame Cathedral caught on fire. After the first fire, a Marshall failed to locate the fire, causing a critical delay. The fire started in the cathedral's attic, potentially from an electrical shortage. The roof was so dense with beams and braces that it was referred to as "the forest."
The Catholic church cathedral is a world-famous landmark to the French capital, second to the Eiffel Tower. Designed in Gothic fashion and built between the 12th and 14th centuries, the cathedral features numerous statues and stained glass windows. It is home to the world's largest organ, and an immense amount of bells. The Bishop of Paris, Maurice de Sully, was the one to order construction in 1160. Actual construction began a few years later in 1163. Maurice de Sully died while the cathedral was still being built. The cathedral was consecrated as complete in 1345.
Caption: The interior of Notre Dame before and after the fire.
Even through numerous years of destruction and restoration, the interior remains true to the original design. The cathedral's architecture is one of the first examples of the flying buttresses. Marius Hauknes, associate professor of art history at Notre Dame, specializes in medieval art history.
"Its gravity-defying vaulted ceilings were meant to imply a kind of transcendence," Hauknes said. "This is a place that you can go into — in a metaphorical way, to enter into a different realm."
More than a week after the fire shook France, investigators are still trying to find the cause of the fire. So far, nothing has been ruled out and investigations could last for many weeks. The fire, as well as the tons of water poured on the cathedral, both played a part in destroying the evidence.
Rebuilding efforts will cost between $330 million and $670 million. More than $1 billion dollars have been pledged. Francois-Henri Pinault, one of France's richest, contributed $112 million. A rival, Bernard Arnault, announced a donation twice as much. The reconstruction of the church will take years to get back to what it used to be. Critics say that the large amounts of money donated to the restoration efforts could have been spent elsewhere. Those collecting the money are encouraging others to keep donating.
Many say the roof should be rebuilt closely to its original state. A major problem in France is a lack of tall oak trees to help rebuild the roof. To some, rebuilding the roof with other material, such as iron, will decrease the chances of the roof catching fire again.
"I would like the cathedral to be able to welcome the world in 2024, since in 2024, Paris will welcome the world," Paris mayor Anne Hidalgo said. The hope is that the cathedral will be finished in 5 years, just in time for the summer Olympics.
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Silk Painting
Silk paintings are made by embroidering on the canvas of silk with sophisticated themes and vibrant colors. Success of silk painting owes much to the quality of the silk because it is used directly as background. Its delicate and refined colors give the picture such harmony with the nature and excellent representation of Vietnamese landscapes and daily life.
The XQ Su Quan in Da Lat is very famous for silky embroidered paintings.
Vietnamese lacquer traditionally comes in only three colors – brown, black and vermilion. During 1930s, artists adopted a new technique called "chiseling" to produce richer color range and sense of distance.
The painting is made on wood. It is covered with a piece of cloth glued to it using the sap of the lacquer tree and then coated with a layer of the sap mixed with earth. The board is then sand papered and recoated with a layer of hot sap. After polishing, this gives a smooth black surface with a brilliant luster.
The painter uses hot lacquer to draw the outline of a picture and the colors are applied one by one, layer upon layer. Each coat dries slowly.
The finishing touches consist of polishing and washing the pictures. This process may seem like brutal treatment for a work of art, but it is done with great care. This process leaves a brilliant surface on a painting.


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Although there is increasing evidence that individuals already infected with human immunodeficiency virus type 1 (HIV-1) can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660 and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time PCR assay, we determined the replication kinetics of the two strains of virus for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4+ T-cell count, CD4+ memory T-cell subsets, cytokine production by virus-specific CD8+ or CD4+ cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral loads of the primary virus and a modest decline in CD4+ T-cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.
Superinfection with human immunodeficiency virus type 1 (HIV-1) is the infection of an HIV-seropositive individual with a second heterologous strain of the virus after infection with the first infecting strain is established. There is accruing evidence for HIV-1 intra- and intersubtype superinfection in settings of intravenous drug use, structured treatment interruptions, and with strains that are resistant to antiretroviral drugs (2, 4, 6, 22, 26, 28, 32, 39, 42, 43, 52, 60, 66). Epidemiologic studies have suggested that the frequency of superinfection ranges from rare to as high as 5% per year in high-risk populations (9, 10, 15, 20, 24, 27, 31, 40, 41, 51, 59, 65, 67). However, it remains unclear how readily superinfections occur after exposure of an infected individual to a heterologous strain of virus. Furthermore, the variables that may contribute to susceptibility or resistance to superinfection, such as the timing of exposure to a second virus or the immunologic status of the exposed individual, have not been well defined. It is also uncertain whether superinfection is invariably associated with the loss of HIV containment and clinical deterioration (8, 17, 21, 23, 26, 27, 30, 60). Understanding the risks for and the biological consequences of HIV superinfection will not only clarify an important clinical problem, it may also provide important insights into the nature of the immune responses that may confer protection against the initial acquisition of HIV.
The nonhuman primate model provides an ideal means of studying the pathogenesis of HIV-1 superinfection. This system allows for control of many important variables, including the dose, strain, route, and timing of infection. However, there have only been a few animal studies that have attempted to explore the biology of superinfection. The implications of these studies are uncertain because they have been done in models in which infected monkeys do not develop AIDS and the viruses used are either replication incompetent or replicate at low levels (11-13, 18, 36-38, 46-48, 53, 56-58, 61-64). Therefore, it is unclear whether we can extrapolate from these studies the frequency HIV-1 superinfection, the implications of superinfection on HIV pathogenesis, and the feasibility of inducing broadly cross-protective immune responses.
In the present study, we have developed a rhesus monkey model of mucosal superinfection to examine whether infection with replication-competent simian immunodeficiency virus (SIV) confers a relative resistance to superinfection and elucidate the factors that influence the clinical course of infection with a second virus. We show that although prior infection with SIV does not protect against subsequent mucosal challenge with a heterologous SIV isolate, the primary infection does attenuate the replication capacity of the second virus.
