AMPL Python API¶
Contents¶
- Introduction
- Getting started
- Class structure
- Python quick start
- Complete listing
- Needed modules and AMPL environment creation
- Load a model from file
- Solve a problem
- Get an AMPL entity in the programming environment (get objective value)
- Modify model data (assign values to parameters)
- Get numeric values from variables
- Get arbitrary values via ampl expressions
- Delete the AMPL object to free the resources
- Python API reference
- Python API examples
- Example 1: First steps
- Example 2: Get and set AMPL options
- Example 3: Use AMPL asynchronously
- Example 4: Get and set data through DataFrames
- Example 5: Operate with multidimensional data
- Example 6: Assign all data to a model and solve it
- Example 7: Build an efficient frontier
- Example 8: Simple heuristic


EOSeoul is a subsidiary of NEOWIZ Co., Ltd., a 22years old top South Korean video game∙tech developer and publisher based in Seoul.
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Gives EOS information, including vote, block producers, website and more. EOStat has many special features to help you find exactly what you're looking for.
With a partnership of NEOWIZ, you can play the first EOS VR rhythm game TAPSONIC. Just play and enjoy the game with blockchain technology!
CODEOS is to develop the global blockchain ecosystem by riding the EOS knowledge gap and supporting EOS developer.
Our EOS mainnet nodes location & specifications
EOSeoul Hybrid-Infrastructure
Our team consists of members from a leading online game company NEOWIZ and its start-up accelerator NEOPLY
Get the latest news from EOSeoul community.


Huahine lies 110 miles NW of Tahiti (or 70 miles from Moorea). Visible from Raiatea, Tahaa and Bora Bora, the two islands of Huahine Nui and Huahine Iti are connected by a bridge and have a combined area of 28 sq.miles.
The road winds 20 miles around the two islands, passing through small villages surrounded by banana, breadfruit, papaya and mango trees. All around both islands are plantations of vanilla, coffee, taro, watermelons and cantaloupe. There are few cars here, so it is perfect to explore on a bicycle. There are many secluded coves and uninhabited islets to visit.
Unmarked Reef south of the entrance to Fare: see details in comments at bottom of page.
Position 16°43'S, 151°02'W.
Fare is the Port of Entry.
It is NO LONGER compulsory for all yachts visiting French Polynesia to visit Papeete Customs and Immigration to complete the clearing-in process.
Yachts can now clear in or out of FP with the “gendarmes” offices (French Police) at the ports of entry in the other islands (Marquesas, Tuamotus, Gambiers, Australs etc.) and complete all customs and immigration formalities in the one place.
The Gendarmerie Nationale in Fare can deal with Immigration and Customs.
Full details can be found at French Polynesia Clearance.
Last updated July 2014.
There is no marina but a public dock in Fare is available for loading/unloading.
Space for anchoring off the village is limited, but there are many attractive anchorages on both west and east coasts of the island.
There are several reports posted adjacent with recommended anchorages.
Motu Murhimahora on the east side of Huahine comes recommended by cruisers as a good anchoring spot. Entry is via Passe Farerea. A local called Paul, whose property fronts onto the beach here, is an incredibly warm and friendly person and will undoubtedly speak with you, not wanting anything other than friendship.
Last updated June 2015.


@InProceedings{Jetley_2016_CVPR,
author = {Jetley, Saumya and Murray, Naila and Vig, Eleonora},
title = {End-To-End Saliency Mapping via Probability Distribution Prediction},
booktitle = {The IEEE Conference on Computer Vision and Pattern Recognition (CVPR)},
month = {June},
year = {2016}
}


Effective Platform Building
with Kubernetes
Wojciech Barczyński - SMACC.io | Hypatos.ai
18 December 2018
Wojciech Barczyński
- Lead Software Developer
& System Engineer
- Organizer Golang Warsaw Meetup
- Visiting Lecturer at WSB and ALK
- 2019 ➡ Trainings & Consultancy
Story
Kubernetes + Go + ...
- SMACC - Fintech / ML - since 2017
- Lyke - Mobile Fashion app - since 2016
I do not like Infra
Kubernetes
- Container management
- Battery for 12factor apps
- ...heading to integration platform
- ...becoming a framework for your Xubernetes
Kubernetes
make docker_push; kubectl create -f app-srv-dpl.yaml
Scale up! Scale down!
kubectl --replicas=3 -f app-srv-dpl.yaml
Deployment and Pods
Service and pods
Service matches pods based on labels
Basic Concepts
| Name | Purpose | |
| Service | Interface | Entry point (Service Name) |
| Deployment | Factory | How many pods, which pods |
| Pod | Implementation | 1+ docker running |
All in git
smacc-platform.git
- branches: dev, staging, master
- Generated k8s files
- ENV variables for each of environment
Similar to the Kelsey Hightower approach
Kubernetes
- Pure and generated
from simple templates with ${ENV_VAR}
- 2x kubernetes operators
Lyke
- E-commerce Indonesia
- Mobile-only
- 50k+ users
- 2M downloads
- Top 10 Fashion Apps
Google Play Store
http://www.news.getlyke.com/single-post/2016/12/02/Introducing-the-New-Beautiful-LYKE
Architecture During Migration
Bridge the new with old
Monitor legacy with new stack
SMACC
- Machine Learning FinTech
- SaaS and API platform
- From Enterprise (Deutsche Bank, AoK) to SME
- Well-known FinTech Startup in Germany
Get to K8S
- Legacy on AWS, experiments with AWS ECS :/
- Self-hosted K8S on ProfitBricks
- ooo... K8S can use Azure Active Directory :D
Run everywhere!
- Get to Microsoft ScaleUp, welcome Azure
- Luckily - Azure Kubernetes Service
- With GPU integration :)
- Our OnPrem = Our OnCloud
Keep it simple
- az aks CLI for setting k8s - README.rst
- Terraform for everything else
- Secrets: 1Password and gopass.pw
Terraform also sets our AWS
Migration was easy...
- Tech-wise yes
- Team-wise no
Difference ☠
- Two teams in Berlin and Warsaw
- Me in Warsaw
Repo .travis.yml
language: go
go:
- '1.16'
services:
- docker
install:
- curl -sL<EMAIL_ADDRESS>| bash
- if [ -f "tools/travis/install.sh" ]; then bash tools/travis/install.sh; fi;
script:
- dep ensure
- make lint
- make test
- if [ -z "${TRAVIS_TAG}" ]; then make snapshot; fi;
deploy:
- provider: script # deploy to docker repo
script: make release
skip_cleanup: true
on:
tags: true
all_branches: true
condition: "$TRAVIS_TAG =~ ^v.*"
- provider: script # to staging cluster
script: curl -sL<EMAIL_ADDRESS>| bash
skip_cleanup: true
on:
tags: true
all_branches: true
condition: "$TRAVIS_TAG =~ ^v.*"
- provider: script # enforce to dev cluster
script: curl -sL<EMAIL_ADDRESS>| bash
skip_cleanup: true
on:
all_branches: true
condition: $TRAVIS_BRANCH =~ ^master|develop$ # squad decide
notifications:
slack:
rooms:
- smaccio:PLAIN_TEXT_HASH
Makefile
|- tools
| |- Makefile
| |- kube-service.yaml
| \- kube-deployment.yaml
|
|- Dockerfile
\- Makefile
Makefile only tasks for dev
Continuous Deployment
- Github
- TravisCI
- hub.docker.com
- AKS
Continuous Deployment
git tag
and push
- smacc-platform.git
- Deploy to staging
- PR to production
What worked
- Hiding k8s
- Understandable CD process
Would do different
- More sensitive to feedback
Next
- Scale our ML trainings on the top of k8s - kubeflow
- Use CRD to bring exteranl tools to k8s
- Keeping an eye on Istio
- Deployment tool based on missy
Effective Kubernetes
- Start with small iterations
- Learn as-you-go
- Keep Kubernetes Understable
Hope the k8s community keep it this way
Effective Kubernetes
- Move configuration to runtime
- Do not terrorize your devs with K8S
- No free lunch... app must be smarter
Big thanks to my colleages in Lyke and SMACC
github.com/wojciech12/talk_effective_kubernetes
Lyke
- E-commerce
- Mobile-only
- 50k+ users
- 2M downloads
- Top 10 Fashion Apps
w Google Play Store
http://www.news.getlyke.com/single-post/2016/12/02/Introducing-the-New-Beautiful-LYKE
Now JollyChic Indonesia
Good parts
- Fast Growth
- A/B Testing
- Data-driven
- Product Manager,
UI Designer,
Mobile Dev,
and tester - one body
Bridge the new with old
Monitor legacy with new stack
Architecture During Migration
Pods
- See each other on localhost
- Live and die together
- Can expose multiple ports
Side-cars


This site isn't ready yet. Check out our Facebook group instead. https://www.facebook.com/groups/1695957843991327/ -MCP


1)
Alia will be celebrating her
birthday next week.
2)
I shall be sleeping by
the time Uncle John’s
flight arrives.
3)
You will be playing tennis
with Peter in the
afternoon.
4)
Mr. Brown will be buying
a new mobile phone for
his wife soon.
5)
We shall be staying with
our grandparents during the
holidays.
6)
Mindy and Megan will be
going on a hiking trip
with the scouts group.
7)
Pat will be feeling extremely
tired after the game of
squash.
8)
Environmentalists say that water
levels of the seas will be rising in twenty
years.
9)
Grandma will be bringing her
delicious-baked cookies for us.
10)
My cousins will be skiing
in the Alps during
their Christmas holidays.


