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tisdag 30 december 2014

BBC kertoo tänään 2014 ebolapurkauksen lähtöpaikan (Sitaatti)


First Ebola boy likely infected by playing in bat tree


Bat being captured to be tested for Ebola Other researchers have been testing bats in West Africa for Ebola virus

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The Ebola victim who is believed to have triggered the current outbreak - a two-year-old boy called Emile Ouamouno from Guinea - may have been infected by playing in a hollow tree housing a colony of bats, say scientists.
They made the connection on an expedition to the boy's village, Meliandou.
They took samples and chatted to locals to find out more about Ebola's source.
The team's findings are published in EMBO Molecular Medicine.
Ebola trail
Meliandou Meliandou is a small village surrounded by farmland and large trees
Meliandou is a small village of 31 houses.
It sits deep within the Guinean forest region, surrounded by towering reeds and oil palm cultivations - these are believed to have attracted the fruit bats carrying the virus passed on to Emile.
During their four-week field trip in April 2014, Dr Fabian Leendertz and colleagues found a large tree stump situated about 50m from Emile's home.
Villagers reported that children used to play frequently in the hollow tree.
Emile - who died of Ebola in December 2013 - used to play there, according to his friends.
The villagers said that the tree burned on March 24, 2014 and that once the tree caught fire, there issued a "rain of bats".
the tree Children from the village used to play in and around the tree
A large number of these insectivorous free-tailed bats - Mops condylurus in Latin - were collected by the villagers for food, but disposed of the next day after a government-led ban on bushmeat consumption was announced.
While bushmeat is thought to be a possible source of Ebola, the scientists believe it didn't trigger the outbreak.
Instead, it was Emile's exposure to the bats and their droppings as he played with his friends in the hollowed tree.
Pest control The scientists took and tested ash samples from the tree and found DNA traces that were a match for the animals.
While they were unable to test any of the bushmeat that the villagers had disposed of, they captured and tested any living bats they could find in and around Meliandou.
No Ebola could be detected in any of these hundred or so animals, however.
But previous tests show this species of bat can carry Ebola.
Dr Leendertz, from the Robert Koch Institute in Germany, and his colleagues say this must be a pretty rare occurrence though.
The expedition team
Dr Leendertz said: "That is also obvious when you think about how many tonnes of bat meat is consumed every year.
"If more bats carried the virus, we would see outbreaks all the time."
He says it is vital to find out more about the bats.
"They have moved into human settlements. They do not just live in the trees but also under the roofs of houses in the villages.
"The Ebola virus must jump through colonies from bat to bat, so we need to know more."
But culling the animals is not the answer.
"We need to find ways to live together with the wildlife. These bats catch insects and pests, such as mosquitoes. They can eat about a quarter of their body weight in insects a day.
"Killing them would not be a solution. You would have more malaria."

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söndag 28 december 2014

Kointähden hohtoa Sierra Leonessa: E-Sierra Leonen KONON alueen 14 päällikkökuntaa organisoituneet. yhteistyöhön


WHO 24. 12. 2014  Yhteiskunnallinen organisoituminen päällikkötasoa myöten tapahtunut Ebolan voittamiseksi!

Sierra Leone communities organize Ebola response

December 2014
Meeting of Ebola Taskforce in Nimiyama Chiefdom, Eastern Sierra Leone, aiming to mobilise all community members in the fight against the disease.
Meeting of Ebola Taskforce in Nimiyama Chiefdom, Eastern Sierra Leone, aiming to mobilise all community members in the fight against the disease.
WHO/Saffea Gborie
“In our chiefdom we have the necessary structures in place— leaders of all the villages are part of the Ebola response and fully involved, we have an active surveillance approach, and the population is well informed,” explains Philip Musa Koroma, Deputy Paramount Chief of Nimiyama Chiefdom in Kono district in Eastern Sierra Leone.

He has just finished a meeting with the chiefdom Ebola Taskforce, where village leaders and representatives of civil society groups proposed and discussed solutions to the current situation. Aiming to mobilize all community members, this Ebola Taskforce embodies local, collective ownership in the fight against the disease.
"After I tested positive for Ebola in the district hospital in Koidu, I was transferred to Kenema for treatment. I think my full recovery was due to the early use of supportive medicines and intravenous fluid. I requested this treatment while still waiting for the transfer."
Umaru Sow, head of Nimiyama community health centre, Sierra Leone
The chiefdom saw its first case of Ebola two months ago. In the last two weeks, more than 20 cases have been reported.
“Our great challenge is logistics. We don’t have ambulances. It takes a long time for teams to arrive while roads are difficult and distances long,” explains Chief Koroma. “The imminent construction of a Community Care Center in our chiefdom and of an Ebola Treatment Center in the district capital Koidu will help patients receive care early.”
One of those who recognises the importance of early care is Umaru Sow, head of Nimiyama main community health centre in the chiefdom. “I got infected when treating a patient who did not give me full information, so I assumed he would not be a possible Ebola case,” recalls Mr Sow. “After I tested positive for Ebola in the district hospital in Koidu, I was transferred to Kenema for treatment. I think my full recovery was due to the early use of supportive medicines and intravenous fluid. I requested this treatment while still waiting for the transfer.”

Timely and coordinated response

Authorities of Kono district were quick to react to the new wave of cases and to mobilise available resources both locally and from international partners. The Kono district has 14 chiefdoms and each one has set up an Ebola Taskforce with regular meetings at District level to coordinate the response.
Daily command centre meetings are chaired by the District Ebola Response Centre Coordinator and aim to coordinate incoming assistance from international partners now on the ground.
The first week of December, the World Health Organisation (WHO) and US Centers for Disease Control joined the Ministry of Health and the National Ebola Response Center in a mission to assist authorities evaluate the situation. With assistance fromm the United Nations Mission for Ebola Emergency Response (UNMEER), supplies and personnel were flown in rapidly. 

The International Federation of Red Cross (IFRC) helped Koidu hospital to better organise its temporary holding centre. The IFRC is currently building a new Ebola Treatment Center (ETC) which will eliminate the need to transfer critically-ill patients via a 4-hour journey on a difficult road to the closest ETC in Kenema.

The United Nations Population Fund (UNFPA) has just completed training more than 300 contact tracers and supervisors.
 UNICEF is supporting social mobilization,
 while the International Rescue Committee supported training 45 hospital staff on safe infection prevention and control measures.
 Other partners such as the World Food Programme,
 Partners in Health and World Vision are also supporting the response.
Umaru Sow, branding the discharge certificate from Ebola treatment centre in front of temporarily closed community health center in Nimiyama chiefdom, Sierra Leone
Umaru Sow, branding the discharge certificate from Ebola treatment centre in front of temporarily closed community health center in Nimiyama chiefdom, Sierra Leone
 
WHO/Saffea Gborie
“The construction of an ETC will improve the situation as we will no longer need to move patients who test positive to Kenema,” explains Mr Michael N`dolie, WHO Team Leader in Koidu. “The biggest issue remains mobility and logistics. We urgently need ambulances for case management teams and vehicles for burial teams and surveillance teams.”

Non-Ebola health needs

Back in Nimiyama Chiefdom, the community health centre run by Umaru Sow is now closed. It needs to be disinfected since seven Ebola patients came through the centre. All of Umaru’s co-workers are quarantined, as they had contact with him and with those seven patients that slept at the centre before they were transferred to a holding centre in Koidu.
“People with non-Ebola needs now must walk more than an hour to get some assistance,” Umaru adds. People are said to fear going to health centers for fear of catching Ebola there. According to a local midwife, a number of deliveries are now being done at homes assisted by traditional birth attendants.
“It takes time, but we hope when we reopen the health centre at the end of the month that people will be using it again.”

lördag 27 december 2014

Suomen THL antaa enterovirus EV D68:sta samanasteisen tiedon . Ei täysin uusi virus kuitenkaan.

Enterovirus D68 voi aiheuttaa vakavia infektioita

SITAATTI Terveyden ja Hyvinvoinnin Laitoksen nettikohdasta  https://blogi.thl.fi/sv/web/infektiouutiset/etusivu/-/blogs/enterovirus-d68-voi-aiheuttaa-vakavia-infektioita


Artikkelin kirjoittanut : Carita Savolainen-Kopra

Enterovirus D68 (EV-D68) -tartunnat jatkuvat edelleen laajasti Yhdysvalloissa ja Kanadassa. EV-D68 on osoitettu jo yli tuhannelta vakavaan hengitystieinfektioon sairastuneelta potilaalta. Sairastuneista suurin osa on lapsia ja tauti on vaatinut sairaalahoitoa varsinkin astmaa sairastavilla. EV-D68 on löytynyt myös muutamilta potilailta, joilla on ollut hengitystieinfektion jälkeen neurologisia, polionkaltaisia halvausoireita. Viruksen ja neurologisen taudin mahdollista yhteyttä selvitetään edelleen.
Euroopassa, ja myös Suomessa, on tänä syksynä aloitettu EV-D68:n tehostettu diagnostiikka, jonka tarkoituksena on kartoittaa tämän virustyypin esiintymistä ja sen aiheuttamaa taudinkuvaa. Viruksen kierrosta väestössä ei ole aikaisemmin ollut kattavaa tietoa. Enterovirukset aiheuttavat yleensä oireiltaan lieviä hengitystieinfektioita, joista näytteitä otetaan harvoin. Vakavimmissakaan infektioissa hengitystienäytteiden enteroviruksia ei aina tyypitetä. EV-D68:n varma tunnistaminen edellyttää aina positiivisen näytteen jatkotutkimuksia ja tyypitys tehdään useimmiten sekvenssianalyysilla.