Animals.Fourteen adult rhesus monkeys (Macaca mulatta) were used in this study. All animals were housed at Bioqual (Rockville, MD) and maintained in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines at the National Institutes of Health.
SIV challenge stocks.The viruses used in this study included cell-free uncloned pathogenic SIVmac251 and pathogenic SIVsmE660 (kindly provided by Vanessa Hirsch, NIAID/NIH). The stock of SIVmac251 was expanded on human peripheral blood mononuclear cells (PBMCs), and the stock of SIVsmE660 was expanded on rhesus monkey PBMCs. To initiate intravenous infections, 2.1 × 105 RNA copies of SIVmac251 and SIVsmE660 were used. Doses of 6.3 × 107 RNA copies of SIVmac251 and 4.3 × 108 copies of SIVsmE660 were used for the intrarectal exposures. These were doses that were previously shown to reproducibly initiate mucosal infections in rhesus monkeys (29).
qRT-PCR.Plasma SIVmac251 and SIVsmE660 RNA levels were determined using a two-step quantitative real-time reverse transcription-PCR (qRT-PCR) assay. Four sets of strain-specific probes and primers for gag and env were used to distinguish and quantify SIVmac251 and SIVsmE660. Viral RNA was extracted and purified from plasma using the QIAmp viral RNA minikit (Qiagen, Valencia, CA). RNAs were subjected to RT with MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) to generate cDNA products for quantitative PCR using the env RT primer, 5′-GAACCCTAGCACAAAGACCCC-3′, and the gag RT primer, 5′-GGTGCAGCAAATCCTCT-3′. These primers were designed to anneal to conserved regions of gag and env that are shared by the two viral strains.
The subsequent qRT-PCRs were set up using TaqmanGold Mastermix (Applied Biosystems, Foster City, CA). cDNAs were amplified with SIVsmE660 TaqMan env and gag probes that were labeled with 6-carboxyfluorescein (FAM) and quencher dye BHQTM1, while the SIVmac251 env and gag TaqMan probes were labeled with Quasar 670 and quencher dye BHQTM2 (Biosearch Technologies, Novato, CA). For each sample, analyses for SIVmac251 and SIVsmE660 were conducted separately for both env and gag. The sequences and annealing temperatures for primers and probes are outlined in Table 1.
The assembled reactions were run on a Stratagene Mx4000 multiplex quantitative PCR system (Stratagene, La Jolla, CA). Thermal cycling conditions consisted of 10 min at 95°C for AmpliTaq activation, followed by 45 cycles of 30 s at 95°C, 35 s at gene- and strain-specific annealing temperatures as described above, and 30 s at 70°C. Triplicate test reactions were performed for each sample. The nominal copy numbers for test samples were determined by interpolation onto standard curves of RNA standards (duplicate reactions for log10 dilutions of 101 to 106 copies eq/ml). All data analysis was performed with the Mx4000 v3.00 software (Stratagene, La Jolla, CA). The threshold sensitivity of this assay is 100 copies eq/ml of plasma. Because a low level of cross-reactivity of probes between the two strains for SIV could not be eliminated, the baseline signal for the heterologous strain was subtracted for all tested samples.
Infection.For intrarectal exposure to SIV, animals were placed in a sternal position after anesthesia (10 mg/kg intramuscular [i.m.] Ketamine and 0.5 mg/kg i.m. Xylazine) with the pelvis propped up at approximately a 45° angle. A lubricated infant feeding catheter was inserted gently into the rectum of the animal approximately 4 to 6 in. without causing any injury. First, 5 ml of diluent (phosphate-buffered saline [PBS], 0.5% human serum albumin) was gently flushed through the catheter and then 1 ml of the virus was injected through the catheter, followed by a 5-ml flush with diluent. The animal was returned to its cage and kept tilted at a 45° angle until it fully recovered from anesthesia. Six weekly, intrarectal challenges were carried out with the heterologous virus.
Antibodies.The antibodies used for surface staining of memory-associated molecules and in the intracellular cytokine staining were purchased from BD Biosciences (BD) and Beckman Coulter (BC). All reagents were validated and titers determined using rhesus monkey PBMCs. The antibodies and conjugates used in memory staining were anti-CD3-peridinin chlorophyll protein (PerCP)-Cy5.5 (SP34.2 from BD), anti-CD4-fluorescein isothiocyanate (19Thy5D7 from BC), anti-CD95-allophycocyanin (APC) (DX2 from BD), and anti-CD28-phycoerythrin (PE) (CD28.2 from BC). For intracellular cytokine staining, the antibodies and conjugates used were anti-tumor necrosis factor alpha (TNF-α)-fluorescein isothiocyanate (FITC) (Mab11 from BD), anti-CD95-PE (DX2 from BD), anti-gamma interferon (IFN-γ)-PE-Cy7 (B27 from BD), anti-CD28-PerCP-Cy5.5 (L293 from BD), anti-interleukin-2 (IL-2)-APC (MQ1-17H12 from BD), anti-CD4-AmCyan (L200 from BD), anti-CD3-Alexa Fluor 700 (SP34.2 from BD), and anti-CD8α-APC-cy7 (SK1 from BD).
CD4+ T-lymphocyte counts and CD4+ memory subsets.Whole blood collected in EDTA was surface stained with anti-CD3-PerCP-Cy5.5, anti-CD4-FITC, anti-CD95-APC, and anti-CD28-PE. Peripheral blood CD4+ T-lymphocyte counts were calculated by multiplying the percentage of CD3+ CD4+ T lymphocytes by the total lymphocyte counts. The percentages of central, naïve, and effector memory cells were calculated by multiplying the percentages of CD28+ CD95+, CD28+ CD95−, and CD28− CD95+ T lymphocytes by the total lymphocyte counts.