Says something:
We just add rules to ".justreq" as follow:
{ ... "rules": [ { "url": "api/post.do", "subs": "api/post.json" }, { "url": "api/user.do", "subs": "api/user.json" } ] }
These json files of this example are located in ".jr/subs/api/"


Heap Sort Algorithm Simulator
This simulator models the behaviour of the heap sort algorithm. For simplicity, only 2 digit numbers are allowed to be sorted (including the - sign on negative numbers.
The Algorithm
| Procedure HeapSort( A ) | |||
| 1 | BuildHeap ( A ) | ||
| 2 | for i ← A.length down to 2 | ||
| 3 | exchange A[ 1 ] ↔ A[ i ] | ||
| 3 | A.heapSize ← A.heapSize - 1 | ||
| 3 | Heapify( A, 1) |
| Procedure BuildHeap( A ) | |||||
| 1 | A.heapSize ← A.length | ||||
| 2 | for i ← ⌊ A.heapSize / 2 ⌋ down to 1 | ||||
| 3 | Heapify ( A , i ) |
| Procedure Heapify( A, i ) | ||
| 1 | l ← leftChild( i ) | |
| 1 | r ← rightChild( i ) | |
| 2 | if l ≤ A.heapSize and A[ l ] > A[ i ] | |
| 3 | largest ← l | |
| 2 | else | |
| 3 | largest ← i | |
| 2 | if r ≤ A.heapSize and A[ r ] > A[ largest ] | |
| 3 | largest ← r | |
| 2 | if largest ≠ i | |
| 3 | exchange A[ i ] ↔ A[ largest ] | |
| 3 | Heapify( A, largest ) |
Enter the number of items to sort (2-20):


This is example application built with meteor-kitchen code generator without manual coding.It demonstrates simple application with user account system and admin panel.Source code (input file for generator) is here.
Continue »


Location
121 Rock Street, 21 Avenue,
New York, NY 92103-9000
Sample text. Clock to selectthe text
box. Click or double click to start
editing the text.
+1<PHONE_NUMBER>
|
|
Location121 Rock Street, 21 Avenue, Sample text. Clock to selectthe text |


This one should return the result 500 since the textarea is longer than that of the content
Scroll width


{% extends "base.html" %} {% block content %}
This web application is based on a 3-layer convolutional neural network (CNN), trained to recognise malaria parasite from human blood sample. The neural network achieved accuracy exceeding 95% on the relevant test set of more than 2700 blood sample images. Please note that the predictive accuracy of the model, depends on the quality and quantity of the training data. Therefore the model has limitations and cannot be used for definite clinical predictions! Instead it can be used for research and comparison purposes (please see notes below).


acting for film
Throughout an honest day of nipple acting work, a career woman reflects on her craft and the industry that surounds her.
Collaborative video with Emily Eddy. Acting for Film premiered on October 19, 2018 at the Portland Unknown Film Festival.
This video is password protected due to nudity, please email<EMAIL_ADDRESS>for access.


But screw your courage
to the sticking-place,
and we’ll not fail.
Give me daggers.
Unsex me here.
Infirm purpose!
You lack the season of all natures, sleep.
Banquo’s buried.
What is done is done.
cannot be undone.
All the perfumes of Arabia will not sweeten this little hand.
My hands are of your colour; but I shame.

Give me daggers.
Unsex me here.
You lack the season of all natures, sleep.
Infirm purpose!
Banquo’s buried.
What is done is done.
Out, I say!
Out, Damned spot!
Nought’s had, all’s spent,
Where our desire is got without content;
’Tis safer to be that which we destroy
Than by destruction dwell in doubtful joy.


the url for the saved report.
raise gen.Return("https://foo.com/%s" % report_id)
def _build_static_dir():
Return the path to lib/streamlit/static.
the url for the saved report.
raise NotImplementedError()
def _get_static_dir():
Return the path to lib/streamlit/static.


New Gateway Job Board Mobile App Coming soon!
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ngAria
The ngAria
module provides support for common
ARIA
attributes that convey state or semantic information about the application for users
of assistive technologies, such as screen readers.
For ngAria to do its magic, simply include the module as a dependency. The directives supported
by ngAria are:
ngModel
, ngDisabled
, ngShow
, ngHide
, ngClick
, ngDblClick
, and ngMessages
.
Below is a more detailed breakdown of the attributes handled by ngAria:
| Directive | Supported Attributes |
|---|---|
| ngDisabled | aria-disabled |
| ngShow | aria-hidden |
| ngHide | aria-hidden |
| ngDblclick | tabindex |
| ngMessages | aria-live |
| ngModel | aria-checked, aria-valuemin, aria-valuemax, aria-valuenow, aria-invalid, aria-required, input roles |
| ngClick | tabindex, keypress event, button role |
Find out more information about each directive by reading the ngAria Developer Guide.
Using ngDisabled with ngAria:
<md-checkbox ng-disabled="disabled">
Becomes:
<md-checkbox ng-disabled="disabled" aria-disabled="true">
It's possible to disable individual attributes added by ngAria with the config method. For more details, see the Developer Guide.
First include angular-aria.js
in your HTML:
<script src="angular.js">
<script src="angular-aria.js">
You can download this file from the following places:
//ajax.googleapis.com/ajax/libs/angularjs/X.Y.Z/angular-aria.js
bower install<EMAIL_ADDRESS>"//code.angularjs.org/X.Y.Z/angular-aria.js"
where X.Y.Z is the AngularJS version you are running.
Then load the module in your application by adding it as a dependent module:
angular.module('app', ['ngAria']);
With that you're ready to get started!
| Name | Description |
|---|---|
| $ariaProvider | Used for configuring the ARIA attributes injected and managed by ngAria. |
| Name | Description |
|---|---|
| $aria |


The HTML5 test score is an indication of how well your browser supports the HTML5 standard and related specifications. Find out which parts of HTML5 are supported by your browser today and compare the results with other browsers.
The HTML5 test does not try to test all of the new features offered by HTML5, nor does it try to test the functionality of each feature it does detect. Despite these shortcomings we hope that by quantifying the level of support users and web developers will get an idea of how hard the browser manufacturers work on improving their browsers and the web as a development platform.
The score is calculated by testing for the many new features of HTML5. Each feature is worth one or more points. Apart from the main HTML5 specification and other specifications created the W3C HTML Working Group or WHATWG, this test also awards points for supporting related drafts and specifications.
Please be aware that although the HTML5 specification is now an official recommendation, other specifications that are being tested are still in development and could change before receiving an official status. In the future new tests will be added for new specifications and existing tests will be updated when the specifications change.
Help us improve HTML5 test by donating
This will allow us to spend more time on HTML5test.com and acquire more devices for our testing lab.


WITNESS LIST
Redistricting, Select Committee
June 12, 2013 - 2:00 PM
For:
Bond, Bruce (Self)
Bowles, Larry (Self)
Cheng, Albert (Self)
Gibson, Michael (Self)
Against:
Arline, Tiffany (Texas Organizing Project)
Blavier, Rhys M. (Self)
Caldwell, James (T.O.P. Texas Organizing Project)
Calvert, Rogene Gee (Texas Asian American Redistricting Iniative)
Gonzalez, Maria (Self)
Honore, Stephan (Self)
Kennedy, Shelley (Self)
Kerner, Debra (Self)
Lewis, Brad (Self)
Lockett, Joseph (Self)
Longhofer, Kyle (Self)
Moffitt, Jill (Self)
Patterson, Andrew (Self)
Sorola-Pohlman, Lenora (Self)
Vogas, James (Self)
On:
Larsen, Yvonne (Self)
Registering, but not testifying:
For:
Bowels, Glenda (Self)
Haehnel, Gaye (Precinct Chair # 3038)
Harnist, Linda (Self)
Raia, Sam (Self)
Sanford, Gary (Self)
Schmid, Judy (Self)
Snyder, Patt (Self)
Against:
Ashley, Larry (Self)
Bell, Tomaro (Self)
Black Navarro, Yolanda (Self)
Brown, Vicki (Self)
Dinh, Cindy (Self)
Etherton, Russell (Self)
Galindo, Mary (Texas Organizing Project)
1
WITNESS LIST
Against:
Hailey, Charles (Self)
Hailey, Mary (Self)
Imam, S. Qaisar (Self)
Makany-Rivera, Tanya (Self)
Presley, Louise (Self)
Shaw, Richard C (Harris County AFL-CIO Council)
Smith, Shaun (Self)
Sterling, SJ (Self)
Tijerina, Joseph (Chair Precinct 0026 Harris County)
Vogas, Mary (Self)
Williams, Daniel (Equality Texas)
Zakes, Tom (Self)
Against:
Blavier, Rhys (Self)
Lockett, Joseph (Self)
Longhofer, Kyle I (Self)
Moffitt, Jill (Self)
Registering, but not testifying:
For:
Presley, Louise (Self)
Tijerina, Joseph (Precinct Chair 0026: Harris County)
Against:
Ashley, Larry (Self)
Brown, Vicki (Self)
Hailey, Mary (Self)
Sterling, SJ (Self)
Williams, Daniel (Equality Texas)
Against:
Blavier, Rhys (Self)
Calvert, Rogene Gee (TX Asian American Redistricting Initiative)
Lockett, Joseph (Self)
Longhofer, Kyle (Self)
Moffitt, Jill (Self)
Vinson, Tommy (Self; Tom Vinson Precinct Chair # 281)
Vogas, James (Self)
Registering, but not testifying:
Against:
Ashley, Larry (Self)
Brown, Vicki (Self)
2
WITNESS LIST
Against:
Hailey, Charles (Self)
Hailey, Mary (Self)
Imam, Qaisar (Self)
Presley, Louise (Self)
Shaw, Richard C (Harris County AFL-CIO Council)
Smith, Shaun (Self)
Sterling, SJ (Self)
Tijerina, Joseph (Precinct Chair 0026; Harris County)
Vogas, Mary (Self)
Williams, Daniel (Equality Texas)
For:
Hamburger, Jay (OSWNA (Old Sixth Ward Neighborhood Assn.)
Against:
Blavier, Rhys (Self)
Davis, Willie (Self; Congresswoman Sheila Jackson Lee)
Horwitz, Noah (Self; Texpatriate)
Lockett, Joseph (Self)
Longhofer, Kyle (Self)
Vogas, James (Self)
Registering, but not testifying:
Against:
Ashley, Larry (Self)
Bell, Tomaro (Self)
Brown, Vicki (Self)
Hailey, Charles (Self)
Hailey, Mary (Self)
Presley, Louise (Self)
Shaw, Richard C (Harris County AFL-CIO Council)
Smith, Shaun (Self)
Sterling, SJ (Self)
Tijerina, Joseph (Precinct Chair 0026 Harris County)
Vogas, Mary (Self)
Against:
Blavier, Rhys (Self)
Lockett, Joseph (Self)
Registering, but not testifying:
Against:
Sterling, SJ (Self)
3
WITNESS LIST
For:
Isbell, Shirley (Self)
Against:
Blavier, Rhys (Self)
Lockett, Joseph (Self)
Registering, but not testifying:
For:
Dinh, Cindy (Self)
Against:
Sterling, SJ (Self)
Against:
Blavier, Rhys (Self)
Isbell, Shirley (Self)
Lockett, Joseph (Self)
Registering, but not testifying:
For:
Dinh, Cindy (Self)
Williams, Daniel (Equality Texas)
Against:
Sterling, SJ (Self)
On:
Rich, Marvin (Self)
For:
Arline, Tiffany (Texas Organizing Project)
Caldwell, James (Texas Organizing Project)
Richards, Assata (Self; Sankofa Research Institute Communities of District D - City of
Houston)
Wright, Carol (Self)
Against:
Blavier, Rhys (Self)
Honore, Stephan (Self)
Lockett, Joseph (Self)
Registering, but not testifying:
For:
Aleman, Alejandra (Texas Organizing Project)
Ashley, Larry W (Self)
4
WITNESS LIST
For:
Cisneros, Alain (Texas Organizing Project)
Garzon, Yoni (Texas Organizing Project)
Gonzales, Roland (Texas Organizing Project)
Perez, Zulema (Texas Organizing Project)
Resendez, Mayra (Texas Organizing Project)
Rich, Marvin (Self)
Sterling, SJ (Self)
Williams, Daniel (Equality Texas)
Against:
Bell, Tomaro (Self)
Brooks, James (Precinct 132)
Fears, Doris (Crestmont Park Civic Association)
Hinojosa, A Robert (Self)
Against:
Blavier, Rhys (Self)
Lockett, Joseph (Self)
Registering, but not testifying:
For:
Dinh, Cindy (Self)
Williams, Daniel (Equality Texas)
Against:
Sterling, SJ (Self)
5


| ZotWeb | book | |
| Src Url | [[Strunk Jr]], [[White]] (2020) | |
'The Elements of Style' (1918), by William Strunk, Jr., is an American English writing style guide. It is the best-known, most influential prescriptive treatment of English grammar and usage, and often is required reading and usage in U.S. high school and university composition classes. This edition of 'The Elements of Style' details eight elementary rules of usage, ten elementary principles of composition, "a few matters of form", and a list of commonly misused words and expressions.
Citer: (Strunk Jr & White, 2020)
FTag: Strunk-Jr-et-White-2020
APA7: Strunk Jr, W., & White, E. B. (2020). The Elements of Style, Fourth Edition. Independently published.
This book is intended for use in English courses in which the practice of composition is combined with the study of literature. It aims to give in brief space the principal requirements of plain English style .