EV-D68 on osoitettu elo-lokakuussa kuudelta hengitystieinfektiota sairastavalta potilaalta Lounais-Suomen alueelta. Euroopan ensimmäinen neurologiseen tautiin yhdistetty EV-D68 -infektio raportoitiin Keski-Ranskasta lokakuun 2014 alussa. EV-D68 todettiin ainoana mikrobiologisena löydöksenä 4-vuotiaalla potilaalla, jolla oli vakavan hengitystieinfektion lisäksi myös mm. aivokalvontulehdus ja halvausoireita. Viruksen esiintymistä seurataan edelleen aktiivisesti sekä suomalaisissa että useissa muissa eurooppalaisissa laboratorioissa.

EV-D68 ei ole Euroopassa täysin uusi virus. Suomessa EV-D68:ia on löydetty satunnaisesti sellaisissa tutkimuksissa, joissa hengitystienäytteiden viruksia on tarkemmin tyypitetty. EV-D68:n tiedetään kiertäneen viime vuosina myös muualla Euroopassa. Esimerkiksi Hollannissa oli EV-D68:n aiheuttama epidemia vuonna 2010, minkä jälkeen EV-D68:ia on löydetty hengitystienäytteistä joka syksy.

Lisätietoa
Non polio enterovirus (CDC)
Continued seasonal circulation of enterovirus D68 in the Netherlands 2011–2014 (Eurosurveillance)
All Known Human Rhinovirus Species Are Present in Sputum Specimens of Military Recruits During Respiratory Infection
Mikä on enterovirus?

Enterovirus D68 mainitaan tämän syksyn seurattavissa asioissa

  • Ruotsin Kansanterveysviraston  palstalla  mainitaan seurattavana asiana  enterovirus D68
Ruotsalainen SITAATTI: kertoo, että elokuun  2014 puolesta välistä on ollut Amerikassa ja Kanadassa enterovirus D68 -purkausta ja virusta on diagnosoitu marraskuun lopulle mennessä  yli 1300 henkilöstä, joilla on esiintynyt vakavaa  tämän viruksen aiheuttamaa keuhkosairautta. Useimmat näistä ovat olleet lapsia. Karolinskassa on havaittu ensimmäiset  tapaukset  Ruotsistakin  marraskuun lopussa. Sen takia on testattu useampikin näyte lapsilta, joilla on ollut  positiivinen PCR enteroviruksille, rhinovirukselle tai  näille molemmille. Kaikenkaikkiaan on  tunnistettu kuusi   EV-D68 positiivista näytettä  Viisi oli alle 4 -vuotiaita lapsia ja yksi hieman vanhempi.  Viidellä oli hengitystieinfektio ja kaksi joutui aivan tehohoito-osastolle asti.  Neljällä lapsella oli  ennestään obstruktiivista keuhkosairautta.  Kenelläkään ei ollut keuhkotulehdusta. Kuudennella lapsella oli enemmänkin yleisen virusinfektion oireita.  Kaikki lapset tervehtyivät eikä kellekään tullut halvausoireita. Muita mikrobiologisia löytöjä ei ollut selittämässä lasten oireita.

Enterovirus D68 påvisat i Sverige
Publicerat
Sedan mitten av augusti 2014 pågår ett utbrott av enterovirus D68 (EV-D68) i USA och Kanada. Hittills har mer än 1300 personer diagnosticerats med allvarlig luftvägssjukdom orsakad av EV-D68 och de allra flesta har varit barn. Nu har de första svenska fallen identifierats på Karolinska Universitetssjukhuset i Solna.
För att få en indikation på om EV-D68 förekommer i Stockholmsområdet har Mikrobiologen på Karolinska Universitetssjukhuset genomfört en undersökning av drygt 20 luftvägsprov från barn som testat positivt i PCR för enterovirus, rhinovirus eller bägge.
Totalt sex fall har identifierats med EV-D68. Fem av barnen var under 4 år och ett var lite äldre. Fem av barnen hade luftvägsinfektion och två barn fick vårdas några dagar på intensivvårdsavdelning. Fyra hade obstruktiv lungsjukdom sedan tidigare. Ingen hade lunginflammation. Det sjätte barnet hade mer allmänna symtom på virusinfektion. Alla barn har tillfrisknat och inget hade förlamningssymptom. Inga andra mikrobiologiska fynd påvisades som förklaring på barnens symtom.
  • Todetut virukset ovat sukua  USA:n purkauksen viruskannoille

Kaikki   testatut näytteet on otettu heinäkuun- lokakuun  ajanjaksona 2014. Tähän mennessä on tyypitetty 21 näytettä ja niistä on osoitettu kuudessa  EV-D68. Tarkempi analyysi virusten sukulaisuussuhteista on osoittanut, että kaikki virustyypit ovat hyvin lähisukuisia keskenään ja  sukua USA:n viruskannoille.

Stammar från utbrottet i USA
Samtliga prov var tagna mellan juli och oktober 2014. Hittills har 21 prover typats och EV-D68 påvisades i 6 av dessa prover. Släktskapsanalys visar att alla sex virusstammarna är mycket nära besläktade med varandra och med stammar från utbrottet i USA 2014.
  •  Löytö ei ole yllättävä

 Löydöt osoittavat, että Ruotsissa kiertelee EV-D68 virusta, mikä ei sinänsä yllätä, koska sitä kiertelee  muuallakin Euroopassa.  Mutta ei tiedetä, miten tavallinen se on ja miten laajalti se on geografisesti levinnyt.  Saattaa olla, että sen leviäminen on rajoittumaan päin, kuten tavallisesti  käy rhinovriuksilla ja enteroviruksilla  loppusyksyä kohden. Tästä huolimatta tulee ottaa huomioon EV-D68 viruksen mahdollisuus, jos  hengitystieinfektio on vakava ja siihen liittyy  lapsen halvauksia.  Ruotsin kansanterveysvirastolle ei ole kuitenkaan tullut raporttia Ruotsin alueella ilmenneestä vakavasta EV-D68 viruksen aiheuttamasta tautitapauksesta.  Tämän viruksen tyypittäminen tehdään kansanterveysvirastossa ja  Karoliinisen Yliopistosairaalan Mikrobiologian laitoksella.

Inte oväntade fynd
Fynden visar att EV-D68 cirkulerar i Sverige, vilket inte är oväntat eftersom viruset påvisats på andra håll i Europa. Det är oklart hur vanligt förekommande och geografiskt utbrett viruset är. Det är möjligt att spridningen kan vara på väg att minska såsom vanligen sker mot slutet av hösten med rhinovirus och enterovirus. Trots detta bör möjligheten av EV-D68 infektion övervägas vid fall av allvarlig luftvägsinfektion eller förlamning hos barn. Folkhälsomyndigheten har inte fått någon rapport av allvarlig sjukdom orsakad av EV-D68 i Sverige.
Typning av EV-D68 görs på Folkhälsomyndigheten och Mikrobiologen på Karolinska Universitetssjukhuset.

Lisätietoa tästä  viruksesta  saa allamainitusta linkistä:
Läs mer
Utbrott av enterovirus D68 i USA, Kanada och Europa

Suomennosta  27.12. 2014

torsdag 25 december 2014

Onko rokotetta uutta A(H5N1) influenssavirusta vastaan? Is there any vaccine against A(H5N1)?

http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm376444.htm

FDA NEWS RELEASE

 
For Immediate Release: Nov. 22, 2013
Media Inquiries: Jennifer Rodriguez, 301-796-8232, jennifer.rodriguez(at)fda.hhs.gov
Consumer Inquiries: 888-INFO-FDA,OCOD(at)fda.hhs.gov
 
FDA approves first adjuvanted vaccine for prevention of H5N1 avian influenza
Vaccine to supplement National Stockpile, not intended for commercial availability
 
The U.S. Food and Drug Administration today approved the first adjuvanted vaccine for the prevention of H5N1 influenza, commonly known as avian or bird flu. The vaccine, Influenza A (H5N1) Virus Monovalent Vaccine, Adjuvanted, is for use in people 18 years of age and older who are at increased risk of exposure to the H5N1 influenza virus.  
 
Avian influenza is an infectious disease of birds caused by certain influenza A viruses. Most avian influenza A viruses do not infect people. However some viruses, such as H5N1, have caused serious illness and death in people outside of the U.S., mostly among people who have been in close contact with infected and ill poultry. When people do become infected with H5N1, about 60 percent die, according to the World Health Organization. H5N1 is an influenza virus with pandemic potential because it continues to infect wild birds with occasional outbreaks of influenza disease in poultry populations, and most humans have no immunity to it.
 
This vaccine could be used in the event that the H5N1 avian influenza virus develops the capability to spread efficiently from human to human, resulting in the rapid spread of disease across the globe,” said Karen Midthun, M.D., director of the FDA’s Center for Biologics Evaluation and Research. “Vaccines are critical to protecting public health by helping to counter the transmission of influenza disease during a pandemic.”
 
The H5N1 avian influenza vaccine is not intended for commercial availability. The U.S. Department of Health and Human Services has purchased the vaccine from the manufacturer, ID Biomedical Corporation of Quebec, Quebec City, Canada (a subsidiary of GlaxoSmithKline Biologicals), for inclusion within the National Stockpile for distribution by public health officials if needed. 
 