IFN-γ ELISPOT assays.Multiscreen 96-well plates were coated overnight with 100 μl per well of 5 μg/ml anti-human IFN-γ antibody (B27; BD Pharmingen) in endotoxin-free Dulbecco's PBS (D-PBS). The plates were then washed three times with D-PBS containing 0.25% Tween 20, blocked for 2 h with D-PBS containing 5% fetal bovine serum to remove the Tween 20, and incubated with peptide pools and 2 × 105 PBMCs in triplicate in 100-μl reaction mixture volumes. The peptide pool used in this study spanning the SIVmac239 Gag protein was comprised of 15-amino-acid peptides overlapping by 11 amino acids. Each peptide in a pool was present at a 1-μg/ml concentration. Following an 18-h incubation at 37°C, the plates were washed nine times with D-PBS containing 0.25% Tween 20 and once with distilled water. The plates were then incubated with 2 μg/ml biotinylated rabbit anti-human IFN-γ (Biosource) for 2 h at room temperature, washed six times with Coulter wash (Beckman Coulter), and incubated for 2.5 h with a 1:500 dilution of streptavidin-alkaline phosphatase (Southern Biotechnology). After five washes with Coulter wash and one with D-PBS, the plates were developed with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) chromogen (Pierce). The process was stopped by washing with tap water, and the plates were air dried and read with an enzyme-linked immunospot (ELISPOT) reader (Hitech Instruments) using Image-Pro Plus image-processing software (version 4.1) (Media Cybernetics, Des Moines, IA).
PBMC stimulation and intracellular cytokine staining.Purified PBMCs were isolated from EDTA-anticoagulated blood and incubated at 37°C in a 5% CO2 environment for 6 h in the presence of RPMI 1640-10% fetal calf serum alone (unstimulated), a pool of 15-mer Gag peptides (5 μg/ml [each peptide]), or staphylococcal enterotoxin B (5 μg/ml; Sigma-Aldrich) as a positive control. All cultures contained monensin (GolgiStop; BD Biosciences) as well as 1 μg/ml of anti-CD49d (BD Biosciences). The cultured cells were stained with monoclonal antibodies specific for cell surface molecules (CD3, CD4, CD8, CD28, and CD95) and with an amine dye (Invitrogen) to discriminate live from dead cells. After being fixed with Cytofix/Cytoperm solution (BD Biosciences), cells were permeabilized and stained with antibodies specific for IFN-γ, TNF-α, and IL-2. Labeled cells were fixed in 1.5% formaldehyde-PBS. Samples were collected on an LSR II instrument (BD Biosceiences) and analyzed using FlowJo software (Tree Star). Approximately 200,000 to 1,000,000 events were collected per sample. The background level of cytokine staining varied within different samples and different cytokine patterns but was typically <0.01% of the level of the CD4+ T cells (median, 0%) and <0.05% of the level of the CD8+ T cells (median, 0.01%). All data are reported after background correction. The only samples considered positive were those in which the percentage of cytokine-staining cells was at least twice that of the background.
Virus neutralization assay.Plasma samples were collected from all 14 infected animals immediately prior to intrarectal exposure to the second virus. Neutralizing antibodies were measured in a luciferase reporter gene assay that utilized either TZM-bl or 5.25.EGFP.Luc.M7 (M7-Luc) cells as described previously (33). The 50% inhibitory dose (ID50) was defined as the plasma dilution that resulted in a 50% reduction in relative luminescence units (RLU) compared to virus control wells after subtraction of background RLU. Assay stocks of uncloned SIVsmE660 were generated in CEMx174 cells. Assay stocks of the Env-pseudotyped virus, SIVmac251/CS.41, were generated by cotransfection of a SIVmac251CS Env plasmid and an Env-deficient HIV backbone plasmid (pSG3ΔEnv) in 293T cells. Both viral stocks were made cell free by filtration through 0.45-μm pores and stored at −70°C until use.
Statistical analyses.Statistical analyses and graphical presentations were computed with GraphPad Prism, using nonparametric Wilcoxon rank sum tests and Mann-Whitney U test. P values of <0.05 were considered significant.
SIVmac251 and SIVsmE660 differ by typical intraclade HIV-1 distance.To evaluate the genetic relatedness of two isolates of SIV that are frequently used in nonhuman primate studies, we compared the genetic distance between SIVmac251 and SIVsmE660 to intraclade and interclade HIV-1 sequence distances. We used HIV clade B and C sequences in the Los Alamos HIV Sequence Database to generate our estimates of HIV-1 interclade and intraclade diversity. We used one sequence per person for these alignments. We analyzed 11,484 pairs of sequences for gag, 21,177 pairs of sequences for env, 32,465 pairs of sequences for nef, and 7,140 pairs of sequences for pol. Figure 1 shows the distribution of normalized frequencies for percent similarity of intraclade and interclade pairwise comparisons. The calculated distances between SIVsmE660 and SIVmac251 at gag, pol, env, and nef are plotted in each panel. As shown in Fig. 1A and B, the distance between gag and env of the two SIV strains is similar to HIV-1 clade B and C intraclade distances, with distances of 0.91 and 0.83, respectively. In contrast, the distances between the two SIV isolates in pol and nef are of the magnitude seen in interclade differences in HIV-1 (Fig. 1C and D). Therefore, these two pathogenic SIV isolates are well suited for use in an SIV model of superinfection because their two key foci, env and gag, have differences that reflect a degree of sequence heterogeneity comparable to different circulating HIV-1 isolates within the same clade.