TOTAL RECALL [Probable Spoilers] Reviewer: Jaye Mathisen Copyright 1990 Jaye Mathisen
Summary: Sometime in the future, Douglas Quaid finds out that he's not who he remembers himself to be. From there, the plot takes a downturn into aliens, violence, Mars,violence, air on Mars, violence, and memory implants.
Up until now, I've always liked Schwarzenegger movies. They're usually not too intellectual, have lots of action, lots of violence, babes, guns, chases, muscles, and all the other good things that make an action film an action film. Until now. While TOTAL RECALL had lots of violence, the special effects got in the way of what could have been a good movie. It doesn't bother me to watch people get hung, shot, karate chopped, or what-have-you, but gallons of blood spraying everywhere tend to be distracting, rather than "entertaining". I know I'm not squeamish about blood (having worked for a fire department and an ambulance company), but slow-mo of globules of blood flying through the air just doesn't do it for me.
The plot of TOTAL RECALL is okay, right up to where Schwarzenegger gets to Mars. (The plot's complicated, I'm not going to attempt to explain it.) The memory implantation stuff reminded me of the many and various SF books that deal with "sensies," or "feelies." (Movies/things that allow you to sense what the characters on the screen sense). Some poor special effects of people in a vacuum, outright ridiculousness of the ending, and the lack of believability in the story-line in general ruined it.
Rating: What's to say?
Enjoyability Bleeech! When I go to a movie, I like to "immerse" myself in what's happening on the screen. Movies like STAR WARS and friends, I can believe, because it's way beyond where we're at, but is nice to think about. TOTAL RECALL on the other hand, was hard to believe in. "Aliens build air-producing reactor, that creates a perfectly breathable atmosphere in every detail for the human life form, on a planet-wide scale, in a matter of seconds, by converting old fashioned ice at the core of planet, into life-giving oxygen. This air-producing reactor hasn't been tested, and the switch to turn it on is a large ball that you put your hand into. Film at 11." Naaaah. 1 star out of 4.
.
The review above was posted to the
rec.arts.movies.reviews newsgroup (de.rec.film.kritiken for German reviews).
The Internet Movie Database accepts no responsibility for the contents of the
review and has no editorial control. Unless stated otherwise, the copyright
belongs to the author.
Please direct comments/criticisms of the review to relevant newsgroups.
Broken URLs inthe reviews are the responsibility of the author.
The formatting of the review is likely to differ from the original due
to ASCII to HTML conversion.
Related links: index of all rec.arts.movies.reviews reviews


{% include 'includes/error_summary.html' %}{# include if this page has validation requirements #}
Select your identity documents
Select 3 documents you can take to the Post Office.
At least one of the documents must show your current address.
You must be able to take the original documents – you can't use photocopies or statements you've printed from online accounts.


Twitter
Insta
LinkedIn
Artist
Artist Info
Artist Info
Album Title
Label and year
© 2009-2019 iroro orife. All rights reserved. | Design:
HTML5 UP


SUN SETS AT DAWN, THE (director/writer: Paul H. Sloane; screenwriter: original story by Sloane; cinematographer: Lionel Lindon; editor: Sherman Todd; cast: Sally Parr (The Girl), Philip Shawn (The Boy), Walter Reed (The Chaplain), Houseley Stevenson (Pops), Howard St. John (The Warden), Percy Helton (Reporter, Feature Syndicate), King Donovan (Reporter, National News Service), Perry Ivins (Forty-Six), Lee Frederick (Blackie), Raymond Bramley (The Deputy Warden); Runtime: 71; Eagle Lion Films; 1951)
Reviewed by Dennis Schwartz
This film is based on a similar case of an innocent boy framed for murder. The Boy (Philip Shawn) is about to be executed today at sunset, as the governor turns down both the sympathetic warden (Howard St. John) and his girlfriend's request for a reprieve.
The film's poetic title can be explained as follows: that the sun always sets instead of rises during the day of an execution. The Girl (Sally Parr) grew up with The Boy and was going to marry him; she breaks down over his death sentence, absolutely sure that he couldn't have done it. He is about to be executed today in the State's brand-new electric chair, which brings reporters from all over the state to cover the event. They are more interested to see how the chair works than in anything else.
The Boy confides to the sympathetic padre (Reed) that he's innocent, as he renacts the crime. He tells the padre that he was an ambitious cub reporter and smelled a big time story around crooked politician Tim Harrah, who befriended the kid and made him his errand boy. The kid always wanted to get rid of the loser image he has; and, he thought if he can get the goods on who was protecting the crooked politician in his illegal trucking scam, that he could break a big story and get promoted to be a star reporter. When he was in a room alone with Tim, someone he couldn't see came up from behind and pumped 6 bullets into the politician and then knocked him out and placed the hot gun in his hand. The jury didn't believe the 'ghost story' he told. The Girl wanting to be near him, moved near the prison three years ago and got work in Pops' Diner as a waitress.
The cynical reporters who gather at the diner to wait for the prison bus to take them to the execution crack jokes about the execution and seemed unmoved that one of their own kind is about to be executed. But luck starts to turn for the kid as a prisoner comes into the diner to get the prison mail. Pops (Houseley Stevenson), not only runs the diner/hotel, but he is also the postmaster. The prisoner recognizes that the patron having coffee is the same criminal who framed him and caused him to get a long jail sentence. His name is now Blackie (Lee Frederick) and he's the head of a trucking firm, but convict 46 (Perry Ivins) says he's really a gangster named Parrot. The only problem is, Parrot's dead body was supposedly hauled out of the ocean three years ago and the Wanted Poster of his that is still hanging in the diner/post office doesn't look like him. But 46 says he can't be fooled, he would know his gestures anywhere.
Warning: spoiler to follow in next paragraph.
When 46 approaches Blackie, he tells him that he knows he's Parrot. This causes the gangster to shoot at 46, but the prison guards having coffee arrest him. Meanwhile the execution took place; but, there must have been Divine intervention, because the chair failed to work. This causes a delay and time for the prison officials to get the fingerprints matching Blackie with Parrot. Since Blackie is being charged with 6 murders he decides to confess to the 7th, the killing of the politician. He relates how he faked his death and got plastic surgery. This saves The Boy from being executed. Incidentally, the vics were called anonymously The Boy and The Girl to show how little the system cared about them.
The story was well told, but the acting left a lot to be desired.  But as far as B-films go, this one is above average.
REVIEWED ON 6/6/2001 GRADE: C+
Dennis Schwartz: "Ozus' World Movie Reviews"
http://www.sover.net/~ozus
<EMAIL_ADDRESS>© ALL RIGHTS RESERVED DENNIS SCHWARTZ
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rec.arts.movies.reviews newsgroup (de.rec.film.kritiken for German reviews).
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Related links: index of all rec.arts.movies.reviews reviews


You need values, empathy and self-awareness do help you get a job. If there is someone better than you at coding (and there always is) you can still get the job if you show these skills because people want people who can work well with other people because you can relate to them. Because of this, you will be more effective in a team and can get much more work done.
The thing that surprised me the most was that I could relate almost everything I learned to past experience I have had, and how I could have used them to better myself and my relationships with others. For example, if I knew about emotional intelligence longer ago I would have handled myself differently when interacting with people I didn't like. I was quite petty with some people I didn't like or would be passive aggressive to another.
I found the meditation the most challenging because I hadn't done it before and as a result of this it was hard to stay still for so long while doing nothing but focusing on my breath, but it did get easier and easier the more I did it.
I think so much time is spent on it because at the end of the Bootcamp we should be able to get a job and if you do not display some of the stuff we learned in core it will be much more difficult to get a job. Some of the core stuff we learned will also help us at Bootcamp, like having a growth mindset. This will help because you understand why you should not give up.
I think it was a good use of time because I knew none of it. It did not feel like a waste of time because I can think of ways that I would use the core skills at old jobs and I do think they will be useful in future jobs and at the Bootcamp.
I would like to use Dec Academy as a way to get into web development and start a career. I think Dev Academy will give me all the skills I need to start and get a job. If I like the job I may decide to go to university to study computer science and become a software developer if I think it will be a good investment at the time, but I don't think I will do this for a good few years after Dev Academy, I also think Dev Academy will give me a feel for the industry and if it is where I want to put all my time and effort or if it isn't suited for me.
I think I still have strengths and weaknesses as before but I also think I do need to be in the right headspace to learn, I have found this when doing the mack-a-mole problem. I did spend a day on the problem and it was not working. Bits of it did work but I was constantly getting stuck and stuff just was not making sense to me. So the next day I left it and did the dress the clown then came back to it in the afternoon, deleted all my code and did it in like an hour which made me feel good. To keep me in a good headspace I think I need to take breaks, but not too frequently because it is hard to get back into my work. Because of this and because I get stuck into my work sometimes I just don't want to take a break so I'll put it off until I have lost concentration.
The two biggest I can think of is staying focused for 9 weeks and being around people so much. I may lose motivation or focus over the 9 weeks so I just need to keep reminding myself why I am here and that I am here my own choice. I also think being around people a lot does drain me, I do like to spend time alone to recharge but I will not have a lot of time to do that because of how long the weeks are. This may make me lose motivation and fall behind work as a result.
I do want to learn how to network mostly. I think this will help me a lot at Bootcamp and more importantly for me in the job market and later on in life. I am also not the best working in teams because I think I work better alone than in a team because I would rather just get stuck in with the work than decide on a plan that works well with the whole team, I am also not very good at communicating my ideas so I don't contribute much to the planning stage of the project. I do like to work with a team when we all get along well. But in teams where there is a clash in personalities like two people trying to lead the project is the most annoying thing for me.
I will try to do all the hard work at the beginning of the week so I have more time at the end of the week to go through the easier work. When I do get stuck I will try to fix it by myself first because if I do I am more likely to remember how I fixed it. If I can not I will ask for help from a facilitator or another student who I think might have it.