The vaccine is made using an egg-based manufacturing process, which is also used for ID Biomedical Corporation’s seasonal influenza vaccine, FluLaval. It contains the adjuvant AS03, an oil-in-water emulsion. An adjuvant is a substance incorporated into some vaccines to enhance or direct the immune response of the vaccinated individual. The adjuvant makes it possible to use a small amount of influenza protein per dose of vaccine to elicit the desired immune response in an individual to prevent influenza disease. Reducing the amount of influenza protein per dose helps to increase the total number of doses of a safe and effective vaccine available for the public during a pandemic. 
 
The H5N1 component and the AS03 adjuvant component are supplied in two separate vials, which must be combined prior to use. The vaccine is administered via intramuscular injection in two doses, 21 days apart.
 
The evaluation of safety compared approximately 3,400 adults 18 years of age and older who received the vaccine to about 1,100 adults who received placebo in a multi-center study. The most common side effect reported during the clinical studies among the vaccine recipients was injection site pain. Muscle aches, headache, fatigue and injection site redness and swelling were also common. To determine how well the vaccine works, the immune response was evaluated in about 2,000 of the vaccinated adults. The results showed that 91 percent of individuals between the ages of 18 and 64 years and 74 percent of individuals 65 years and older who received the two-dose regimen developed antibodies at a level that is expected to reduce the risk of getting influenza.
 
The manufacturer will collaborate with the FDA and other U.S. governmental agencies on plans to collect additional safety and effectiveness data through U.S. government-sponsored studies of the vaccine, in the event that it is used during an H5N1 influenza virus pandemic.
 
For more information:
 
The FDA, an agency within the U.S. Department of Health and Human Services, protects the public health by assuring the safety, effectiveness, and security of human and veterinary drugs, vaccines and other biological products for human use, and medical devices. The agency also is responsible for the safety and security of our nation’s food supply, cosmetics, dietary supplements, products that give off electronic radiation, and for regulating tobacco products.
 
# # #

onsdag 24 december 2014

A H5N1 influenssassa on ongelmana HLH:n kaltainen reaktiotapa

  • Pahamaineinen HLH on harvinainen, letaali solutason immunologinen häiriötila. Siinä on vikaa immuunisysteemin normaalissa ylläpidossa säätelyteitten kriittisissä kontrollipisteissä ja silloin immunologisen vasteen luonnollisten päätöstapahtumien varmistuminen estyy.
Haemophagocytic lymphohistiocytosis (HLH, OMIM #267700, #603553) is a rare, lethal, cellular immunological disorder that draws attention to critical, genetically determined checkpoints in the regulatory pathways that normally maintain homeostasis within the immune system and ensure the natural termination of effector immune responses. 
  • HLH, hemofagosyyttinen lymfohistiosytoosi,  käsittää useita erilaisia tautikokonaisuuksia kuten primäärin tai perinnöllisen muodon ja sekundäärisen muodon, jota liittyy infektioihin tai maligniteetteihin.
  •  ( Huom: lintuinfluenssan H5N1 yhteydessä on mainittu HLH:n kaltainen reaktiotapa yhtenä komplikaationa)

Kanadassa lintuinfluenssaa A(H5N1). Myös yksi nuori nainen menehtynyt siihen.

http://www.calgaryherald.com/news/metro/Avian+detected+Fraser+Valley+farms+CFIA/10433853/story.html
Tämä lintuvirus A(H5N1) on  tarkasti seurannassa, koska se on interfaasissa  ihmiskuntaan. Ihmistapaukset ovat olleet vaikeita.

Hollannin lintuinfluenssa A(H5N8)

 A(H5N8) lintuinfluenssa tapaus ei jäänyt yhdeksi, vaan samaa esiintyi toisellakin farmilla OIE:n tiedon mukaan. 

Two Outbreaks of Bird Flu Confirmed in the Netherlands

05 December 2014
THE NETHERLANDS - The World Organisation for Animal Health (OIE) confirms the two outbreaks of highly pathogenic avian influenza, strain H5N8 in Kamperveen and Zoeterwoude.
The outbreak in Kamperveen in Overijssel started on 21 November, and involved 14,600 birds. Around 100 died and the rest were destroyed. The farm is situated within the 1-km zone of the previously reported outbreak in Kamperveen.
The second outbreak started on 29 November at Zoeterwoude in Zuid-Holland (South Holland). It hit a flock of 28,000 layer and breeding hens, of which, 25 died and the rest were destroyed. All premises in the 10-km zone have been screened for the virus.

tisdag 23 december 2014

ROKOTUKSISTA selventävää

http://www.helsinki.fi/palmenia/helsinki/farmasia/pd/2014/PD%20lopputy%C3%B6%20Tiina%20Tuononen.pdf

Pegivirus Persistent GB virus HPgV, Flaviviridae Pegivirushistoriaa

J Gen Virol. 2011 Feb;92(Pt 2):233-46. doi: 10.1099/vir.0.027490-0. Epub 2010 Nov 17.The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae.Stapleton JT1, Foung S, Muerhoff AS, Bukh J, Simmonds P.
Abstract
In 1967, it was reported that experimental inoculation of serum from a surgeon (G.B.) with acute hepatitis into tamarins resulted in hepatitis.
 In 1995, two new members of the family Flaviviridae, named GBV-A and GBV-B, were identified in tamarins that developed hepatitis following inoculation with the 11th GB passage. either virus infects humans, and a number of GBV-A variants were identified in wild New World monkeys that were captured.
Subsequently, a related human virus was identified [named GBV-C or hepatitis G virus (HGV)], and recently a more distantly related virus (named GBV-D) was discovered in bats.
Only GBV-B, a second species within the genus Hepacivirus (type species hepatitis C virus), has been shown to cause hepatitis; it causes acute hepatitis in experimentally infected tamarins.
 The other GB viruses have however not been assigned to a genus within the family Flaviviridae.
 Based on phylogenetic relationships, genome organization and pathogenic features of the GB viruses, we propose to classify GBV-A-like viruses, GBV-C and GBV-D as members of a fourth genus in the family Flaviviridae, named Pegivirus (pe, persistent; g, GB or G). We also propose renaming 'GB' viruses within the tentative genus Pegivirus to reflect their host origin.
PMID:
21084497
[PubMed - indexed for MEDLINE]

PMCID:
PMC3081076

Free PMC Article

HCV ja HPgV katsaus . Ehkä tässä on uusi HCV genotyyppi 7 mainittuna, jota on vain yksi maailmassa.

 LÄHDE:
Infect Genet Evol. 2013 Oct;19:386-94. doi: 10.1016/j.meegid.2013.01.021. Epub 2013 Feb 16.
Hepatitis C virus infections in the Democratic Republic of Congo exhibit a cohort effect.
Abstract
The prevalence and genetic diversity of hepatitis C virus (HCV) and human pegivirus (HPgV) in many regions of sub-Saharan Africa is poorly characterized, including in the Democratic Republic of Congo--the largest country in the region and one of the most populous.

To address this situation we conducted a molecular epidemiological survey of HCV and HPgV (previously named GB Virus C or hepatitis G virus) in samples collected in 2007 from 299 males from the DRC, whose ages ranged from 21 to 71 years old. Samples were tested for the presence of HCV antibodies by ELISA and reactive samples were subsequently tested for HCV RNA using RT-PCR in which both the HCV Core and NS5B genome regions were amplified. Remaining samples were tested for HPgV RNA and the HPgV NS3 genome region of positive samples was amplified.

 For HCV, 13.7% of the samples were seropositive (41/299) but only 3.7% were viremic (11/299).
 HPgV RNA was found in 12.7% (33/259) of samples.
HCV viremia was strongly associated with age; the percentage of samples that contained detectable HCV RNA was ~0.5% in those younger than 50 and 13% in those older than 50.
Our study represents the first systematic survey of HCV genetic diversity in the DRC.
 HCV sequences obtained belonged to diverse lineages of genotype 4, including subtypes 4c, 4 k, 4 l and 4r, plus one unclassified lineage that may constitute a new subtype.

These data suggest that HCV in the DRC exhibits an age 'cohort effect', as has been recently reported in neighbouring countries, and are consistent with the hypothesis that HCV transmission rates were higher in the mid-twentieth century, possibly as a result of parenteral, iatrogenic, or other unidentified factors.

 Different HCV subtypes were associated with individuals of different ages, implying that HCV infection in the DRC may have arisen through multiple separate HCV epidemics with different causes.
Copyright © 2013 Elsevier B.V. All rights reserved.KEYWORDS:Democratic Republic of Congo; Hepatitis C; Human pegivirus; Phylogeny; Prevalence; RNAPMID:23419346[PubMed - indexed for MEDLINE]

Ihmisen pegivirusta HPgV on kuusi eri tyyppiä. Prevalenssi Afrikassa korkein.

J Gen Virol. 2013 Dec;94(Pt 12):2670-8. doi: 10.1099/vir.0.055509-0. Epub 2013 Sep 28. Deep sequencing identifies two genotypes and high viral genetic diversity of human pegivirus (GB virus C) in rural Ugandan patients Ghai RR1, Sibley SD, Lauck M, Dinis JM, Bailey AL, Chapman CA, Omeja P, Friedrich TC, O'Connor DH, Goldberg TL.