Plasma SIV RNA levels following primary infection.We then established cohorts of rhesus monkeys that were infected with one or the other of these two strains of SIV. The viruses and routes of administration used to initiate these infections are summarized in Table 2. Eight animals were initially infected with SIVmac251 (Fig. 2A), and six animals were initially infected with SIVsmE660 (Fig. 2B). Infection was successfully established in 9 of these 14 monkeys via the intrarectal route. However, 5 of 14 monkeys did not exhibit detectable viremia after 18 sequential intrarectal inoculations and had to be inoculated intravenously to initiate the primary infection (CR53, AV74, and CG5G with SIVmac251 and CR54 and CP37 with SIVsmE660).
Viral replication during primary infection occurred with kinetics typical of SIV replication in naïve rhesus monkeys. Moreover, SIV replication kinetics did not differ significantly between animals that became infected by mucosal or intravenous routes. Monkeys that were infected with SIVmac251 all developed uniform peak plasma viral RNA levels of 6 to 7 logs at 14 days after virus inoculation followed by a sustained viremia of 4 to 6 logs of plasma viral RNA, with the exception of one monkey (CT76) who had undetectable viremia by 700 days postinfection.
In the cohort of monkeys infected by SIVsmE660, monkeys had peak plasma viral RNA levels of 5 to 8 logs at 14 days after virus inoculation, followed by sustained viremia of 5 to 7 logs of plasma viral RNA in animals CP37 and CP23. However, three of the monkeys infected with SIVsmE660 (CP3C, CG7G, and AK9F) had undetectable plasma viral RNA levels by 700 days postinfection, while monkey CR54 had an undetectable plasma viral RNA levels by 85 days postinfection. This wide range in peak and set point viremias in monkeys infected with SIVsmE660 has been previously described (7, 19, 35). Since plasma viral RNA levels at peak and set point in some of the SIVsmE660-infected monkeys (CP37, CP23, and CG7G) were of a magnitude comparable to that seen in monkeys following SIVmac251 infection, the variability in SIVsmE660 replication levels in monkeys likely reflects a host factor effect rather than an intrinsic lack of replicative capacity of the SIVsmE660 strain.
Plasma SIV RNA levels following superinfection.Once set point plasma virus RNA levels were reached, all monkeys were exposed to the heterologous virus by 6 weekly intrarectal inoculations. The duration of primary infection and plasma virus RNA levels at time of exposure to the second virus are summarized in Table 2. The eight SIVmac251-infected monkeys and six SIVsmE660-infected monkeys were then monitored for evidence of superinfection by assessing plasma SIVmac251 and SIVsmE660 RNA weekly for 20 weeks.
To monitor the viral replication dynamics for each SIV strain in the dually infected monkeys, we developed a qRT-PCR assay using strain-specific probes. Figure 3 shows the replication kinetics of the two strains of SIV following the first and second infections. As depicted in Fig. 3A, six of six monkeys that were initially infected with SIVsmE660 became superinfected with SIVmac251. Of the eight monkeys that were initially infected with SIVmac251, six became superinfected with SIVsmE660 (Fig. 3B). Viral RNA of the heterologous SIV strain was detected by 14 to 21 days after challenge. In 11 of 12 superinfected animals, with the exception of AK9F, the levels of plasma viral RNA of the second virus at peak viremia were 1 to 4 logs lower than the peak viremia of the first virus. In addition, the levels of plasma viral RNA of the second virus also declined rapidly to undetectable levels in six animals (CR54, CP23, CR53, PBE, AH4X, and CG71), while the viral load persisted at low levels in the remaining six animals (CP37, CG7G, CP3C, AK9F, CP1W, and CT76). The presence of the superinfecting virus at multiple time points was confirmed in each animal by direct sequencing.
Of the 14 infected animals that were exposed to a heterologous virus, only 2 (AV74 and CG5G) that were initially infected with SIVmac251 resisted superinfection with the heterologous virus (Fig. 3C). There was no detectable SIVsmE660 viral RNA in these animals for 20 weeks after exposure. The absence of replication by the second virus was verified by direct sequencing (data not shown).
No apparent acceleration in disease progression after superinfection.Interestingly, we observed an increase in plasma viral RNA levels of the primary virus (Fig. 3A and B) and a transient decline in CD4+ T cells following superinfection in all of the animals, except AH4X (Fig. 4A), CP3C, and AK9F (Fig. 4B). This finding is consistent with case reports of HIV superinfection in which superinfected individuals developed a transient perturbation in total plasma viral RNA levels in association with a clinical prodrome that aroused suspicion that an intervening event might have caused a sudden rise in viral load (2, 26, 27, 42, 60, 67). The CD4+ T-cell counts re-equilibrated 2 to 6 weeks after superinfection, and a small increase in the CD4+ T-cell counts in some of the animals was observed from 42 to 126 days after superinfection (CT76, CP1W, CG71, CP3C, AK9F, and CG7G). We did not perform statistical analyses on the differences in the CD4+ T-cell decline between superinfected and nonsuperinfected animals due to the small sample size of animals that resisted superinfection, but the trends in changes of CD4+ T-cell counts were indistinguishable between all animals. Therefore, there appeared to be no acceleration in disease progress in the superinfected monkeys as a consequence of superinfection.
Peak viral replication following the second infection was lower than peak viral replication following the first infection.Of the 12 monkeys that became superinfected, 11 animals efficiently controlled the second virus at peak viremia, with the exception of AK9F. Peak replication following the second virus infection was lower than peak replication after the first infection in each monkey (Fig. 5A). The decrease in peak viremia was statistically significant as determined by the paired Wilcoxon rank sum test (P = 0.001). Furthermore, when considered as a cohort, the median peak viral load value following the second infection was lower than that observed following the first infection (Fig. 5B). The difference in the median values and interquartile ranges of peak viremia between the first and second infections was statistically significant as determined by the unpaired Mann-Whitney U test (P < 0.0001).