What a week of news! Today we are covering SpaceX Starship, Starlinks Record-Breaking Mission, Crew Dragon and Boeing Starliner updates.


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A custom element for iOS style swipeout actions, backed by Hammer.js
GitHub npm
${ email.author }
${ email.date }
${ email.title }
…


Configuration reader and writer implementation with
Commons Betwixt
.
Related Documentation
Commons Betwixt


| 1217 | (A) | Burgesse and baylȝes als beralles þay lukede |
| 1220 | (C) | And burgeys with balyes . as barellys somme tyme |
| 1203 | (D) | The burgeys with belyes as barels byfor |
| 1180 | (E) | burgeyses with belyes . as barellys þat hadde |
| (Ex) | ---- no text ---- | |
| 1245 | (L) | burges with balies as barels or þat tyme |
| (P) | ---- no text ---- | |
| 1188 | (U) | And burgeys witħ her belyes / as aforne that |
| 281 | (V) | Burgeis with belies . as barełł or þat tyme |


--- title: The Biology of Sloppy Code layout: post ---
I’m watching the Future of Programming Languages panel and a point came up that really resonated with me. To the question, “what is the next big trend in programming”, Guy Steele said, “Maybe it’s sloppy programming.”
Fascinating thoughts on the future of programming.


Spending vs. GDP per person (GEOMAP)
GEOMAP
This is a GeoMap of the expenditures and GDP for the years 2013
and 2017. Select the link below:
This is a GeoMap of the expenditures and GDP for the years 2013
and 2017. Select the link below:


Sara Wallis: 'Olivia Coleman, Suranne Jones and Helena Bonham Carter were inspired casting'
Of course they could sing! After apologising in advance for their terrible voices, ten British actors proved their talent as they recorded an album for Children In Need
by Sara Wallis, https://www.facebook.com/SaraWallisMirror/This week we witnessed a BBC1 show that could have been called Oh My God, I Didn’t Know They Could Sing! For that is what we all said repeatedly for the entire hour-and-a-half.
Celebrities trooped into the legendary Abbey Road recording studio for Children In Need: We Got It Covered, full of apologies about their terrible voices.
LIES, it was all lies.
In the latest idea for the charity, ten British actors all agreed to record an album. Not just a Band Aid-style song, singing a line or two, but a track each, inspired by their own lives.
Never mind the songs, the casting was pretty inspired. Deep breath: Helena Bonham Carter, Jim Broadbent, Olivia Colman, Shaun Dooley, Luke Evans, Suranne Jones, Adrian Lester, Himesh Patel, David Tennant and Jodie Whittaker.
Clearly TV producers wrote their wishlist and got it – the power of Pudsey at play. There were so many great moments. The Crown buddies Olivia and Helena casually discussing bathroom singing.
“You always think you sound different than you actually do. A bit like how I get dressed in the morning,” said Helena.
Olivia, who was either crying or on the verge of crying throughout, was nervous as hell.
“I’d rather give birth again with no drugs,” she said, before quickly telling producers she was having a lovely time.
“I don’t want to be rude,” explained the most polite woman in telly.
Oh and of course, she can sing. She can do anything, she’s a goddess.
And then... Phoebe Waller-Bridge and her sister Isobel on ukuleles? This is why we pay our licence fees.
It still hadn’t peaked. Taylor Swift sauntered in to surprise Shaun, who was mid-record of her hit Never Grow Up.
Olivia starred in The Favourite with Taylor’s boyfriend Joe Alwyn and had texted in the favour. This was actors pulling strings live in action and I loved it. Who else does Olivia have in her contacts book? Who does she invite for dinner parties?
Jodie lived out a teen fantasy when Coldplay’s drummer Will Champion and lead guitarist Jonny Buckland interrupted her “northern twang” rendition of Yellow.
“Oh my god, I fully stalked you!” she squeaked.
When they started playing, she giggled. But later, as she sang for her nephew Harry, who died aged three, she cried. Harry’s parents cried. We all cried.
This film yo-yoed the emotions about from one extreme to the other.
Adrian was beatboxing, Helena drank weird herbal teas and David told us he used to busk Proclaimers songs.
They saved the best for last. Suranne giving it full on musical theatre lungs, with a backing choir of kids from a children’s theatre. Hearts fit to burst.


PivotTreeMap showing customer count over different customer geographic locations across a period of fiscal years.
Color Mapping


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Companies still have choices to secure and control access to file shares, even if you use a shared key to connect to them.
The finance company has stated that they'd like to implement security best practices. One approach is to lock down access to the file shares to a specific range of IP addresses. They'd also like to understand their options to use Azure Active Directory Domain Services (Azure AD DS) authentication.
In this unit, you'll investigate ways to secure access to Azure file shares from on-premises machines. You'll check to ensure that data is encrypted in transit to Azure. With secure access in place, you'll look at how the finance company can use Azure AD to control access to the file shares. As an extra level of protection for the shared files, you'll also explore the snapshot feature.
Azure file shares only support mounting them from SMB version 3.0. There are known vulnerabilities with SMB 1.0. Microsoft recommends you either uninstall or disable this version from all the machines you're responsible for.
If you need to remove SMB 1, use this PowerShell command: Remove-WindowsFeature -Name FS-SMB1
.
When you mount Azure file shares, Windows needs to communicate over port 445. Organizations can block network access over 445 by default. If your organization has this policy, consider requesting to open the port. There's nothing inherently insecure about this specific port. With SMB 1 disabled, the risk of an SMB-based attack dramatically reduces.
To limit access to your on-premises networks, you'll need to know your public-facing IP address. With this information, enable the storage account's firewall to allow access from selected networks. You can then add your public IP address to the list of allowed addresses, either in the Azure portal, or by using a PowerShell command.
Add-AzStorageAccountNetworkRule `
-ResourceGroupName "myresourcegroup" `
-AccountName "mystorageaccount" `
-IPAddressOrRange "NNN.NNN.NNN.NNN"
Or, you could use a CLI command.
"az storage account network-rule add \"
--resource-group "myresourcegroup" \
--account-name "mystorageaccount" \
--ip-address "NNN.NNN.NNN.NNN"
where NNN.NNN.NNN.NNN
is your public-facing IP address.
Running the commands in the previous exercise creates an Azure storage account with secure transfer enabled by default. If your file shares were created without this feature enabled, it's easy to do that in the portal.
Or you can automate it with a PowerShell command.
Set-AzStorageAccount `
-Name "StorageAccountName" `
-ResourceGroupName "ResourceGroupName" `
-EnableHttpsTrafficOnly $True
Or you could use an Azure CLI command.
az storage account update \
--resource-group ResourceGroupName \
--name StorageAccountName \
--https-only true
The secure transfer option only allows requests to the storage account over a secure HTTPS connection. Any requests that use HTTP will be rejected. When you use Azure Files, connections without encryption will fail, even when you use SMB 2.1, SMB 3.0 without encryption, or some flavors of the Linux SMB client.
The finance company is running their new reporting application on a VM hosted on Azure. That's why they'd like to set up Azure AD DS authentication to access the file shares. It's something they want to do before they move more of their processing into the cloud.
The benefits of Azure AD DS are that you can manage access with role-based access controls. Files inherit their existing NTFS DACLs.
The first step in setting up Azure AD DS is to enable Azure AD Domain Services for the tenant and have the VM reside in the same virtual network. With these things in place, you'll enable Azure AD DS authentication on the storage account. You can then grant access permissions to a share in the storage account. These permissions can be at the user, group, or service principal level.
Microsoft has created three new roles for permissions levels of access to SMB file shares for users:
Share snapshots give you an extra level of security and help reduce the risk of data corruption or accidental deletion. You can also use them as a general backup for disaster recovery.
The snapshots are easy to create in the Azure portal or with the REST API, client libraries, the Azure CLI, and PowerShell.
Snapshots are at the root level of a file share and apply to all the folders and files contained in it.


unset - Delete variables
unset ?-nocomplain? ?--? ?name name name ...?
This command removes one or more variables.
Each name is a variable name, specified in any of the
ways acceptable to the set command.
If a name refers to an element of an array then that
element is removed without affecting the rest of the array.
If a name consists of an array name with no parenthesized
index, then the entire array is deleted.
The unset command returns an empty string as result.
If -nocomplain is specified as the first argument, any possible
errors are suppressed. The option may not be abbreviated, in order to
disambiguate it from possible variable names. The option --
indicates the end of the options, and should be used if you wish to
remove a variable with the same name as any of the options.
If an error occurs, any variables after the named one causing the error
are not
deleted. An error can occur when the named variable does not exist, or the
name refers to an array element but the variable is a scalar, or the name
refers to a variable in a non-existent namespace.
Create an array containing a mapping from some numbers to their
squares and remove the array elements for non-prime numbers:
array set squares {
1 1 6 36
2 4 7 49
3 9 8 64
4 16 9 81
5 25 10 100
}
puts "The squares are:"
parray squares
unset squares(1) squares(4) squares(6)
unset squares(8) squares(9) squares(10)
puts "The prime squares are:"
parray squares
set, trace, upvar
remove, variable
Copyright © 1995-1997 Roger E. Critchlow Jr.
Copyright © 1993 The Regents of the University of California.
Copyright © 1994-1996 Sun Microsystems, Inc.
Copyright © 2000 Ajuba Solutions.