Abstract

Human pegivirus (HPgV), formerly 'GB virus C' or 'hepatitis G virus', is a member of the genus Flavivirus (Flaviviridae) that has garnered significant attention due to its inhibition of HIV, including slowing disease progression and prolonging survival in HIV-infected patients. Currently, there are six proposed HPgV genotypes that have roughly distinct geographical distributions.
Genotypes 2 and 3 are the most comprehensively characterized, whereas those genotypes occurring on the African continent, where HPgV prevalence is highest, are less well studied.
Using deep sequencing methods, we identified complete coding HPgV sequences in four of 28 patients (14.3%) in rural Uganda, east Africa. One of these sequences corresponds to genotype 1 and is the first complete genome of this genotype from east Africa. The remaining three sequences correspond to genotype 5, a genotype that was previously considered exclusively South African.
All four positive samples were collected within a geographical area of less than 25 km(2), showing that multiple HPgV genotypes co-circulate in this area. Analysis of intra-host viral genetic diversity revealed that total single-nucleotide polymorphism frequency was approximately tenfold lower in HPgV than in hepatitis C virus. Finally, one patient was co-infected with HPgV and HIV, which, in combination with the high prevalence of HIV, suggests that this region would be a useful locale to study the interactions and co-evolution of these viruses.PMID:24077364 [PubMed - indexed for MEDLINE] PMCID: PMC3836497 Free PMC Article

Ihmisen pegivirus HPgV Argentiinassa

J Med Virol. 2014 Dec;86(12):2076-83. doi: 10.1002/jmv.23918. Epub 2014 Mar 10. Human pegivirus molecular epidemiology in Argentina: potential contribution of Latin American migration to genotype 3 circulation. Trinks J1, Maestri M, Oliveto F, Del Pino N, Weissenbacher M, Torres OW, Oubiña JR.

Abstract

In order to determine the human pegivirus (HPgV) genotypic diversity in Argentina taking into account the potential contribution of human migration from neighboring countries, samples from 130 Argentine injecting drug users, 116 Argentine- and 50 immigrant-pregnant women were analyzed. HPgV RNA prevalence among human immunodeficiency virus (HIV)-positive injecting drug users was similar to HIV-positive pregnant women, as was the case when comparing HIV-negative injecting drug users and HIV-negative pregnant women (P > 0.05). HPgV genotype 2 (HPgV/2) was prevalent among both Argentine injecting drug users and pregnant women, in contrast to HPgV/3 observed among pregnant women from Latin American countries with predominant indigenous populations and who had experienced their initial sexual intercourses--and possibly their source of infection--in those countries (P < 0.01). In addition, HPgV vertical and horizontal transmission was proven by molecular analysis of E2 gene and construction of identity matrixes with epidemiologically non-related isolates. This study shows that human migration from neighboring Latin American countries with predominant indigenous populations might contribute to HPgV/3 circulation in Argentina. © 2014 Wiley Periodicals, Inc. KEYWORDS:
Argentina; human pegivirus (HPgV); injecting drug users; pregnant women; viral genotypes

Ihmisen pegivius HPgV, myös kutsuttu hepatiitti G virukseksi joskus , pinttynyt virus

J Gen Virol. 2014 Jun;95(Pt 6):1307-19. doi: 10.1099/vir.0.063016-0. Epub 2014 Mar 25. Human pegivirus RNA is found in multiple blood mononuclear cells in vivo and serum-derived viral RNA-containing particles are infectious in vitro.

Abstract

Human pegivirus (HPgV; previously called GB virus C/hepatitis G virus) has limited pathogenicity, despite causing persistent infection, and is associated with prolonged survival in human immunodeficiency virus-infected individuals. Although HPgV RNA is found in and produced by T- and B-lymphocytes, the primary permissive cell type(s) are unknown. We quantified HPgV RNA in highly purified CD4(+) and CD8(+) T-cells, including naïve, central memory and effector memory populations, and in B-cells (CD19(+)), NK cells (CD56(+)) and monocytes (CD14(+)) using real-time reverse transcription-PCR. Single-genome sequencing was performed on viruses within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4(+) and CD8(+) T-lymphocytes (nine of nine subjects), B-lymphocytes (seven of ten subjects), NK cells and monocytes (both four of five). HPgV RNA levels were higher in naïve (CD45RA(+)) CD4(+) cells than in central memory and effector memory cells (P<0 .01="" 0.006-0.010="" 0.07="" 0.58-0.14="" a="" among="" and="" apparent="" b-lymphocytes="" broad="" by="" carboxyfluorescein="" cells="" class="highlight" commercial="" conserved="" contribute="" csfe="" delivered="" delivery="" ester="" evidence="" exosome="" explaining="" highly="" hpgv="" in="" including="" isolation="" labelled="" low="" may="" microvesicles="" monocytes="" nk="" non-synonymous="" of="" particles="" pbmcs="" per="" persistent="" precipitated="" pressure.="" range="" ratio="" reagent="" replication.="" rna-containing="" rna="" selective="" sequences="" serum="" site="" span="" subjects="" subsequent="" substitution="" substitutions="" succinimidyl="" suggesting="" synonymous="" t-="" the="" this="" thus="" to="" transferred="" tropism="" uninfected="" virus="" vivo="" was="" were="" with="" within="">human
RNA virus.PMID:24668525[PubMed - indexed for MEDLINE]PMCID:PMC4027039[Available on 2015/6/1]

Ihmisen pegivirus HPgV

Virology. 2014 May;456-457:300-9. doi: 10.1016/j.virol.2014.03.018. Epub 2014 Apr 24.Human pegivirus (GB virus C) NS3 protease activity inhibits induction of the type I interferon response and is not inhibited by HCV NS3 protease inhibitors.Chowdhury AY1, Tavis JE2, George SL3.

Abstract

We previously found that human pegivirus (HPgV; formerly GBV-C) NS3 protease activity inhibits Human Immunodeficiency Virus (HIV) replication in a CD4+ T cell line.
 Given the protease׳s similarity to the Hepatitis C virus (HCV) NS3 protease, we characterized HPgV protease activity and asked whether it affects the type I interferon response or is inhibited by HCV protease antagonists.
We characterized the activity of proteases with mutations in the catalytic triad and demonstrated that the HCV protease inhibitors Telaprevir, Boceprevir, and Danoprevir do not affect HPgV protease activity. HPgV NS3 protease cleaved MAVS but not TRIF, and it inhibited interferon responses sufficiently to enhance growth of an interferon-sensitive virus. Therefore, HPgV׳s inhibition of the interferon response could help promote HPgV persistence, which is associated with clinical benefits in HIV-infected patients. Our results also imply that HCV protease inhibitors should not interfere with the beneficial effects of HPgV in HPgV/HCV/HIV infected patients.
Published by Elsevier Inc.

KEYWORDS: GB Virus-C; Hepatitis C virus; Human pegivirus; MAVS; Protease inhibitors; Serine protease; TRIF; Type I interferon

Ihmisen pegiviruksen HPgV kaltaisia löydetty eläimistä , mm vanhan maailman apinoista

PLoS One. 2014 Jun 11;9(2):e98569. doi: 10.1371/journal.pone.0098569. eCollection 2014.Discovery and characterization of distinct simian pegiviruses in three wild African Old World monkey species.

Abstract

Within the Flaviviridae, the recently designated genus Pegivirus has expanded greatly due to new discoveries in bats, horses, and rodents. Here we report the discovery and characterization of three simian pegiviruses (SPgV) that resemble human pegivirus (HPgV) and infect red colobus monkeys (Procolobus tephrosceles), red-tailed guenons (Cercopithecus ascanius) and an olive baboon (Papio anubis). We have designated these viruses SPgVkrc, SPgVkrtg and SPgVkbab, reflecting their host species' common names, which include reference to their location of origin in Kibale National Park, Uganda. SPgVkrc and SPgVkrtg were detected in 47% (28/60) of red colobus and 42% (5/12) red-tailed guenons, respectively, while SPgVkbab infection was observed in 1 of 23 olive baboons tested. Infections were not associated with any apparent disease, despite the generally high viral loads observed for each variant. These viruses were monophyletic and equally divergent from HPgV and pegiviruses previously identified in chimpanzees (SPgVcpz). Overall, the high degree of conservation of genetic features among the novel SPgVs, HPgV and SPgVcpz suggests conservation of function among these closely related viruses. Our study describes the first primate pegiviruses detected in Old World monkeys, expanding the known genetic diversity and host range of pegiviruses and providing insight into the natural history of this genus.PMID:24918769[PubMed - in process]PMCID:PMC4053331

Immunomoduloivan pegiviruksen merkitys ebolainfektiosta selviytymisessä

J Virol. 2014 Dec 3. pii: JVI.02752-14. [Epub ahead of print] GB virus C co-infections in West African Ebola patients. Lauck M1, Bailey AL1, Andersen KG2, Goldberg TL3, Sabeti PC2, O'Connor DH4.
Abstract
 Yhteenveto:
Tutkimusaineisto:  Sierra Leone, 49 ebolapotilasta.
Näistä  13 oli positiivista myös pegi-C-viruksen suhteen (GBC+). (Siis negatiivisia 36)
53% niistä jotka olivat GBV-C-positiivisia ebolapotilaita, selvisi hengissä.  6-7 henkilöä. 13:stä (14% koko ryhmästä 49 )
 22% GBV-C  negatiivisista  ebolapotilaista  selvisi hengissä.7-8 henkilöä  36:sta. (14% koko ryhmästä 49)
Sekä hengissäpysyminen ebolainfektiossa ja  GBV-C status olivat riippuvaisia iästä. iäkkäämmät selviytyivät huonommin. 21- 45 vuotiailla esiintyi  enemmän pegi-positiivisuutta.
Noin 14 eloonjäänyttä 49:stä on  noin 29%. 
Ymmärtämys  GBV-C viruksen   ja iän eriillisistä  ja yhteisvaikutuksista   ebolasta selviytyjillä  saattaa johtaa uuteen terapia- ja preventiostrategiaan ehkä immuuniaktivaatioon iästä riippuvien teitten kautta.