Susceptibility to superinfection was not associated with time after the first infection or persistence of the primary virus.In these two cohorts of monkeys, superinfection was initiated between 3 and 20 months after the primary infection (Table 2). This large window of susceptibility suggests that infected individuals are likely susceptible to superinfection regardless of the state of immune competence of the host or the maturity of the immune response to the initial virus. Superinfection can occur after the immune response against the initial infection has had time to develop and mature. In addition, since 10 of 12 superinfected animals harbored the Mamu-A*01, -B*08, and -B*17 alleles (Table 2), susceptibility to superinfection appears not to be a consequence of major histocompatibility complex alleles that are associated with relatively efficient viral control.
Furthermore, the likelihood of acquiring a second virus appears not to be correlated with the persistence of replication of the primary virus at the time of exposure to the heterologous virus (Table 2). Some animals became superinfected despite relatively high levels of replication of the primary virus, ranging from 104 to 106 RNA copies/ml in the plasma (CP23, CP37, CP1W, PBE, CG71, AH4X, and CR53), while others became superinfected in the setting of undetectable or low-level replication of the primary virus, ranging from 102 to 103 RNA copies/ml in the plasma (CP3C, CG7G, AK9F, CR54, and CT76).
Interestingly, in animals that had a high-set-point viremia following exposure to the first virus, either SIVmac251 (CP1W, CR53, PBE, AH4X, and CG71) or SIVsmE660 (CP37 and CP23), the second virus was efficiently controlled after superinfection while the first infecting virus remained the predominant viral quasispecies in the plasma. In contrast, in animals that had undetectable plasma viral RNA levels following exposure to SIVsmE660 (CG7G, CP3C, and AK9F) or SIVmac251 (CT76) prior to superinfection, the heterologous virus replaced the first viral strain after superinfection even in monkeys with blunted peak replication of the second virus. Only one monkey in the cohort, CR54, was able to control both viruses to undetectable levels. These data suggest that, although direct viral interference did not contribute to susceptibility to superinfection, it may have influenced the viral replication dynamics of the second virus relative to the primary virus after superinfection.
Susceptibility to superinfection was not associated with absolute CD4+ T-cell counts or percent central memory CD4+ T cells.To determine if there were any clinical parameters associated with relative susceptibility to superinfection in these cohorts of monkeys, we assessed the absolute CD4+ T-cell counts and the percentage of CD4+ T lymphocytes that were central memory cells immediately prior to the exposure of these animals to the heterologous virus. There was no difference between absolute CD4+ T-cell counts or the percentage of CD4+ central memory T cells in the animals that became superinfected and those that resisted superinfection (Fig. 6A and B). Although a statistical analysis could not be performed to validate this observation due to the small sample size of animals that resisted superinfection, the absolute CD4+ T-cell counts and the percentage of central memory CD4+ T cells of animals that resisted superinfection were within the range of the corresponding parameters in animals that became superinfected. In addition, we also analyzed the percentages of effector and naïve memory CD4+ T cells and found that there were no differences in these values between the two groups of monkeys (data not shown). Together, these data indicate that animals with immune systems that are more damaged by a prior SIV infection appeared not to have an increased susceptibility to superinfection.
Susceptibility to superinfection was not associated with virus-specific cellular immune responses.To determine whether systemic virus-specific cellular immune responses conferred protection against heterologous virus in the monkeys that resisted superinfection, all rhesus monkeys were evaluated for SIV-specific cellular immunity immediately prior to exposure to the heterologous virus. Cellular immunity to SIV was first evaluated using an ELISPOT assay to assess PBMC IFN-γ responses following exposure to a pool of SIV Gag peptides (Fig. 7A). SIV-specific T-cell responses were indistinguishable between the animals that became superinfected and those that resisted superinfection.
SIV-specific CD4+ and CD8+ T-lymphocyte function was further evaluated by intracellular cytokine staining. Immediately prior to exposure to the heterologous virus, PBMC production of IFN-γ, TNF-α, and IL-2 was assessed after stimulation with SIV Gag peptide pools. We were able to detect virus-specific CD4+ (Fig. 7B) and CD8+ (Fig. 7C) T-lymphocyte responses in PBMCs of all monkeys. We did not perform statistical analyses on the differences in cytokine secretion between the two groups of monkeys due to the small sample size of animals that resisted superinfection. However, the cytokine responses of the two animals that resisted superinfection were within the range of the corresponding parameters in animals that became superinfected. Therefore, the qualitative and quantitative cell-mediated SIV-specific immune responses of monkeys that became superinfected and those that resisted superinfection appeared to be indistinguishable. These findings suggest that SIV-specific cellular immune responses likely did not account for the variability in the susceptibility of these monkeys to superinfection.
Antibody responses did not protect against superinfection.The role of neutralizing antibody responses in protecting against HIV superinfection is not clear (5, 49, 50). To assess whether SIV-specific antibodies played a role in the resistance to superinfection in these cohorts of animals, plasma samples harvested just prior to the heterologous viral challenge were assayed for neutralizing antibody responses elicited by the primary SIV infection. The ability of plasma antibody to neutralize SIVsmE660 and SIVmac251 was measured in luciferase reporter gene neutralizing antibody assays using uncloned SIVsmE660 and pseudoviruses expressing viral Envelope cloned from SIVmac251CS.41 (33). The serum ID50 neutralizing titers against both viruses are shown in Table 3, Plasma from five of six monkeys (except CR54) that were first infected with SIVsmE660 neutralized the homologous SIVsmE660 (1:62 to 1:508), while plasma from five of eight SIVmac251-infected monkeys neutralized homologous SIVmac251 (1:33 to 1:215).
To investigate whether the antibodies generated by these animals following primary infection have the ability to neutralize the heterologous virus, we assayed the plasma of the monkeys for neutralization activity against the second virus before their exposure to that virus. As shown in Table 3, animals initially infected with SIVsmE660 generated undetectable or low titers of neutralizing antibodies to SIVmac251 (ranging from undetectable to 1:41). We also detected neutralizing antibodies against SIVsmE660 in six of eight animals that were initially infected with SIVmac251 (ranging from 1:73 to 1:245). The titers against the heterologous SIVsmE660 in the SIVmac251-infected animals were not significantly lower than the titers against the homologous SIVsmE660 in SIVsmE660-infected animals (P = 0.95, Mann-Whitney test).