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Clickers are HTML elements that have been associated with a click event through the Game object! Only the Game() object comes with an addClicker(...) method.
Let's see, if we wanted to make our 'Earn Points' button clickable and actually earn us points, we can just do:
//add clickers
game.addClicker("#earn",function() {
game.attributes.Points+=2;
});
3b. How to use a clicker [Refer to the documentation for additional parameter]
Essentially, you provide the identifier/class/name of the element you want to make clickable, and give it a function to execute on click. We provided an
anonymous function function with our code that will henceforth always be executed whenever #earn is clicked.
Great, click-event handling taken care of. What about the other button?
The syntax for
addClicker stays the same, but the code inside our anonymous function will, obviously, be a bit more complex.
game.addClicker("#getSome", function() {
//this function also shows another way to access attributes
var p=game.getAttribute("Points");
var cost=10;
var increase=2;
if (p>=cost)
{
//we can buy
p-=cost;
game.setAttribute("Points",p);
var pps=game.getAttribute("PointsPerSecond");
pps+=increase;
game.setAttribute("PointsPerSecond",pps);
}
});
3b. Code to actually buy stuff - this also shows a different way to access attributes
Great, so we have our clickers in place! Let's put it all together!
<script>
//the init() function initializes or sets up our game
function init()
{
//create Game() instance
game=new Game();
//add attributes
game.addAttribute("Points",0).track("Points","#points", function(value) { return value.toFixed(0); });
game.addAttribute("PointsPerSecond",0,"#points_per_second");
//add clickers
game.addClicker("#earn",function() {
game.attributes.Points+=2;
});
game.addClicker("#getSome", function() {
//this function also shows another way to access attributes
var p=game.getAttribute("Points");
var cost=10;
var increase=2;
if (p>=cost)
{
//we can buy
p-=cost;
game.setAttribute("Points",p);
var pps=game.getAttribute("PointsPerSecond");
pps+=increase;
game.setAttribute("PointsPerSecond",pps);
}
});
//play
game.play(play);
}
</script>
3c. Our Init function
You'll notice I added an anonymous function to the track() method for the first attribute, Points. If you recall from the sub-section on trackers (
2c), this is an optional function that takes in one parameter, the value of the attribute being tracked, and returns the way it should be displayed. I have simply set it to return no decimals, only the integer itself, in case I decide to change cost from 2 to some other decimal value.
If you refresh your game and try playing it, you'll notice you can
earn points and
buy more 'Points Per Second'. However, nothing actually happens 'per second'. This is where our
play() function comes in handy.
//the play() function contains essential game code
function play()
{
//here we see another way to get/update attributes more quickly
var g=game.attributes;
g["Points"]+=g["PointsPerSecond"]/game.getFPS();
//divide by FPS because we have to take into account the Frames Per Second when adding anything per Second,
}
3c. the play() function - also demonstrating yet another way to access attributes
Fairly straightforward, right?
We're accessing the
Points attribute
directly and adding to it the
PointsPerSecond attribute, since it does make sense.
But wait, why are we dividing the points added per second by the game's FPS - the game's FPS is 1 by default, so it doesn't matter right?
Yes, in its current state - the FPS of the game is 1 and so the division is unnecessary. But what if you decide to set the FPS to 10, or 15, or 30?
Let's do the math to see what happens if we exclude the divided-by-game.getFPS() and the FPS is 10
Suppose the
PPS (points per second) is
2. The
play() loops runs
10 times (frames) per second.
Expected Behaviour: In
1 second, the
Points attribute should increase by 2.
Actual Behaviour: In
1 second, the
Points attribute increases by 20 per second, because 2 was added 10 times per second.
Hence, in such operations, make sure you factor in the FPS, whether it is 30 or 1 (to be safe). By dividing the FPS here, we are basically adding up 10 parts of 2 in the span of 1 second. 1/10th of 2 is 1/5. So by adding 1/5
ten times to the Points attribute, we get 2 in one second!
10-15 FPS is a good choice because it also updates the tracked variables quicker. Currently, clicking on either button doesn't update the
displayed corresponding attribute immediately - the trackers waits until the next loop iteration to update, so we would have to manually throw in a Javascript/HTML text manipulation in our click functions.
Increasing the FPS would alleviate this problem at this scale.