In 49 patients with known Ebolavirus (EBOV) outcomes during the ongoing outbreak in Sierra Leone, 13 were co-infected with the immunomodulatory pegivirus GB virus C (GBV-C).
 53% of these GBV-C+ patients survived; in contrast, only 22% of GBV-C- patients survived. Both survival and GBV-C status were associated with age, with older patients having lower survival rates and intermediate-age patients (21-45 years) having the highest rate of GBV-C infection. Understanding the separate and combined effects of GBV-C and age on EBOV survival could lead to new treatment and prevention strategies, perhaps through age-related pathways of immune activation.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

måndag 22 december 2014

HAI (Human Animal interface): H5N1

HAI
http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_04December2014.pdf


Pandemia Alert- vaiheessa  edelleen näistä on AH5N1.
 http://www.who.int/influenza/preparedness/pandemic/h5n1phase/en/
Tällä hetkellä sen surmaamia on    kolmesataa  kuudestasadasta sairastuneesta, joten  kuolleisuus on miltei 60% tässä vaiheesa, mutta  virus ei ole hankkinut vielä nopeaa siirtymiskykyä ihmiskunnassa.   Sehän on kytenyt siellä Egyptissä jo kauan.

Mutta toinen  Kiinassa, idässä kytevä,  on tarkassa WHO seurannassa A(H7N9). Se on nopeammin kehkeytyvä ilmeisesti.

Lisäksi on havaittu USA.ssa kolme  ihmis-influenssaa A (H3N2)v. ( variantteja)

 Ruotsissa on havaittu viime vuodesta asti sikakarjan hoitajilla  varianttia  A(H1N2) ja jos se aiheuttaa ihmis-influenssaa  sen patogeenisuus on matala, sillä  edellisen sikainfluenssarokotuksen antama suoja osin  tehoaa sitäkin vastaan.

OIE  seuraa  karjaeläimissä esiintyviä kantoja ja on havainnut mm  A(H5N6), A (H5N9) ja A(H5N3) siipikarjassa.

fredag 19 december 2014

Hyttysen kuurnien

People infected with dengue while traveling abroad can spread the disease at home when mosquitos bite them, and then bite other people, Leggiadro said.

Tämän lauseen löysin lähteestä
 http://www.livescience.com/47340-viruses-scarier-than-ebola.html
Etsin vastausta tällaiseen ajatukseen:

Denguealue kattaa samat alueet kuin ebola_alue.

Jos nyt itkka joka välittää denguetautia  (jotain sen neljästä kannasta) -  puree ihmistä, jolla on ebolavirusta veressä ja  sitten puree seuraavaa ihmistä, jlla ei ollut ebolavirusta veressä, mikä vaikutus tällä mikroinjektiolla on   ebolaterveen henkilön immuunivasteeseen, jos hän sitten sairastuu ebolaan myöhemmin?
 Seuraako jonkinlainen parempi  selviytyminen ebolasta ja jonkinasteien dendriittisolujen valmius ettei kaikki aivan katoa siinä yhdessä hujauksessa?

En kyllä nyt löydä  denguesta muuta kuin yleiskartan joka vaikutaa  samantapaiselta kuin ebolan.
Denguerokotetta alkaa kohta olla, tetravalenttia, nyt jo hiirillä testataan. 

Otsikoksi asetin hyttysen kuurninnan- sillä vaikka  hemorrhagiset virukset surmaavatkin Afrikassa, suurempi ongelma sittenkin on  julmat  genosidiset  aallot.  Ilman  afrikkalais ja islamilaisgenosidejä voitaisiin auttaa  infektiosairauksienkin torjunnassa paljon tehokkaammin.




Ebolavirus AND Arthropoda

Results: 11

1.
Hu L, Trefethen JM, Zeng Y, Yee L, Ohtake S, Lechuga-Ballesteros D, Warfield KL, Aman MJ, Shulenin S, Unfer R, Enterlein SG, Truong-Le V, Volkin DB, Joshi SB, Middaugh CR.
J Pharm Sci. 2011 Dec;100(12):5156-73. doi: 10.1002/jps.22724. Epub 2011 Aug 19.
PMID:
21858822
[PubMed - indexed for MEDLINE]
2.
Smither SJ, Piercy TJ, Eastaugh L, Steward JA, Lever MS.
J Virol Methods. 2011 Oct;177(1):123-7. doi: 10.1016/j.jviromet.2011.06.021. Epub 2011 Jul 5.
PMID:
21762730
[PubMed - indexed for MEDLINE]
3.
Shelemba-Chepurnova AA, Omel'ianchuk LV, Chepurnov AA.
Vopr Virusol. 2011 Jan-Feb;56(1):37-40. Russian.
PMID:
21427954
[PubMed - indexed for MEDLINE]
4.
Opsenica I, Burnett JC, Gussio R, Opsenica D, Todorović N, Lanteri CA, Sciotti RJ, Gettayacamin M, Basilico N, Taramelli D, Nuss JE, Wanner L, Panchal RG, Solaja BA, Bavari S.
J Med Chem. 2011 Mar 10;54(5):1157-69. doi: 10.1021/jm100938u. Epub 2011 Jan 25.
PMID:
21265542
[PubMed - indexed for MEDLINE]
Free PMC Article
5.
Sun Y, Carrion R Jr, Ye L, Wen Z, Ro YT, Brasky K, Ticer AE, Schwegler EE, Patterson JL, Compans RW, Yang C.
Virology. 2009 Jan 5;383(1):12-21. doi: 10.1016/j.virol.2008.09.020. Epub 2008 Nov 4. Erratum in: Virology. 2010 Mar 30;399(1):186.
PMID:
18986663
[PubMed - indexed for MEDLINE]
Free PMC Article
6.
Ye L, Lin J, Sun Y, Bennouna S, Lo M, Wu Q, Bu Z, Pulendran B, Compans RW, Yang C.
Virology. 2006 Aug 1;351(2):260-70. Epub 2006 May 4.
PMID:
16678231
[PubMed - indexed for MEDLINE]
Free Article
7.
Darriet F.
Med Trop (Mars). 2000;60(3):303-4. French. No abstract available.
PMID:
11258069
[PubMed - indexed for MEDLINE]
8.
Reiter P, Turell M, Coleman R, Miller B, Maupin G, Liz J, Kuehne A, Barth J, Geisbert J, Dohm D, Glick J, Pecor J, Robbins R, Jahrling P, Peters C, Ksiazek T.
J Infect Dis. 1999 Feb;179 Suppl 1:S148-54.
PMID:
9988178
[PubMed - indexed for MEDLINE]
Free Article
9.
Monath TP.
J Infect Dis. 1999 Feb;179 Suppl 1:S127-38.
PMID:
9988176
[PubMed - indexed for MEDLINE]
Free Article
10.
Swanepoel R, Leman PA, Burt FJ, Zachariades NA, Braack LE, Ksiazek TG, Rollin PE, Zaki SR, Peters CJ.
Emerg Infect Dis. 1996 Oct-Dec;2(4):321-5.
PMID:
8969248
[PubMed - indexed for MEDLINE]
Free PMC Article
11.
Turell MJ, Bressler DS, Rossi CA.
Am J Trop Med Hyg. 1996 Jul;55(1):89-90.
PMID:
8702028
[PubMed - indexed for MEDLINE]

Denguealue Afrikassa

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DENGUE rokotekehittely

LÄHDE:  
BMC Microbiol. 2014 Dec 18;14(1):233. [Epub ahead of print]
Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties.

Abstract

Background
Currently, a licensed vaccine for Dengue Virus (DENV) is not yet available.
Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens.
Results
In the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-¿ and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS.
 The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera.
Conclusions
Our results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.
PMID:
25520151
[PubMed - as supplied by publisher]

Millä tavalla denguevirus vikuuttaa endoteeliä?

 Denguen patogeneesi  on huonosti tunnettua runsaista tutkimuksista huolimatta.  Tässä uusinta katsausta: Denguevirusekn primääri kohdesolu on dendriittisolu ja monosyytti/makrofagi, joista tulee runsaat kemokiinit ja sytokiinit infektion aikana.  Niistä taas endoteeli aktivoituu ja suonisto alkaa tulla läpivuotavaksi. 
 
(Kommenttini: Ilmiö on tältä alkuosalta ja kohdesoluvalinnalta    samantyyppistä kuin ebolassakin, mutta ebola on  rajumpi kulkuinen eikä säästä soluja elossa ja iskee koko immuunivasteen alueen) . 
 
DENV lisääntyy, replikoituu, myös endoteelisoluissa, jotka osallistuvat suoraan viremiaan, immuuniaktivaatioon, suonen läpäisevyyteen ja vasta-aineitten kohdistautumiseen suonen sisäpintaan. Myös viruksen  NS proteiinin 1 ja  erittyneen proteiinin  vasta-aineet ovat osaltaan syynä  endoteelin  toiminnanhäiriössä.
 