Interestingly, animals AV74 and CG5G, who were initially infected with SIVmac251 and subsequently resisted superinfection with SIVsmE660, had neutralizing antibodies against SIVsmE660 prior to exposure to this heterologous virus. However, the titers of these antibodies were within the range of antibody titers against SIVsmE660 that were generated by other SIVmac251-infected animals that became superinfected following exposure to SIVsmE660. We did not perform statistical analyses of the differences in antibody titers against SIVsmE660 between the SIVmac251-infected monkeys that resisted superinfection and the SIVmac251- infected monkeys that became superinfected because of the small number of animals that resisted superinfection. Nevertheless, the titers of neutralizing antibodies specific for the heterologous viruses that were elicited during primary infection appear to not have influenced the susceptibility of monkeys to superinfection.
HIV superinfection has important implications for vaccine prevention of HIV infection and the global genetic diversity of HIV. In this study, we used intrarectal inoculations of two replication-competent strains of SIV to simulate HIV-1 superinfection and employed quantitative analyses of viral RNA using strain-specific primers to define the replication dynamics of each virus over time. We demonstrated that immune responses generated during primary infection that are capable of controlling one strain of SIV do not preclude subsequent infection with a second strain of SIV. Superinfection occurred as early as 3 months and as late as 2 years following primary infection, and susceptibilities to superinfection appeared to be independent of classical adaptive immune responses or the level of replication of the primary virus, even though we were not able to evaluate the statistical significance of these parameters because of the small number of animals that resisted superinfection in this study. Importantly, the replication of the superinfecting virus during the first days following exposure was attenuated compared with the replication of the primary virus. The relative susceptibility of monkeys to superinfection in the present study could not be attributed to a difference in the replication capacities of these two strains of SIV, since superinfection occurred in both cohorts of animals regardless of which virus was used to establish the first infection. Furthermore, the ability of both SIVmac251 and SIVsmE660 to maintain dominance in superinfected monkeys suggests that these two SIV strains are comparable in their fitness.
Previous nonhuman primate studies using a live attenuated immunodeficiency virus to generate protection against a pathogenic immunodeficiency virus challenge provide an important context for the present findings. Although such live attenuated viruses can confer protection against a homologous virus challenge (11, 14, 25, 36, 57, 64), they provide only partial protection against a heterologous virus infection (16, 34, 44, 63). The results of the present study are consistent with those findings in that prior infection did not prevent superinfection with a heterologous virus, but did damp replication of the second virus at peak and in the post-acute phase of superinfection. Interestingly, the two animals that resisted superinfection had also resisted 18 attempts at the first infection by the intrarectal route and required intravenous inoculation to establish primary infection. This finding raises the possibility that variations in the mucosal barrier rather than specific immunological mechanisms may have contributed to differences in susceptibility to mucosal infection in this cohort of animals (29).
Just as the correlates of protective immunity have not yet been defined for the protection observed in monkeys that have received a live attenuated SIV vaccine (1, 3, 11, 44, 45, 54, 55), the mechanisms accounting for the partial protection observed against superinfection in our study are not clear. We used pooled peptides corresponding to SIVmac239 Gag to evaluate virus-specific cellular immune responses because the cross-reactive responses are likely the most germane to controlling the replication of the heterologous virus. Nevertheless, there may be additional T-cell responses that contribute to controlling the second virus that are not detected using SIVmac239 peptides. A recent study by Reynolds et al. examining the ability of live-attenuated SIV to protect macaques against heterologous virus challenge implicated major histocompatibility complex (MHC) class I-restricted CD8+ cellular responses in reducing heterologous viral replication during the chronic phase of infection (44). However, further studies are needed to elucidate the relative contributions of CD8+ T cells and other factors, including CD4+ T cells, antibodies, and NK cells, in the acute phase of replication of the second virus. A decrease in the number of potential target cells as a result of depletion of memory CD4+ T cells in the lamina propria in the gut and lymph nodes following the first infection may have contributed to the reduction and magnitude of peak viremia observed following the second infection. Further detailed characterization of CCR5+ transitional and effector memory T cells in mucosal effector sites is needed to determine the availability of target cells. Other factors, such as innate immune responses or viral interference, may have also contributed to the relative protection observed against the superinfecting virus.
The present study of superinfection in the SIV/rhesus monkey model has important implications for HIV pathogenesis and vaccine development. Although this SIV model of superinfection utilized a higher-dose mucosal challenge to establish superinfection than likely occurs in human cases of HIV superinfection, the findings in the present study suggest that HIV superinfection can occur readily throughout the course of infection. Therefore, the prevalence of HIV superinfection is likely underestimated, especially in cases whose only clinical manifestation is transient low-level replication of the second virus. Similar to human cases of HIV superinfection described by Casado and Piantadosi et al. (8, 40), SIV superinfection also did not necessarily lead to increases in viral load and clinical deterioration. This could be because both SIV strains that were used in this study are comparably fit, and therefore the persistence of either one or both may not dramatically affect disease progression. In contrast, the clinical sequelae in HIV superinfection may have more variable outcomes than what we observed in this study, since the relative dynamics of the two viruses may be markedly different as a consequence of their relative replication fitness. Interestingly, although superinfection is likely a common phenomenon in HIV-1 infections, it may not have clinical consequences if the two viruses are equivalent in their fitness or if the superinfecting virus transiently replicates at a low level. In contrast to this, superinfection likely has a more profound impact on the sensitivity of circulating viruses to antiretroviral therapy and global HIV genetic diversity as a consequence of viral recombination.