The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.
A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.
Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.
The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.
The genus Flavivirus of the family Flaviviridae consists of more than 70 virus species including many arthropod-borne viruses. It contains highly pathogenic agents such as the name-giving member yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and dengue virus (DENV), which can cause encephalitis or hemorrhagic fever and pose some of the most challenging emerging diseases in humans [ 1].
Flaviviruses comprise three epidemiologically distinct groups; the mosquito-borne group, the tick-borne group and the unknown vector viruses [ 2]. They are enveloped positive-stranded RNA viruses with a genome of approximately 11 kb. The viral genome encodes a large single polyprotein from which three structural proteins, Capsid (C), Envelope (E) and Membrane (M) and seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, are produced [ 3].
Flaviviruses pose a threat to public health worldwide, especially in developing countries. In recent years, there has been a dramatic increase in cases of DENV infections globally. The disease is endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-east Asia, and the Western Pacific. About 50–100 million cases of DENV infection are reported worldwide every year, resulting in 500,000 cases of hemorrhagic fever requiring hospitalization and 25,000 fatalities [ 4]. Yellow Fever is endemic in 45 countries throughout Africa and Latin America, with approximately 200,000 cases of human infections worldwide, and 30,000 mortalities [ 5]. WNV is one of the most widespread flaviviruses. WNV lineage 1 is endemic in the Americas, India, and Australia. Both lineages 1 and 2 are found in Africa and Europe [ 6- 8]. The recent WNV outbreak reported in August 2010 in Greece was the first large outbreak in humans in Europe since the Romanian outbreak in 1996–1997 [ 9]. JEV is the leading cause of viral encephalitis in Asia with 30,000–50,000 clinical cases reported annually and 10,000 deaths [ 10]. TBEV, virus-causing infection of the central nervous system, has spread extensively in some European regions. According to ECDC [ 11], human cases of TEBV infections have increased in the past 30 years dramatically posing a danger to public health in European countries.
A number of flaviviruses have repeatedly demonstrated their ability to expand their geographical range. Some current examples are the introduction of Usutu virus and Bagaza virus into central Europe [ 12, 13], or Zika virus into Yap island and Cambodia [ 14]. In recent years, also a high number of mosquito-only flaviviruses including Culex and Aedes flaviviruses were discovered throughout the world [ 15- 18]. Apart from the human pathogenic and mosquito-only flaviviruses, several novel flaviviruses were latterly discovered: Nounane in Cote d’Ivoire [ 19], T’Ho virus in Mexico [ 20], Lammi virus in Finnland [ 21], and Duck tembusu virus in China [ 22]. In contrast to mosquito-only flaviviruses, these novel flaviviruses were shown to be phylogenetically related to human pathogenic flaviviruses.
The clinical diagnosis of flavivirus infections is not unambiguous due to unspecific symptoms varying from mild, febrile illness to viral hemorrhagic fever. Many of these viruses have a common transmission vector and can co-circulate in the same area. All these factors make a precise identification of the pathogen difficult.
Today most diagnostic laboratories use serological assays for flavivirus testing. These tests are commonly based on the enzyme-linked immunosorbent assay (ELISA) and detect virus-specific IgM and IgG antibodies. Antibodies are undetectable prior to 5–7 days after onset of infection which hampers the usefulness of the serological methods. Molecular techniques, in contrast, can be used already in the acute phase and are known to be rapid, highly specific and sensitive.
Since the early 1990s, several group-specific and generic molecular assays for detection of flaviviruses have been developed as reviewed previously [ 23- 25]. A number of attempts to detect several flaviviruses in a single reverse transcription-polymerase chain reaction (RT-PCR) assay have been made [ 26]. These assays vary in the selected target region, assay format and detection method. The highly conserved NS3 and NS5 genes have been used predominantly for flavivirus testing, mainly as nested, hemi-nested RT-PCR or SYBR green real-time PCR, without using a probe for sequence verification.
The aim of this study was to develop a rapid, sensitive and reliable TaqMan probe-based quantitative RT-PCR (qRT-PCR) assay for simultaneous detection of several flaviviruses, using the conserved NS5 gene region. The introduction of Locked-Nucleic Acid (LNA) bases in the probe increases robustness, specificity and sensitivity of the assay and has been shown to allow quantification of the viral load.
Primers from publication [ 26- 28] were aligned against genomic sequences of all flaviviruses deposited in the NCBI database to verify their flavivirus-generic amplification capacity. Degenerate primers were selected based on these published sequences, but modified in order to increase the spectrum of flaviviruses included. As alternative approach individual specific flavivirus species-specific primers were combined to avoid unspecificity due to base combinations contained in the degenerate primers not matching exisiting viral sequences. In particular we used 17 forward and 15 reverse primers and another four reverse primers targeting different locations. Testing of the primer mix with viral RNA revealed that the performance of the primer mixes was not improved compared to the results obtained with the degenerate primers. Also for the probes we tested 22 individual flavivirus-specific sequences covering all known sequence variants at the binding site and established mixtures yielding comparable threshold cycle (Ct) values for all virus species tested, but the signal level was significant lower than for degenerate LNA-probes (data not shown). In conclusion, neither the set of individual primers nor the set of probes improved the detection limit nor the range of viruses detected and we continued the evaluation with the degenerate set of primers combined with one of the three LNA-probes.
In total, six combinations assembled from two degenerate primer sets (Flavi all S & Flavi all AS1 and Flavi all S & Flavi all AS2) and three probes (Flavi all probe 1, Flavi all probe 2 and Flavi all probe 3) were tested using RNA from flaviviruses and other viruses to check the performance of these Pan-Flavi assays. Combinations with Flavi all probe 3 showed earlier Ct values (Table 1). Best results among these six combinations were obtained with Flavi all S, Flavi all AS2 primers and Flavi all probe 3, but detection of DENV type 4 was insufficient. Analysis by gel electrophoresis revealed missing amplification, except a faint band for primer combination Flavi all S and AS1, correlating to a low signal and very late Ct value (Table 1).
Comparison of designed two degenerate primer sets and three probes containing wobble and LNA bases in six combinations for detection of the different flavivirus RNA
| leftbottom 1 | leftbottomDENV-1 | centerbottom31.50 | centerbottom17.57 | centerbottom15.66 | centerbottom27.95 | centerbottom17.84 | centerbottom15.61 |
| leftbottom 2 | leftbottomDENV-2 | centerbottom22.69 | centerbottom22.21 | centerbottom19.76 | centerbottom22.27 | centerbottom22.19 | centerbottom19.92 |
| leftbottom 3 | leftbottomDENV-3 | centerbottomNo Ct | centerbottom21.25 | centerbottom19.14 | centerbottomNo Ct | centerbottom20.63 | centerbottom18.57 |
| leftbottom 4 | leftbottomDENV-4 | centerbottomNo Ct | centerbottom35.50 | centerbottom35.48 | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct |
| leftbottom 5 | leftbottomJEV | centerbottom20.40 | centerbottom21.63 | centerbottom20.93 | centerbottom18.99 | centerbottom19.98 | centerbottom19.27 |
| leftbottom 6 | leftbottomRSSEV | centerbottom27.69 | centerbottom29.38 | centerbottom27.31 | centerbottom24.67 | centerbottom26.13 | centerbottom23.81 |
| leftbottom 7 | leftbottomTBEV K23 | centerbottom17.68 | centerbottom21.81 | centerbottom16.99 | centerbottom16.73 | centerbottom18.63 | centerbottom16.32 |
| leftbottom 8 | leftbottomTBEV Louping ill | centerbottom26.00 | centerbottom28.75 | centerbottom23.78 | centerbottom22.13 | centerbottom24.8 | centerbottom21.22 |
| leftbottom 9 | leftbottomUsutu virus | centerbottom25.63 | centerbottom33.96 | centerbottom25.91 | centerbottom22.96 | centerbottom30.3 | centerbottom23.48 |
| leftbottom 10 | leftbottomWNV Uganda | centerbottom30.22 | centerbottom30.27 | centerbottom30.68 | centerbottom28.46 | centerbottom28.45 | centerbottom28.26 |
| leftbottom 11 | leftbottomWNV Israel | centerbottom21.76 | centerbottom21.83 | centerbottom20.78 | centerbottom20.06 | centerbottom21.68 | centerbottom19.8 |
| leftbottom 12 | leftbottomSLEV | centerbottom25.85 | centerbottom26.95 | centerbottom25.57 | centerbottom24.41 | centerbottom24.90 | centerbottom23.49 |
| leftbottom 13 | leftbottomYFV 17D | centerbottom19.82 | centerbottom22.48 | centerbottom18.41 | centerbottom20.03 | centerbottom22.12 | centerbottom18.92 |
| leftbottom 14 | leftbottomYFV Asibi | centerbottom23.71 | centerbottom26.98 | centerbottom22.82 | centerbottom24.17 | centerbottom26.83 | centerbottom23.53 |
| leftbottom 15 | leftbottomYFV Brasil | centerbottom21.35 | centerbottom23.73 | centerbottom20.48 | centerbottom21.75 | centerbottom23.82 | centerbottom21.43 |
| leftbottom 16 | leftbottomYFV Ivory Coast | centerbottom21.35 | centerbottom23.98 | centerbottom20.36 | centerbottom21.47 | centerbottom23.94 | centerbottom21.44 |
| leftbottom 18 | leftbottomChikungunya virus | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct |
| leftbottom 19 | leftbottomRVFV | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct |
| leftbottom 20 | leftbottomSindbis virus | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct |
| leftbottom 21 | leftbottomH1N1 Influenza A | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct |
| leftbottom 22 | leftbottomH5N1 Influenza A | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct | centerbottomNo Ct |
| left 17 | leftNegative control | centerNo Ct | centerNo Ct | centerNo Ct | centerNo Ct | centerNo Ct | centerNo Ct |
DENV: Dengue virus, JEV: Japanese encephalitis virus, RSSEV: Russian spring-summer encephalitis virus, WNV: West Nile virus, TBEV: Tick-borne encephalitis virus, SLEV: Saint Louis encephalitis virus, RVFV: Rift valley fever virus, YFV: Yellow fever virus.
S: Flavi all S, AS1: Falvi all AS1, AS2: Flavi all AS2, probe1: Flavi all probe 1, probe 2: Flavi all probe 2, probe 3: Flavi all probe 3.
To overcome this deficit the combination Flavi all S/AS2/probe 3 was supplemented with an additional DEN4 F primer and all tests were repeated. DENV type 4 was now detectable with Ct values similar to the specific assay. The additional DEN4 F primer had no influence on the detection of all other flaviviruses tested (Table 2). Evaluation of the Pan-Flavi assay was performed using primers Flavi all S/DEN4 F and Flavi all AS2. Changing primers to Flavi all S/Flavi all S2/ Flavi all AS4 allowed to detect also Nounane virus (Table 3), without changing specificity and sensitivity of Pan-Flavi assay for the other viruses (data not shown).
Representative results of the Pan-Flavi assay to detect different flaviviruses, and its comparison with flavivirus species-specific assays
| leftbottom Flavivirus | ||||||||
| leftbottomDENV-1 | leftbottomATCC VR-344 | centerbottom15.64 | centerbottom0.02 | centerbottom2.0 × 10 9 | centerbottom15.93 | centerbottom0.03 | centerbottom4.6 × 10 9 | centerbottom[ 29] |
| leftbottomDENV-2 | leftbottomATCC VR-345 | centerbottom20.76 | centerbottom0.20 | centerbottom6.6 × 10 7 | centerbottom18.77 | centerbottom0.11 | centerbottom6.7 × 10 8 | centerbottom |
| leftbottomDENV-3 | leftbottomATCC VR-1256 | centerbottom19.36 | centerbottom0.01 | centerbottom1.7 × 10 8 | centerbottom20.30 | centerbottom0.01 | centerbottom2.4 × 10 8 | centerbottom |
| leftbottomDENV-4 | leftbottomATCC VR-1257 | centerbottom16.33 | centerbottom0.19 | centerbottom1.3 × 10 9 | centerbottom16.18 | centerbottom0.29 | centerbottom3.9 × 10 9 | centerbottom |
| leftbottomYFV 17D | leftbottomX03700 | centerbottom19.17 | centerbottom0.17 | centerbottom1.9 × 10 8 | centerbottom20.51 | centerbottom0.17 | centerbottom5.6 × 10 8 | centerbottom[ 30] |
| leftbottomYFV ASIBI | leftbottomRKI reference strain | centerbottom23.73 | centerbottom0.05 | centerbottom9.1 × 10 6 | centerbottom23.72 | centerbottom0.28 | centerbottom4.7 × 10 7 | centerbottom |
| leftbottomYFV Brazil | leftbottomRKI reference strain | centerbottom21.18 | centerbottom0.07 | centerbottom5.0 × 10 7 | centerbottom22.50 | centerbottom0.10 | centerbottom1.2 × 10 8 | centerbottom |