Rev Med Virol. 2014 Nov 27. doi: 10.1002/rmv.1818. [Epub ahead of print]
Endothelial dysfunction in dengue virus pathology.
Abstract
Dengue virus (DENV) is a leading cause of illness and death, mainly in the (sub)tropics, where it causes dengue fever and/or the more serious diseases dengue hemorrhagic fever and dengue shock syndrome that are associated with changes in vascular permeability. Despite extensive research, the pathogenesis of DENV is still poorly understood and, although endothelial cells represent the primary fluid barrier of the blood vessels, the extent to which these cells contribute to DENV pathology is still under debate. The primary target cells for DENV are dendritic cells and monocytes/macrophages that release various chemokines and cytokines upon infection, which can activate the endothelium and are thought to play a major role in DENV-induced vascular permeability. However, recent studies indicate that DENV also replicates in endothelial cells and that DENV-infected endothelial cells may directly contribute to viremia, immune activation, vascular permeability and immune targeting of the endothelium. Also, the viral non-structural protein-1 and antibodies directed against this secreted protein have been reported to be involved in endothelial cell dysfunction. This review provides an extensive overview of the effects of DENV infection on endothelial cell physiology and barrier function. Copyright © 2014 John Wiley & Sons, Ltd.Copyright © 2014 John Wiley & Sons, Ltd.
PMID:
25430853
[PubMed - as supplied by publisher]

torsdag 18 december 2014

Pitäisi löytää mikä ultaäänifrekvenssi stimuloi ebolaviruksen sisään menonisäntäsoluun

Ebolviruksesta ( sen  uhrista, kuten  ihmisestä )hyötyy  moni eläin joka myös äyttää  biosooreitaan ja ultraääntä.
Muta tuo äänten maailma  ei ole ihmisen korvien havaintoalueella.  Vain tekniikalla voi katsoa  eri frekvenssien ja  säteilylajien  vaikutukset ebolaviruksen  elinsykliin.

Hyperfysiikka selvittää millä frekvenssillä lepakot  lähettävät ultraääntä.
http://hyperphysics.phy-astr.gsu.edu/hbase/sound/usound.html

Bats and Ultrasound

Bats use ultrasonic sound for navigation. Their ability to catch flying insects while flying full speed in pitch darkness is astounding. Their sophisticated echolocation permits them to distinguish between a moth (supper) and a falling leaf.
About 800 species of bats grouped into 17 families.
 The ultrasonic signals utilized by these bats fall into three main categories.
 1. short clicks, 
2. Frequency-swept pulses,
and 3. constant frequency pulses.
 There are two suborders, Megachiroptera and Microchiroptera.
Megas use short clicks, Micros use the other two.
 Tongue clicks produce click pairs separated by about 30ms, with 140-430 ms between pairs. (Sales and Pye, Ultrasonic Communication by Animals).
 10-60 kHz in frequency swept clicks. One kind of bat, the verspertilionidae, have frequency swept pulses 78 kHz to 39 kHz in 2.3 ms.
 Emits pulses 8 to 15 times a second, but can increase to 150-200/s when there is a tricky maneuver to be made.

Ultraääni stimuloinee ebolan soluunmenovaiheessa sitä funktiota mikä on retrovirukselta kaapattu ?

Virol J. 2011 Sep 22;8:446. doi: 10.1186/1743-422X-8-446.

Effect of ultrasound on herpes simplex virus infection in cell culture.

Abstract

BACKGROUND:

Ultrasound has been shown to increase the efficiency of gene expression from retroviruses, adenoviruses and adeno-associated viruses.
 The effect of ultrasound to stimulate cell membrane permeabilization on infection with an oncolytic herpes simplex virus type 1 (HSV-1) was examined.

RESULTS:

Vero monkey kidney cells were infected with HSV-1 and exposed to 1 MHz ultrasound after an adsorption period. The number of plaques was significantly greater than that of the untreated control. A combination of ultrasound and microbubbles further increased the plaque number. Similar results were obtained using a different type of HSV-1 and oral squamous cell carcinoma (SCC) cells. The appropriate intensity, duty cycle and time of ultrasound to increase the plaque number were 0.5 W/cm², 20% duty cycle and 10 sec, respectively.
Ultrasound with microbubbles at an intensity of 2.0 W/cm², at 50% duty cycle, or for 40 sec reduced cell viability.

CONCLUSION:

These results indicate that ultrasound promotes the entry of oncolytic HSV-1 into cells. It may be useful to enhance the efficiency of HSV-1 infection in oncolytic virotherapy.
PMID:
21939524
[PubMed - indexed for MEDLINE]

PMCID:
PMC3189159

Free PMC Article

UVC germicidinen UV säteily ja ebolavirus

Arch Virol. 2011 Mar;156(3):489-94. doi: 10.1007/s00705-010-0847-1. Epub 2010 Nov 23.

Sensitivity to ultraviolet radiation of Lassa, vaccinia, and Ebola viruses dried on surfaces.

Abstract

Germicidal UV (also known as UVC) provides a means to decontaminate infected environments as well as a measure of viral sensitivity to sunlight. The present study determined UVC inactivation slopes (and derived D(37) values) of viruses dried onto nonporous (glass) surfaces. The data obtained indicate that the UV resistance of Lassa virus is higher than that of Ebola virus. The UV sensitivity of vaccinia virus (a surrogate for variola virus) appeared intermediate between that of the two virulent viruses studied. In addition, the three viruses dried on surfaces showed a relatively small but significant population of virions (from 3 to 10 % of virus in the inoculum) that appeared substantially more protected by their environment from the effect of UV than the majority of virions tested. The findings reported in this study should assist in estimating the threat posed by the persistence of virus in environments contaminated during epidemics or after an accidental or intentional release.
PMID:
21104283
[PubMed - indexed for MEDLINE]

fredag 12 december 2014

Yksi Ebolarokotuskokeilu keskeytetty nivelkipujen takia

http://www.bbc.com/news/health-30432079
11.12.2014

The clinical trial of an Ebola vaccine in Switzerland has been interrupted after some patients complained of joint pains in their hands and feet.
The trial was stopped one week early in all 59 volunteers "as a measure of precaution", the University of Geneva Hospital said.
The vaccine being tested is one developed by NewLink, and recently bought by Merck.
There is currently no vaccine licensed for use to protect against Ebola.
Two vaccines are currently being tested in humans in a number of countries.
The side-effects in the Geneva trial were experienced by four volunteers.
The hospital said human safety trials would resume on 5 January in up to 15 volunteers after checks had taken place to ensure the joint pain symptoms were "benign and temporary".
"They are all fine and being monitored regularly by the medical team leading the study," the hospital said.

Four trials of another Ebola vaccine developed by GlaxoSmithKline, in collaboration with the United States National Institute of Allergy and Infectious Diseases, are also underway.

måndag 1 december 2014

Ebolaviruksen sofistinen suhde monosyytteihin ( Tässä on alku sytökiinimyrskyn kehkeytymiseen) Pohdittavaksi.






Ebola Virus Exploits a Monocyte Differentiation Program To Promote Its Entry

Osvaldo Martinez, Joshua C. Johnson, [...], and Christopher F. Basler

Abstract

Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo. However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. 
Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.

INTRODUCTION

Zaire Ebola virus (EBOV) is an emerging zoonotic pathogen that has caused outbreaks of viral hemorrhagic fever in humans with fatality rates approaching 90% (1). EBOV tropism toward antigen-presenting cells (APCs) is thought to play an important role in viral pathogenesis, contributing to the establishment of infection and to the development of hemorrhagic fever (2). EBOV productively infects APCs, including monocytes, macrophages, and dendritic cells (DCs), in vitro (39), and tissue sections from EBOV-infected humans and nonhuman primates contain APCs positive for EBOV antigen/nucleic acid, demonstrating APC infection in vivo (1016). Although APCs serve as sites of virus amplification, their infection also deregulates APC function (2, 17, 18). This deregulation may contribute to the development of an ineffective antiviral immune response, as well as an intense and deregulated inflammatory response (4, 19).
Although monocytes, the most abundant blood-borne APCs, likely contribute to EBOV pathogenesis, an apparent discrepancy exists in our understanding of monocyte infection by EBOV (18). Specifically, although EBOV productively infects human blood-derived monocytes (4, 20), several other studies demonstrate limited or restricted EBOV GP-mediated entry into monocytes (2123).
EBOV entry, which includes attachment and penetration into the target cell cytoplasm, is mediated by the surface glycoprotein (GP) (24). GP likely mediates viral attachment through the receptor-binding domain (RBD) located at its N terminus (2528). Subsequent viral uptake likely occurs via macropinocytosis (2932). A number of cell surface molecules, including C-type lectins, have been identified as attachment or entry factors (3338).

 However, none of these factors appears to function as an essential cell surface receptor. Additional host factors have also been implicated as regulating entry, including components of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, the cysteine proteases cathepsin L and cathepsin B and signaling molecules, including acid sphingomyelinase and phosphoinositide-3 kinase (25, 26, 3944). Recently, Niemann-Pick C1 (NPC1), an endosomal protein involved in cholesterol transport and storage, was identified as an essential EBOV entry receptor (39, 45, 46). Negatively acting entry restriction factors have also been identified, including the interferon-inducible transmembrane proteins (IFITMs) (47, 48).
Here we sought to define the basis of EBOV tropism toward monocytes, macrophages, and DCs. The data indicate that undifferentiated monocytes are indeed refractory to EBOV entry, whereas macrophages and DCs are fully permissive. However, EBOV can associate with undifferentiated monocytes and can complete the entry process as the cells spontaneously differentiate into macrophages or DCs. This results in substantially delayed entry kinetics and less robust cytokine responses of monocytes relative to differentiated macrophages or DCs.
Profiling an array of genes previously implicated in EBOV entry demonstrates that during monocyte differentiation essential entry factors NPC1 and cathepsin B increase, while the restriction factors IFITM1, -2, and -3 decrease. Furthermore, although monocyte infection with EBOV accelerates the rate of IFITM downregulation, the kinetics of upregulation of cathepsin B and NPC1 expression remain largely unchanged.
 Lastly, overexpression of NPC1 in THP-1 monocytes, which are nonpermissive for EBOV GP-mediated entry unless differentiated into macrophage-like cells, partially rescued EBOV GP-mediated entry.