Creating a vaccine that can protect against infection by a virus with the genetic heterogeneity of HIV is a daunting challenge, given that immune responses generated after live SIV infection do not prevent infection of macaques by a heterologous SIV isolate in the nonhuman primate model. Nevertheless, the phenomenon of HIV/SIV superinfection should not discourage the pursuit of an AIDS vaccine, since effective vaccines for viruses such as the mumps and measles viruses also do not prevent entry of virus into the body. While the immune system does not prevent new strains of virus from establishing infections, it can limit the spread of those viruses and attenuate the pathogenic sequelae of infection. Further dissection of the virologic and immune correlates of protection against superinfection in monkeys may provide important insights into the nature of immune responses that are required to provide protective immunity against an immunodeficiency virus infection.
We thank Vanessa Hirsch for providing virus stock SIVsmE660.
This work was supported by NIH NIAID PHS grants K08-AI069995 (W.W.Y.) and AI-067854 (W.W.Y. and N.L.L.) and the Center for HIV/AIDS Vaccine Immunology.
- Received 23 October 2008.
- Accepted 23 December 2008.
- ↵*Corresponding author. Mailing address: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, CLS 1043, 3 Blackfan Circle, Boston, MA 02115. Phone: (617) 735-4400. Fax: (617) 735-4527. E-mail: nletvin{at}bidmc.harvard.edu
↵▿ Published ahead of print on 7 January 2009.
- American Society for Microbiology


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OmniJoy by Cerulean Submergence, a multipurpose Raspberry Pi powered robot controller and gamepad, funding on Kickstarter through Fri, February 2 2018 10:45 AM EST.
Friday, January 19, 2018 - 7:00pm -- Raleigh's Quail Ridge Books hosts Lyndsay Ely for her new young adult novel Gunslinger Girl, "a thrilling dystopian Western starring a teenage sharpshooter to rival Annie Oakley."
Tuesday, February 13, 2018 - 7:00pm -- Chapel Hill's Flyleaf Books hosts John Darnielle for the paperback release of his latest novel Universal Harvester. "Life in a small town takes a dark turn when mysterious footage begins appearing on VHS cassettes at the local Video Hut."
Thursday, February 15, 2018 - 7:00pm -- Raleigh's Quail Ridge Books hosts John Kessel for his new novel Pride and Prometheus. "Pride and Prejudice meets Frankenstein in this NCSU English professor's literary mash-up of the classics as Mary Bennet falls for Victor Frankenstein and befriends his monstrous creature."
- May Newsletter: Zeno Alexander, Valerie Nieman, Lewis Shiner, Free Comic Book Day, Star Wars Day, and more
- January Newsletter: Robin Kirk, Holly Black, illogiCon with Annalee Newitz, and more
- February Newsletter: Colson Whitehead, John Darnielle, John Kessel, the Playthrough Gaming Convention, and more
- The year in NC science fiction and fantasy, part 3 of 3: Beyond the Triangle
- The year in NC science fiction and fantasy, part 2 of 3: The Rest of What's Best from the Triangle
Tag Archives: poetry
Poetry poetry poetry!
Here's some speculative poetry news, both relating to Bull Spec. First, as Bull Spec poetry editor Dan Campbell notes: Issue 7 of Bull Spec is now for sale! It has poetry by alexa_seidel, Daniel A. Rabuzzi, thunderpigeon, Deborah Walker, Athena Andreadis, Mari Ness, … Continue reading
Posted in poetry, Uncategorized Tagged poetry 1 Comment


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Sweaty Tee Review !
Sweaty - Tee Review
By: Christian Mosi
Sweaty Tee is A sweat resistant shirt that is available in a tee, tank top, or sleeveless shirt. It's built in with a sweat-absorbing terry cloth mini towel to wipe away sweat. You are able to choose your own design and color ! What I personally love about this tee is how lightweight it is ! This material is perfect for your sportswear attire. The tee is also very affordable starting at $17.99 ! Check out my review below to see my final thoughts ! A special thank you to the founder Joan for this great Tee !


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Spotlight on Innovation: Building Resilient Systems with Heidi Waterhouse
Untangling features and making sense of microservices
Join us for this edition of Spotlight on Innovation to hear from LaunchDarkly's Heidi Waterhouse, who will share some of the biggest challenges in feature management. In a perfect world, we'd develop tidy, discrete features that are easy to manage. But in reality, we're often struggling with a complicated snarl of features that are difficult to test and deploy. Heidi explains how you can use feature management design to build modular, testable, resilient systems—topics which we'll also be covering at our Velocity and Software Architecture conferences this June in San Jose.
O'Reilly Spotlight explores emerging business and technology topics and ideas through a series of one-hour interactive events. You'll engage in a live conversation with experts, sharing your questions and ideas while hearing their unique perspectives, insights, fears, and predictions for the future.
In every edition of Spotlight on Innovation, you'll learn about, discuss, and debate the future. Best of all, you'll discover what successful companies have in common and how you can follow their lead with small practical steps to transform your organization and prepare for the Next Economy.
Want to learn more about how to design, build, and manage resilient systems? Attend the O'Reilly Velocity and Software Architecture Conferences in San Jose, CA, June 10–13, to get the insights you need to drive results for your business. O'Reilly online learning members receive a 25% discount when they register using promo code LEARN.
What you'll learn-and how you can apply it
By the end of this live show, you'll better understand:
- How to design for feature management
- How to start migrating existing features and services to a more modular, testable, and resilient system
- How to make changes to containers without needing to rebuild them entirely
- How to test a distributed architecture on your laptop
This event is for you because:
- You want to identify the fundamental shifts that are transforming the business landscape and customer needs and address them early.
- You want to learn from other businesses that have successfully transformed and found solutions to customer problems.
This training course is for you because...
- You want to identify the fundamental shifts that are transforming the business landscape and customer needs and address them early.
- You want to learn from other businesses that have successfully transformed and found solutions to customer problems.