| leftbottomYFV Ivory Cost | leftbottomRKI reference strain | centerbottom21.44 | centerbottom0.21 | centerbottom4.2 × 10 7 | centerbottom21.29 | centerbottom0.05 | centerbottom3.1 × 10 8 | centerbottom |
| leftbottomWNV Uganda | leftbottomAY532665, lineage 1 | centerbottom28.74 | centerbottom0.06 | centerbottom3.2 × 10 5 | centerbottom26.83 | centerbottom0.27 | centerbottom3.4 × 10 6 | centerbottom[ 31] |
| leftbottomWNV Israel | leftbottomlineage 2 | centerbottom19.81 | centerbottom0.15 | centerbottom1.2 × 10 8 | centerbottom19.86 | centerbottom0.01 | centerbottom4.0 × 10 8 | centerbottom |
| leftbottomTBEV K23 | leftbottomAF091010 | centerbottom16.33 | centerbottom0.03 | centerbottom1.3 × 10 9 | centerbottom16.78 | centerbottom0.14 | centerbottom2.1 × 10 8 | centerbottom[ 32] |
| leftbottomRSSEV | leftbottomRKI reference strain | centerbottom24.20 | centerbottom0.32 | centerbottom6.7 × 10 6 | centerbottom19.35 | centerbottom0.01 | centerbottom4.1 × 10 6 | centerbottom |
| leftbottomTBEV Louping ill | leftbottomRKI reference strain | centerbottom20.59 | centerbottom0.22 | centerbottom7.4 × 10 7 | centerbottom20.43 | centerbottom0.06 | centerbottom2.0 × 10 7 | centerbottom |
| leftJEV | leftATCC SA14-14-2 | center19.36 | center0.01 | center2.4 × 10 8 | center16.73 | center0.01 | center7.7 × 10 8 | centerIn house assay |
GC: genome copies, Ct: threshold cycle, SD: standard deviation.
DENV: Dengue virus, JEV: Japanese encephalitis virus, RSSEV: Russian spring-summer encephalitis virus, WNV: West Nile virus, TBEV: Tick-borne encephalitis virus, YFV: Yellow fever virus.
*Pan-Flavi assay was performed using Flavi-all S, Flavi all AS2, DEN4 F primer and Flavi all probe3 mix.
#The same amount of RNA (5 μl) was applied to both pan-Flavi assay and virus species-specific one-step qRT-PCR assays.
Performance of the Pan-Flavi assay to detect different flaviviruses. Five μl of viral RNA extracted from virus cell culture supernatant were applied to both pan-Flavi assay and RT-PCR
| leftbottom Flaviviridae | ||||
| leftbottom St. Louis Encephalitis virus | centerbottomSLEV | centerbottomATCC VR-1265 | centerbottom22.19 | centerbottomN.d. |
| leftbottom Usutu virus | centerbottomUSUV | centerbottomAY453411 | centerbottom22.48 | centerbottomN.d. |
| leftbottom Kunjin virus | centerbottomKUNV | centerbottomPI.France | centerbottom24.97 | centerbottomN.d. |
| leftbottom Kedougou virus | centerbottomKEDV | centerbottomArD 14701 | centerbottom29.62 | centerbottomPositive |
| leftbottom Koutango virus | centerbottomKOUV | centerbottomAnD 5443 | centerbottom30.29 | centerbottomPositive |
| leftbottom Spondweni virus | centerbottomSPOV | centerbottomSA Ar 94 | centerbottom26.02 | centerbottomNegative |
| leftbottom Uganda S virus | centerbottomUGSV | centerbottomArD 109325 | centerbottom29.48 | centerbottomPositive |
| leftbottom Wesselsbron | centerbottomWESSV | centerbottomArB 4177 | centerbottom24.73 | centerbottomPositive |
| leftbottom Zika virus | centerbottomZIKV | centerbottomPI, Senegal | centerbottom22.45 | centerbottomPositive |
| leftbottom Sepik virus | centerbottomSEPV | centerbottomMK 7148 | centerbottom16.77 | centerbottomPositive |
| leftbottom Dakar bat virus | centerbottomDBV | centerbottomAnD 249 | centerbottom32.57 | centerbottomPositive |
| leftbottom Yaounade virus | centerbottomYAOV | centerbottomArY 276/68 | centerbottom17.52 | centerbottomPositive |
| leftbottom Bouboui virus | centerbottomBOUV | centerbottomArB 490 | centerbottom31.88 | centerbottomNegative |
| leftbottom Saboya virus | centerbottomSABV | centerbottomAnD 4600 | centerbottom27.50 | centerbottomNegative |
| leftbottom Nounane virus # | centerbottomNOUV | centerbottomB3 isolate | centerbottomNo ct | centerbottomN.d. |
| leftbottom Bunya viridae | ||||
| leftbottom Rift valley fever virus | centerbottomRVFV | centerbottomRKI | centerbottomNo ct | centerbottomN.d. |
| leftbottom Togaviridae | ||||
| leftbottom Chikungunya virus | centerbottom | centerbottomLR 2006 | centerbottomNo ct | centerbottomN.d. |
| leftbottom Chikungunya virus | centerbottom | centerbottomST05,African isolate | centerbottomNo ct | centerbottomN.d. |
| leftbottom Sindbis virus | centerbottom | centerbottomRKI | centerbottomNo ct | centerbottomN.d. |
| leftbottom Influneza virus | ||||
| leftbottom H1N1 Hamburg | centerbottom | centerbottomA/Hamburg/04/2009 H1N1 | centerbottomNo ct | centerbottomN.d. |
| leftbottom H1N1 California | centerbottom | centerbottomA/California/04/2009 H1N1 | centerbottomNo ct | centerbottomN.d. |
| left H5N1 | center | centerA/dk/Germany R603/06 H5N1 | centerNo ct | centerN.d. |
N.d.: not done, Ct: threshold cycle, * RNA of African flaviviruses were detected using reference method described previously [ 33]. # Nounane virus was detected using final optimized primer sets Flavi all S/Flavi all S2/Flavi all AS4 and Flavi all probe 3 mix.
The specificity of the final version of the Pan-Flavi assay was tested using a panel of mosquito- and tick-borne flaviviruses, one insect-only novel flavivirus and one flavivirus with unknown vector, one virus of the Bunyaviridae family, three of the Togaviridae family and three of the Orthomyxoviridae family . Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. However, one insect-only novel flavivirus was not detected. Neither non-flaviviruses nor negative controls resulted in signals, demonstrating the absence of cross-reactivity of the Pan-Flavi assay with non-flaviviruses (Tables 2 and 3).
To determine the linearity of the assay, 10-fold serial dilutions of the standard plasmid were tested. The Pan-Flavi assay was shown to be linear over a range of 5 log
Standard curve constructed using ten-fold serial dilution of standard plasmid containing NS5 gene of yellow fever virus. The detection thresholds (Ct values) are plotted against the log of known standard plasmid (pYFV) copy numbers.
The Pan-Flavi assay was performed as a one-step qRT-PCR. The sensitivity of the Pan-Flavi assay to detect viral RNA was compared to three virus species-specific one-step qRT-PCR assays for DENV, TBEV and YFV, all assays could be thus compared in one run. The RNA stocks of DENV-1, YFV strain 17D and TBEV strain K23 were quantified by virus species-specific qRT-PCR as described previously [ 29, 30, 32]. Then, 10-fold serial dilution of the RNA was prepared from quantified stocks to cover a range of 10 1 to 10 7 genome copies/reaction. The detection limit of Pan-Flavi assay was determined using these pre-quantified RNA dilutions. Detection limit of the Pan-Flavi assay was 100 genome copies for YFV 17D, 10 genome copies for DENV-1 and 10 genome copies for TBEV K23 (Table 4).
Test of sensitivity of Pan-Flavi assay with pre-quantified viral RNA dilutions
| leftbottom1 × 10 7 | centerbottom16.8 | centerbottom16.6 | centerbottom- |
| leftbottom1 × 10 6 | centerbottom20.5 | centerbottom19.5 | centerbottom20.8 |
| leftbottom1 × 10 5 | centerbottom23.4 | centerbottom23.0 | centerbottom24.2 |
| leftbottom1 × 10 4 | centerbottom27.4 | centerbottom26.2 | centerbottom27.6 |
| leftbottom1 × 10 3 | centerbottom30.2 | centerbottom29.4 | centerbottom30.1 |
| leftbottom1 × 10 2 | centerbottom37.1 | centerbottom31.7 | centerbottom36.4 |
| left1 × 10 1 | center39.7 | center33.0 | centerNo Ct |
GC: genome copies, Ct: threshold cycle.
YFV: Yellow fever virus, TBE: Tick-borne encephalitis virus, DENV: Dengue virus.
The same amounts of RNA (5μl) from DENV, WNV, YFV, TBEV and JEV were detected quantitatively by using virus species-specific qRT-PCR assays, and the results were compared with those obtained by the Pan-Flavi assay for these viruses (Table 2). All four serotypes of DENVs were detected by the Pan-Flavi assay, with almost identical Ct values and GC/ml; although for DENV the Pan-Flavi assay showed delayed Ct values corresponding to one log dilution. For all YFV isolates tested the results were similar to those with the YFV-specific assay. Also WNV lineage 2 gave the same results as the WNV-specific assay, while the Ct values for lineage 1 virus were delayed. All three subtypes of TBEV were detected by both the Pan-Flavi assay and a TBEV-specific assay. The Ct values and genome copies/ml determined by both methods were identical except for RSSEV. For the detection of JEV, the Pan-Flavi assay had half a log lower sensitivity compared to the JEV-specific assay. WNV- and JEV-specific assays were performed in a two-step format which could explain their higher sensitivity compared to the Pan-Flavi assay.
In summary, the performance of the Pan-Flavi assay was comparable to virus species-specific assays except for minor differences for some strains of flaviviruses.
The ongoing emergence of flaviviruses poses major public health concerns worldwide. Techniques used for molecular testing and surveillance of flaviviruses should be able to detect and identify a wide range of flavivirus species rapidly, with a high level of specificity and sensitivity. For this purpose, an LNA probe-based qRT-PCR (Pan-Flavi assay) for detection of RNA of various flaviviruses was developed. We tested several important human-pathogenic flaviviruses to demonstrate the ability to detect relevant members of the flavivirus genus. The Pan-Flavi assay was also tested with other viruses, in particular phleboviruses, alphaviruses and influenza viruses, to determine absence of cross-reactivity.
In recent years, generic approaches targeting highly conserved NS3 and NS5 genes have been described for the detection of flaviviruses as summarized previously [ 24, 26]. These methods were predominantly in the format of nested RT-PCR or SYBR green qRT-PCR which required post-amplification methods such as sequencing or melting temperature analysis to verify the result. In general, these methods have shown variable or lower sensitivity than species-specific approaches when evaluated during EQA exercises for the molecular diagnosis of different flavivirus [ 29, 34]. Only in some cases assays were comparable to those from the species-specific methods [ 27].
In the past 10 years, the primer sets targeting a conserved region of NS5 have been developed and optimized for a broad detection of flaviviruses. This approach was not only useful to detect known flaviviruses, but has also led to discovery of novel flaviviruses [ 18, 35, 36].
For the novel Pan-Flavi assay, the degenerate primer sequences share the conserved NS5 region (Figure 2) targeted by the previous published primers (Figure 3). These primers exhibited good efficiency in detecting a broad range of common and exotic flaviviruses (Tables 2 and 3) but failed to detect the RNA of DENV type 4 or Nounane virus. The alignment for the Flavi all S primer revealed mismatches at 6th and 8th position from the 3′-end which would have been rated as non-critical (Figure 2). However, only addition of DEN4 F primer improved the assay for DENV type 4 (Tables 1 and 2). Remarkably, the 3rd position from 3′-end mismatch in the reverse primer Flavi all AS2 for YFV, DENV type 2 and Usutu virus did not have any impact as these three viruses were detected well by Pan-Flavi assay (Figure 2). These results underline that the in-silico analysis provides only a limited prediction for the primer specificity, which still requires to be confirmed by laboratory testing. Designing of a single probe for detection of all flaviviruses is challenging because of the genetic diversity and the restricted length of conserved regions. One approach is the use of oligonucleotide mixtures covering many target sequence variants, which was applied for detection of DENV and Crimean-Congo Hemorrhagic fever virus (CCHF) [ 37, 38]. Other approaches to design broad range probes are the use of a short conserved sequence in combination of minor groove binding (MGB) or LNA modifications [ 39, 40]. First, we used 22 specific probes for flaviviruses generic detection, but the amplification signals were insufficient. To overcome this problem, probes with LNA bases were designed for the Pan-Flavi assay. Major advantages of LNA probes in comparison to other real-time PCR formats are the shorter probe length, stability, increased sensitivity, specificity and quenching efficiency which have been demonstrated by many studies [ 30, 41- 45]. One of three designed and tested LNA-based probes (Flavi all probe) successfully targeted a broad spectrum of different flaviviruses. The signal for detection of YFV and DENV type 4 were optimized by adding small fractions of specific probes (Table 5).
Sequence alignment of primers and probe with 26 flaviviruses conserved region of NS5 gene. + Symbol under oligonucleotides indicates position of locked nucleotides.
The published and Flavi-all primer sites in the Yellow fever 17D genome (NC 002031) with locations of genes. In dark green: forward primer, in light green: reverse primer, in red: site of probe used in this study. The figure is prepared using Geneious pro version (5.4.3) created by Biomatters.
Oligonucleotide sequence of primers and probes used in Pan-Flavi qRT-PCR assay
| leftbottom Flavi all S | leftbottomTACAACATgATggggAARAgAgARAA | centerbottom10 pmol | centerbottomS | centerbottom8993–9019 | centerbottom54.7 |