MATERIALS AND METHODS

Cell culture, vectors, and plasmids.

HEK293T (293T) cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% l-glutamine. The plasmids used in transfections included (i) pcDNA BLA-VP40, which expresses a β-lactamase-Zaire EBOV VP40 chimera (49, 50), (ii) pCAGGS GFP-VP40, which expresses a VP40-GFP chimera (51), (iii) pcDNA EBGP, which expresses wild-type Zaire EBOV strain Mayinga GP, (iv) pcDNA EBGPF88A (50, 52, 53), (v) pcDNA EBGPF159A (53), or (vi) pcDNA VSVG, which expresses vesicular stomatitis virus glycoprotein (VSV G). NPC1 was expressed using the pBABE expression retroviral vector kindly provided by Kartik Chandran (Albert Einstein College of Medicine).

Isolation and culture of human monocytes, macrophages, and DCs.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (Histopaque; Sigma-Aldrich, St. Louis, MO) from the buffy coats of healthy human donors (New York Blood Center). CD14+ monocytes were purified using anti-human CD14 antibody-labeled magnetic beads and iron-based LS columns (Miltenyi Biotec, Auburn, CA) and used immediately or further differentiated into macrophages or DCs. Stains of isolated monocytes typically showed >90% positivity for CD14 staining. Monocytes were plated (0.7 × 106 to 1 × 106 cells/ml) in DC medium (RPMI [Invitrogen, Carlsbad, CA] supplemented with 100 U of penicillin/ml, 10 μg of streptomycin/ml, 55 μM β-mercaptoethanol, and 4% human serum AB [GemCell; Gemini Bio-Products, West Sacramento, CA]) supplemented with either 500 U of human granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech, Rocky Hill, NJ)/ml, 500 U of human interleukin-4 (IL-4; Peprotech)/ml to differentiate into DCs (54), or 20 to 50 ng of macrophage colony-stimulating factor (M-CSF; Peprotech)/ml to differentiate into macrophages. By day 5, immature DCs expressed surface CD11c, CD1c, and HLA-DR (major histocompatibility complex [MHC] II) and low/negative levels of CD14, whereas macrophages retained high levels of CD14 and were HLA-DR positive (55, 56) and were permissive for EBOV entry and infection (50).

VLP production and purification.

For virus-like particle (VLP) production, 293T cells were cotransfected with a combination of two plasmids using Lipofectamine 2000 (DNA/Lipofectamine 2000 ratio of 1:1): either pcDNA BLA-VP40 or pCAGGS GFP-VP40, and either pcDNA expressing EBGP or VSV G at a ratio of 3:2 for VP40 to envelope glycoprotein. The VLPs were harvested at 3 days posttransfection; the 293T cell spent supernatant was layered over 10 ml of 20% sucrose in NTE buffer (100 mM NaCl, 20 mM Tris-hydrochloride [pH 7.5], and 1 mM EDTA [EDTA]), and VLPs were pelleted at 25,000 rpm in an SW-28 rotor (∼80,000 × g) for 2 h at 4°C. The VLPs were then gently washed without resuspension with 25 ml of cold NTE or phosphate-buffered saline (PBS) and centrifuged again. The VLPs were finally resuspended in a total of 150 μl of NTE and stored on ice at 4°C.

Flow cytometry.

Flow cytometry was performed using an LSR II flow cytometer equipped with a violet laser (BD Bioscience). The data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Mouse macrophages and dendritic cells.

C57/BL6 mice were sacrificed in order to obtain peritoneal macrophages (large CD11b+ cells) or bone marrow cells according to standard techniques. In order to induce monocyte differentiation into dendritic cells (DCs) or macrophages, mouse bone-marrow cells grown in DMEM plus 10% FBS were supplemented with mouse GM-CSF (50 ng/ml; Peprotech) or M-CSF (20 ng/ml; Peprotech), respectively. All animal procedures were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines and have been approved by the IACUC of Mount Sinai School of Medicine.

Determining EBOV infectious titers.

EBOV titers were determined using an agarose plaque assay. Briefly, VeroE6 cells were plated in six-well plates to 90 to 100% confluence. Fivefold dilutions of stock virus were prepared (10−1 to 10−6.5). Medium samples were removed from the six-well plates, and 200 μl of inoculum for each dilution was added to each well of the plate (six replicates), rocked, and allowed to incubate for 1 h at 37°C and 5% CO2, with rocking every 15 min to prevent the cell layer from drying. At the end of the absorption period, each well was overlaid with 2 ml of a 1× agarose overlay (1% agarose mixed with 2× Eagle modified essential medium [EMEM; Gibco], 2× Anti-Anti [Gibco], 2× GlutaMAX [Gibco], and 10% fetal calf serum [FCS; Gibco] to yield a final overlay of 0.5% agarose [SeaKem ME agarose; Lonza], 1× EMEM, 1× Anti-Anti, 1× GlutaMAX, and 5% FCS). The agarose was allowed to solidify, and the plates were returned to an incubator. On day 9 after the primary overlay, a secondary overlay of the primary overlay mixture plus 4% neutral red (Gibco) was added, the samples were allowed to solidify, and the cells were again returned to the incubator for an additional night. Plaques were counted the following day, and the titers were determined.

Entry assays.

Entry assays were performed as outlined in a previous study (50). Different VLP preparations were used such that equivalent levels of β-lactamase activity were present for the entry assays. VLPs were mixed with target cells and centrifuged (“spinoculated”) at 1,850 rpm for 45 min at 4°C before incubation at 37°C in 2% RPMI medium for the indicated times. Target cells were then loaded with fluorescent CCF2-AM substrate for 1 h at room temperature. An LSR II (BD Bioscience) flow cytometer equipped with a violet laser was used to excite the CCF2-AM substrate, and events (cells) were assayed for green and blue fluorescence after sorting for live cells. Live cells were sorted using side-scatter and forward-scatter properties (low side scatter and high forward scatter [data not shown]) and for their ability to retain the CCF2-AM substrate (cells that fluoresce green) since at least a subset of dead cells are permeable and therefore leaky. Cells fluorescing blue compared to the control (for example, mock control [results not shown]) were scored as entry positive.

EBOV infections.

All EBOV infections were performed at the U.S. Army Medical Research Institute of Infectious Diseases at biosafety level 4. Monocytes, macrophages, and DCs were infected with EBOVGFP (50) or EBOV (Kikwit 1995) at the indicated multiplicities of infection (MOIs) for 1 to 2 h with rocking. When infecting human cells with the EBOVGFP, we used MOIs of 5 and 10 because this consistently results in a significant level of infection (>20%). After infection, cells were washed three times with PBS, resuspended, and plated in triplicate. To enumerate the percentages of cells infected by EBOVGFP, the cells were gently scraped off the plates on at 2 and 3 days postinfection and analyzed by flow cytometry after gating out dead cells using side-scatter and forward-scatter properties (side scatter low, forward scatter high). To identify secreted cytokines induced after EBOVGFP infection, day 3 spent supernatants from infected and noninfected control cells were analyzed as described below. To determine the gene expression (mRNA) levels, total RNA was first harvested using Tri-Reagent, which was added to each well containing (infected) cells, and then pipetted to make sure the cells were dissociated and lysed, and the cell lysate was further processed as outlined below.

RNA isolation and cDNA synthesis.

RNA was extracted from the aqueous phase of the Tri-Reagent (Sigma-Aldrich) samples using an RNeasy micro kit (Qiagen), including a DNase step as recommended by the manufacturer. cDNA was generated from isolated mRNA using a Superscript III first-strand synthesis kit (Invitrogen) using an oligo(dT) primer as outlined by the manufacturer.

Quantitative reverse transcription-PCR (RT-PCR).

Primers were designed by using online real-time PCR primer design software and selecting intron-spanning primers (Roche Applied Science [http://www.rocheappliedscience.com/sis/rtpcr/upl/index.jsp?id=uplct_000000]). Since IFITM genes show high sequence identity, rather than just relying on the PCR primer design software, the IFITM primers were modified to span nucleotides that allowed for specific IFITM type amplification. EBOV NP primers were previously used to assay for EBOV NP expression (57). Table 1 provides the nucleotide sequences (5′ to 3′) for the forward and reverse primers used, respectively, for each human and EBOV gene.
Table 1
Primersused for quantitative RT-PCR
Relative gene expression was determined using the iQ SYBR green Supermix (Bio-Rad) according to the manufacturer's instructions. The PCR temperature profile was 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 60°C for 60 s. All of the reactions were performed in triplicate, and standard error bars were added to graphs (see Fig. 7). CXF Manager software (Bio-Rad) was used to analyze the relative mRNA expression levels by the change in threshold cycle (ΔCT) method using the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene to normalize the results. To determine the NP mRNA levels in EBOV-infected monocytes and DCs, a standard curve generated by using an NP expression plasmid as a template was used to calculate the relative copy numbers of NP mRNA from equal numbers of cells.
Fig 7
mRNA levels of factors that regulate EBOV entry in plated monocytes with or without EBOV infection. Freshly isolated blood monocytes were plated or infected with EBOV at an MOI of 1 before plating. RNA from infected and noninfected monocytes was harvested ...

Fluorescence microscopy.