- Come with your questions for Heidi Waterhouse
- Have a pen and paper handy to capture notes, insights, and inspiration
Recommended follow-up:
- Read Building Microservices (book)
- Take Microservice Fundamentals (live online training course with Sam Newman)
- Explore Scaling Microservices (learning path)
- Read Production-Ready Microservices (book)
About your instructor
Heidi Waterhouse is a developer advocate at LaunchDarkly. She delights in working at the intersection of usability, risk reduction, and cutting-edge technology. One of her favorite hobbies is talking to developers about things they already knew but had never thought of that way before. She sews all her conference dresses so that she's sure there's a pocket for the mic.
The timeframes are only estimates and may vary according to how the class is progressing
Tuesday, April 23, 2019 at 9:00am PT / 12:00pm ET
- Introduction and presentation (15 minutes)
- Interactive discussion and Q&A (45 minutes)


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Restaurant Review: Landry's Seafood
May 16, 2017
Overlooking the Waterway in The Woodlands, Landry's Seafood restaurant has one of the best locations in the area. At the patio, patrons can enjoy their meal with a view of the water and walking trail plus a constant breeze.
For those looking for a great lunch or happy hour, Landry's offers an extensive menu selection with something for everyone. However, one of the best deals on the menu is the Lunch Box for $15.00, which includes an appetizer, salad and main course.
If you are more of a taco person, the beer batter fried fish tacos with pepper jack and chili aioli, served with French fries and onion rings, are a great option in a shareable portion. A gluten free menu is available upon request.
During March and April, Landry's Seafood will offer a crawfish boil every Saturday on the patio with live music, from 1 pm till 5 pm at $5.00 per pound.
Landry's Seafood in The Woodlands offers not only great food, but a great value with a beautiful view and ambiance.


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Pythagoria Stores
No deals
No prices to compare at the moment.
What is Pythagoria about?
Pythagoria is a brain-teasing number puzzle game. The puzzle is simple to explain: find the missing value, which is denoted by a question mark highlighted in color. The only mathematics you need to know to get started is that the area of a rectangle is the length multiplied by the width. Only basic math knowledge is required to understand and solve the puzzles.
Here's what makes the puzzle hard: you are not allowed to use fractions in the solutions. All area puzzles can be solved using only whole numbers.
Pythagoria is fun, brain-teasing puzzle game made for all ages. Solve the area puzzles from easier ones to harder ones, and remember that some of problems need kind of inspiration to solve.
Title: PythagoriaTime: 1-2 hoursFirst of all: Thanks to the developer who provided me with the game.
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Today is my birthday!!!!! In honor of me turning 22 I've decided to share with you 22 reasons why I fell in love and am currently in love with fashion photography.Read More
the capture blog
7 Fashion Photography Blogs That Will Benefit You
I decided to share with you guys a few blogs that I find inspirational and beneficial to fashion photographers and those that appreciate the medium.Read More
Interview: SCAD ATL Alumna, Claudia Bost
Claudia Bost is a fashion photographer and another fellow SCAD Atlanta alumna that is rising the ranks of the local fashion scene in Atlanta. I met up with her on a busy Saturday morning at Dancing Goats at the buzzworthy Ponce City Market to chat about the Atlanta fashion scene, the internet famous Asiyami Gold and being a young creative.Read More
Interview: SCAD ATL Alumna, Brittany Wages
Brittany Wages is a lifestyle, fashion and portrait photographer and fellow SCAD Atlanta alumna. We sat down at Alon's Bakery in Highlands, after an early morning test shoot with Spin Style, to chat about gaining photo experience post-college and what we didn't learn in art school.Read More
Interview: Forrest Aguar Photography
Forrest Aguar is a working photographer and photo assistant here in Atlanta. After graduation from the University of Georgia in 2013, he has been steadily gaining his footing in the photo industry. With a unique style based in minimalist compositions and flat, yet vibrant color palettes, Aguar is a creative image maker that's worth watching out for.Read More
Interview: Jamie Hopper Photography
Jamie Hopper is a local fashion photographer based here in Atlanta. I sat down with her at a cool hybrid coffee shop and retail store here in Atlanta called Chrome Yellow Trading Company to pick her brain.Read More
Interview: Cameron Adams of Atlanta Street Fashion
Atlanta is home to a bevy of talented photographers of all genres. Here we have Cameron Adam of the blog Atlanta Street Fashion.Read More
POC in the Industry: Coreen Simpson
As a person of color (POC) in an industry that used to and still kind of is dominated by white males, I would love to take the time to recognize great fashion photographers, models and more that contribute to the art.Read More


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The program was developed in 2007 and was made available in 2008 sponsored by Citi Foundation, with the goal of identifying role models for women-led SMEs and enhancing their competitiveness through trainings and business women networks. In 2012, the fund is to be used for Citi-KOSBI women-owned enterprise academy, Citi-KOSBI women entrepreneurs' award, and networking between women business owners and college students aspiring to run startups at home and abroad.
"I expect this program will continue to help foster as many competitive women-owned businesses as possible," said Chairperson Jeon, Soo-hye of Korean Women Entrepreneurs Association showing her expectations of this 5-year long program. "I hope that the business knowledge and networks you gain from this program will help you grow your business and become role-models of women entrepreneurs," said CEO Ha to encourage CEOs of women-led SMEs.
The signing ceremony was followed by the entrance ceremony of the "5th Citi-KOSBI women-owned enterprise academy." The program catered to CEOs and middle managers of women-led SMEs will last from April 3 till Jul 10 at the KOSBI office located in Yoido. In this 14-week course, 21 subjects including leadership, global competence, fun management, business innovation, SME governmental finance, etc. are to be provided to enhance job capabilities and innovative business ideas of women CEOs. Tuition fees are approximately KRW 3 million per person, but the full amount will be sponsored by Citi Foundation.
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