| leftbottom DEN4 F | leftbottomTACAACATgATgggRAAACgTgAGAA | centerbottom10 pmol | centerbottomS | centerbottom8996–9019 | centerbottom60.18 |
| leftbottom Flavi all AS 1 | leftbottomgTCCCANCCDgCKgTRTC | centerbottom10 pmol | centerbottomAS | centerbottom9236–9253 | centerbottom52.5 |
| leftbottom | leftbottomgTCCCATCCAgCKgTRTCATC | centerbottom5 pmol | centerbottomAS | centerbottom9236–9256 | centerbottom57.1 |
| leftbottom Flavi all AS 2 | leftbottomgTgTCCCAgCCNgCKgTgTCATCWgC | centerbottom10 pmol | centerbottomAS | centerbottom9232–9260 | centerbottom69.6 |
| leftbottom Flavi all probe 1 | leftbottomFAM-AARggHAgYMgNgCCA + TH + T + g + g + T-BBQ | centerbottom5 pmol | centerbottomS | centerbottom9044–9065 | centerbottom69–83 |
| leftbottom Flavi all probe 2 | leftbottomFAM-Tg + gTWYATgTggYTNg + gRgC--BBQ | centerbottom5 pmol | centerbottomS | centerbottom9062–9082 | centerbottom62–75 |
| leftbottom Flavi all probe 3 mix | leftbottomFAM-Tg + gTWYATgT + ggYTNg + gRgC—BBQ c | centerbottom5 pmol | centerbottomS | centerbottom9062–9082 | centerbottom66-79 |
| leftbottom | leftbottomFAM-CCgTgCCATATggTATATgTggCTgggAgC-BBQ d | centerbottom0.5 pmol | centerbottomS | centerbottom9052–9081 | centerbottom74.7 |
| leftbottom | leftbottomFAM-TTTCTggAATTTgAAgCCCTgggTTT-BBQ e | centerbottom0.5 pmol | centerbottomS | centerbottom9086–9012 | centerbottom68 |
| leftbottom Flavi all S2 | leftbottomTACAACATgATgggMAAACgYgARAA | centerbottom10 pmol | centerbottomS | centerbottom8996–9019 | centerbottom58.2 |
| left Flavi all AS4 | leftgTgTCCCAGCCNgCKgTRTCRTC | center10 pmol | centerS | center9235–9260 | center64.1 |
Tm: temperature, Locked-Nucleic Acid bases are written as ‘ + _’, e.g., +A.
Degenerate bases: R = (A/G), W = (A/T), K = (T/G), Y = (C/T), N = (A/G/T/C).
a S: sense orientation, AS: antisense orientation.
b YFV strain; accession no: NC 002031.
c Flavi all probe, d Flavi probe YFV, e Flavi probe DEN4.
Final Pan-Flavi assay comprise of Flavi all S, Flavi all S2 & Flavi all AS4 primer and Flavi all probe 3 mix.
At present, RT-PCR in nested or hemi-nested format is used most frequently. It requires sequencing for the identification of viruses and needs approximately one day for experimentation. In addition, this carries a high risk of contamination caused by open handling of PCR products, increasing the potential for false positives. In contrast, the Pan-Flavi assay presented here requires only some 50 minutes for specific and sensitive detection of several flaviviruses in one reaction. The detection sensitivity of YFV, DENV, TBEV, JEV and WNV, using the described Pan-Flavi assay, was close to or as good as the species-specific qRT-PCR assays. These differences may have resulted from deviant reactions conditions, enzyme kits and instruments, which were used as published or in-house established, and it is to mention, that WNV and JEV assays were performed in a two-step PCR which is known to be more sensitive than the one-step procedure [ 46].
The sensitivity of the Pan-Flavi assay to detect RNA was demonstrated by using 10-fold serial dilutions of viral RNA from DENV-1, YFV and TBEV. The assay was able to detect 10–100 viral RNA copies per reaction, depending on the respective flavivirus. The assay allows the quantification of flavivirus RNA by use of standard plasmids or standardised in-vitro RNA, providing information regarding viral load and can be easily established in other laboratories. Therefore, the Pan-Flavi assay could be used to confirm the presence of flaviviruses in a sample without post amplification steps or complement species-specific flavivirus assays. The amplicon length is about 260 bp, which is sufficient to distinguish and identify different the virus by (pyro) sequencing or using for example an array.
The Pan-Flavi assay allows rapid detection of a broad range of flaviviruses including local-endemic and exotic species with a high level of sensitivity and is cost-effective, making it suitable for screening of livestock samples, monitoring viral activity, outbreak investigation as well as surveillance studies of flaviviruses, and might help finding and identifying novel flaviviruses. We expect that the assay can be also helpful for testing clinical samples from patients with symptoms especially in an early phase of infection and without knowledge about locally prevalent flaviviruses or travel history in order to avoid parallel testing on several flaviviruses, although a negative test will of course not prove absence of a flavivirus infection.
Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. The Pan-Flavi assay presented here has been developed and evaluated in comparison with flavivirus species-specific qPCR assays to detect a broad range of flaviviruses simultaneously in a single-tube. This assay represents a valuable tool for the detection of flaviviruses in livestock samples, and will support molecular diagnosis as well as surveillance studies of various vectors and non-human hosts.
All flavivirus and non-flavivirus strains listed below were derived from cell culture and provided by the Robert Koch Institute, Berlin, Germany. The following inactivated and stable virus preparations were used in this study: DENV-1 VR344 (Thai 1958); DENV-2 VR345 (TH-36 strain); DENV-3 VR216 (H87 strain); DENV-4 VR217 (H241 strain); WNV Uganda strain (AY532665); WNV Israel (H. Bin, Sheba Medical Center, Israel), kunjin virus (Institute Pasteur, France), usutu virus (AY453411); JEV (ATCC SA14-14-2); Saint Louis encephalitis virus (SLEV) (ATCC VR-1265); TBEV strain K23 (AF091010); YFV strain 17D (X03700); YFV strain ASIBI (AY640589); YFV strain Brazil; YFV strain Ivory Coast; Russian Spring Summer Encephalitis virus (RSSEV); chikungunya virus (LR 2006); chikungunya virus African isolate; sindbis virus; Rift Valley Fever virus and influenza A virus subtype H5N1 (A/dk/Germany R603/06 H5N1).
Two different standard preparations of H1N1 influenza viruses (A/California/04/2009 and A/Hamburg/04/2009) used in this study were provided by the European Network for Diagnostics of Imported Viral Diseases (ENIVD). RNA of Nounane virus was kindly provided by Dr. Fabian Leendertz from NG2, Robert Koch Institute, Berlin. RNA of eleven African flaviviruses (Table 3) were kindly provided by Dr. Amadou A Sall and testing of these viruses was done at Institut Pastuer Dakar, Senegal.
Viral RNA from above described viruses was isolated from 140 μl aliquots of cell culture supernatants, using the QIAamp Viral Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted in 100 μl of elution buffer and stored at -80°C until further use.
The flaviviral NS5 gene contains conserved regions often used for PCR testing. Our laboratory results but also the literature [ 27] revealed that the commonly used primers [ 26- 28] exhibit a low sensitivity and we failed to detect rare flaviviruses such as Nounane virus. The primer binding region were aligned against all Genbank (NCBI) deposited flavivirus sequences including partial entries for the NS5 region only using the MEGA5 software. Based on the alignment, 32 primers and 22 probes representing all known variants of flavivirus sequences were selected to be used as mixture in one reaction (data not shown). In an alternative approach two degenerate generic primer sets and three degenerate probes containing LNA nucleotides were designed to match ‘all’ flaviviruses. LNA nucleotides contain a 2 ′–5 ′ bridge, increasing the duplex stability, which in turn allows the use of shorter sequence probes matching the conserved sequence motif.
The Pan-Flavi qRT-PCR assay (Pan-Flavi assay) was run with the Transcriptor One-Step RT-PCR Kit (Roche Diagnostics Mannheim, Germany) according to the manufacturer’s instructions. In brief, 5 μl of RNA, 10 pmol of primers Flavi all sense, Flavi all antisense 2 and DEN4 F, and a mixture of three probes (Table 5) were added to the master mix. Optimized run conditions were as follows: reverse transcription at 55°C for 2 min, denaturation at 95°C for 30 s and 45 cycles of 95°C for 10 s and 60°C for 25 s. The amplification was performed on a Roche LightCycler 480 instrument, software version 1.5. The spectrum of the assay can be expanded by using Flavi all S2 instead of DEN4 F primer (Table 5).
Serial dilutions of a standard (10–10 6 copies/μl) were run in duplicate for quantification. Standard plasmid was prepared by cloning the PCR product obtained from YFV using a TOPO TA Cloning Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions and quantified by spectrophotometric method.
Except for DENV and JEV we used published group-specific assays for comparison.
For TBEV, we used an assay targeting the NS1 gene as described previously [ 32]. qRT- PCR was carried out in one-step format on an ABI 7500 cycler (Applied Biosystems, California, USA), using the Superscript III Platinum OneStep qRT-PCR Kit (Invitrogen).
A WNV-specific qRT-PCR assay for detection of lineages 1 and 2 was used to detect and quantify genome copies (GC) of WNV as described previously [ 31]. qRT-PCR was performed in two-step format on a Stratagene MX 3000 cycler (Agilent Technologies, Santa Clara, USA).
YFV-specific primer combination YFV FP/RP and probe YFV LNA2 were used to detect and quantify genomic RNA of YFV as described previously [ 30]. The assay was performed in one-step format on the LightCycler 480 instrument using the QuantiTect Virus Kit (Qiagen, Hilden, Germany).
DENV-genomic RNA was tested and quantified by an in-house qRT-PCR as described previously [ 29]. The assay is a DENV group-specific assay and was carried out in one-step format on an ABI 7500 real-time PCR system using the AgPath-ID One-Step RT-PCR Kit. Plasmid standards were used for the quantification of the DENV GE.
A JEV-specific in-house qPCR assay was used for detection and quantification of JEV GE. qPCR was performed in two-step format on a Stratagene MX 3000 cycler. cDNA was generated using SuperScript II (Invitrogen) and was stored at -20°C until use. The test was performed using Platinum Taq polymerase (Invitrogen) in a total volume of 25 μl containing 5 μl of cDNA, 2 μl of 10x Reaction Mix, 4 mM MgCl
The pan-flavi assay was evaluated using forward primers Flavi all S and DEN4 F, reverse primer Flavi all AS2 and Flavi all probe 3 mix. Based on an alignment of latterly available NS5 gene flavivirus sequences, we substituted primer DEN4F by Flavi all S2 and Flavi all AS2 by Flavi all AS4 to include some novel flaviviruses (Table 3).
RT: Reverse transcription; Tm: Melting temperature; Ct: Threshold cycle; GC: Genome copies; DENV: Dengue virus; YFV: Yellow fever virus; TBEV: Tick-borne encephalitis virus; WNV: West nile virus; JEV: Japanese encephalitis virus; LNA: Locked-nucleic acids.
The authors declare that they have no competing interests.
PP, MK conceived and designed the experiments; OL, MK, PP participated in the sequence alignment/assay design; PP, TK, UL, OF performed the Experiments; PP, OL, MK analysed the data; AS, OL, MN contributed reagents/materials/analysis tool, PP wrote paper. All authors have read and approved this manuscript.
Pranav Patel is working as a scientist at Department of Centre for Biological Security 1, Robert Koch Institute, Berlin, Germany and has vast experience on developing rapid diagnostic assays and platforms for detection of emerging pathogens.
Oligonucleotides were kindly provided by the Tib-Molbiol, Berlin. The authors are grateful to Anette Teichmann and Patricia Bußmann for technical support. We also thank Eduardo Thuroff and Regina Schädler for their critical review of the manuscript and Ursula Erikli for copy editing.
This work was supported by the consultant laboratory for tick-borne encephalitis virus at Center for Biological Security 1 (ZBS1), Robert Koch institute as a part of research activities.


Erica Daborn
Erica Daborn 'Hecho en Mexico'
A collection of paintings made during a sabbatical year from the School of the Museum of Fine Arts, Boston, eight months of which was spent in San Miguel de Allende, Mexico
We all enjoy stories. From mythology to the movies on a Saturday night. They are the way we all connect to the bigger picture; the way we experiment with a response to challenging events. Through stories we experience the exotic, the traumatic, the unfamiliar, all from the safety of a parent's lap, a comfy sofa, or the anonymity of a darkened theatre.
But unlike fairy stories or moral tales, myths or movies, the stories in these paintings have no beginning or end. They show only fragments of the human drama, neither disclosing what has preceded the moment they capture, nor indicating what will follow. Some appear to be accidental encounters, others family dramas, still others homely sagas poised precariously between Mother Goose and the Twilight Zone. Each image emerges out of an intuitive drawing process. I wait for interesting characters to enter the frame; then I follow their cues. Many are erased, discarded; the most persuasive remain.


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