Monocytes were infected with green fluorescent protein (GFP)-tagged VLPs pseudotyped with EBGP as described above except that the cells were spinoculated at 1,850 rpm for 45 min at 4°C onto coverslips. Monocyte VLPs were gently washed twice with PBS supplemented with calcium and magnesium (PBS-CM) before incubation at 37°C in 2% RPMI medium for 0, 1.5, and 4 h. The cells were fixed in 4% paraformaldehyde in PBS-CM and cell surface stained with fluorescently labeled wheat germ agglutinin (Invitrogen). Coverslips were washed in PBS-CM twice and once in water and then mounted in antifade reagent (ProLong Gold; Invitrogen) before they were viewed using a confocal Leica SP5 DMI microscope. At least five different random fields were used to determine the number of fluorescent VLPs that were taken up by monocytes.

Measurement of cytokines.

Cytokine measurements were conducted in triplicate using a human cytokine 30-plex panel (Invitrogen) in accordance with the manufacturer's instructions. The data were acquired using a LuminexFlexMAP 3D system (Bio-Rad) and exported to Bio-Rad Bioplex Manager 6.0 for data analysis. Standard curves were optimized by using a software algorithm based on a five-parameter logarithmic curve fit.

RESULTS

EBOV GP-mediated entry into monocytes is restricted.

Several published studies report the productive replication of EBOV in primary human monocytes (4, 20), while others report that EBOV GP pseudotyped viruses are unable to enter monocytes (2123). To begin to address this apparent discrepancy, we used an established virus-like particle (VLP) entry assay in which VLPs pseudotyped with either EBOV GP or control VSV glycoprotein (VSV-G), either of which can mediate entry (50), contain β-lactamase fused to VP40 (BLA-VP40). Purified VLPs are incubated with a target cell for a defined period of time after which entry is assayed. Successful entry results in the delivery of β-lactamase into the target cell cytoplasm where it cleaves a preloaded cytosolic fluorescent substrate (Fig. 1A). Once cleaved by β-lactamase the substrate-loaded cells fluoresce blue, while uncleaved substrate-loaded cells fluoresce green (50, 58).
Fig 1
EBOV entry into monocytes is restricted. (A) Schematic representation of the entry assay protocol. β-Lactamase-VP40 (BLA-VP40) containing VLPs pseudotyped with either EBOV GP or VSV G is incubated with APCs for 3.5 h. Penetration into the cytoplasm ...
We first tested whether EBOV VLP entry into freshly isolated undifferentiated monocytes was restricted compared to macrophages and DCs that were differentiated from M-CSF or GM-CSF+IL-4 cytokine-treated monocytes, respectively (54, 5961). VLPs were normalized for total β-lactamase activity from purified VLP preparations and were used to infect both human (Fig. 1B, ,D,D, and andE)E) and mouse (Fig. 1C) monocytes and macrophages. EBOV GP-mediated entry was restricted in human (Fig. 1B) and mouse (Fig. 1C) monocytes. In contrast, equivalent levels of VLPs entered human (Fig. 1B and andD)D) and mouse peritoneal (Fig. 1B) macrophages. Although EBOV GP-mediated entry into monocytes was restricted (consistently ≤10% [data not shown]), control VSV G-mediated entry into human monocytes was efficient (less so in mouse monocytes), demonstrating that the BLA-VP40 assay can detect entry into human (Fig. 1B) and mouse (Fig. 1C) monocytes. Assays testing entry restriction into monocytes compared to DCs were repeated three times and gave results similar to those shown in Fig. 1B and andC.C. Since macrophages can be differentiated from monocytes, these data suggest that entry permissiveness is associated with a differentiated phenotype.

Efficient EBOV entry into differentiated monocytes.

We also found that entry into macrophages differentiated from monocytes by M-CSF treatment was not significantly altered or restricted if we also included IL-4, transforming growth factor β (TGF-β), or gamma interferon (IFN-γ)—cytokines that regulate the expression of proteins, such as DC-SIGN, implicated in EBOV entry (Fig. 1D and data not shown) (6264). Similarly, entry into human blood monocyte-derived DCs differentiated from monocytes by GM-CSF treatment (Fig. 1E) or mouse bone marrow-derived DCs (not shown) was not significantly affected by IL-4, TGF-β, or IFN-γ treatment.

Kinetics of EBOV entry into differentiating monocytes.

Next, we determined at what point differentiating monocytes become permissive for entry. Monocytes are typically differentiated into immature DCs by incubation with GM-CSF plus IL-4 (54) for 5 to 7 days (50, 51, 55, 56). Therefore, we treated monocytes with these cytokines for periods of 0, 18, 48, 72, 96, and 120 h before testing for entry (Fig. 2A). As seen above (Fig. 1B), VSV G-mediated entry into monocytes was highly efficient and did not require differentiation-inducing cytokine treatment (Fig. 2B). On the other hand, increased EBOV GP-mediated entry efficiency was associated with increased duration of cytokine exposure. At 18 h after cytokine treatment, <10 48="" a="" and="" by="" class="fig-table-link fig figpopup" detected="" differentiating="" entry="" h="" href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624207/figure/F2/?report=objectonly" into="" monocytes="" of="" only="" permitted="" substantial="" target="_blank" the="" was="">Fig. 2B
). This experiment was repeated twice with similar results.
Fig 2
EBOV entry and infection occurs during monocyte differentiation. A schematic representation of the experimental protocol used to test entry after induction of monocyte differentiation is shown in panel A. (A and B) Freshly isolated blood monocytes were ...

EBOV infection of monocytes is delayed compared to DCs.

A recombinant EBOV expressing GFP (EBOVGFP [50]) was used to test infection efficiency in monocytes and DCs. Using EBOVGFP at MOIs of 5 and 10, monocytes and DCs were incubated with virus for 2 h, washed, and incubated at 37°C. Few cells exhibited GFP fluorescence by 24 h postinfection (data not shown). At 48 h postinfection, there was an ∼3-fold difference in the number of GFP+ monocytes and DCs (Fig. 2C), but this difference was no longer apparent by 72 h postinfection (Fig. 2D) when the percentage of infected cells was comparable. The difference in the percentages of GFP+ cells indicates that undifferentiated monocytes are permissive for EBOV infection, but the kinetics of replication is slower in monocytes compared to DCs. The 72-h time point from this experiment was repeated, confirming the reproducibility of these results.

Cultured monocytes become permissive for EBOV entry in the absence of supplemental cytokines.

Freshly plated and cultured ex vivo monocytes slowly and spontaneously differentiate (6567). Therefore, we hypothesized that monocytes become permissive for EBOV entry as they differentiate. We tested the kinetics of EBOV entry when EBOV VLPs were added to plated monocytes and cultured for up to 48 h in media not supplemented with cytokines. In the experiments described above (Fig. 1A), VLPs were incubated with target cells for 3.5 h before testing for cytoplasmic penetration. In this experiment (Fig. 3A), VLPs were added to monocytes or DCs, gently washed, and the VLP target cell mixture cultured for up to 2 days. At 2, 4, 6, 12, 24, and 48 h after the addition of VLPs, monocytes and DCs were tested for entry (Fig. 3B). We observed significant entry into DCs by 2 h after VLP incubation, but significant entry into monocytes did not occur until the 12-h time point. In both cases, the percentage of entry-positive cells reached a plateau at ∼24 h after VLP treatment; however, monocytes never achieved the same level of infection as the DCs. This suggests that entry into monocytes was not only delayed but that entry into undifferentiated monocytes is less efficient than entry into fully differentiated DCs. This experiment has been repeated with similar results.
Fig 3
Delayed EBOV entry into monocytes compared to DCs. (A) Schematic representation of a modified entry protocol used. Instead of incubating VLPs with target host cells for 3.5 h (as in Fig. 1A), target cells and VLPs were incubated together for 2, 4, 6, ...
Since significant entry into monocytes occurred after 24 h of culture, we tested whether there was a significant difference in the levels of the cell surface differentiation and adhesion markers CD1c, CD14, CD11b, CD11c, ICAM-1, integrin β2, CD44, MHC1, or MHC2 expressed between freshly isolated monocytes and monocytes cultured for 24 h (Fig. 3C). Control DC cells, but not monocytes, expressed significant cell surface levels of DC marker CD1c, whereas the monocytes expressed higher levels of CD14. However, there were no significant differences in the expression levels on freshly isolated monocytes compared to monocytes cultured for 24 h of any of the markers tested, including ICAM-1 and CD44 (data not shown and Fig. 3C).
Because primary monocytes spontaneously differentiate in culture, it is difficult to determine whether differentiation is required for the delayed EBOV VLP entry. As an alternative, the human monocyte-like cell line THP-1 was used in experiments to address the role of differentiation in EBOV infection of monocytes. This cell line is commonly used as an experimental model for human monocytes, but these cells do not spontaneously differentiate in culture. However, THP-1 cells can be induced to differentiate by the addition of phorbol myristate acetate (PMA) (68). This results in an adherent macrophage-like phenotype and renders the cells permissive for EBOV GP-mediated entry (22). We therefore tested for entry into THP-1 cells treated or not with PMA (Fig. 4A). As with primary human monocytes, the undifferentiated THP-1 cells were permissive for entry by VSV G, but not by EBOV GP VLPs, whereas the differentiated THP1s allowed entry (Fig. 4B). We again tested for the expression of CD1c, CD14, CD11b, CD11c, ICAM-1, integrin β2, CD44, MHC1, or MHC2 on untreated and PMA-treated THP-1. Of all the markers tested, only expression of ICAM-1, previously shown to be upregulated on monocytes cultured for several days (69), was significantly upregulated on PMA-treated THP-1 cells (Fig. 4C). The combination of significant ICAM-1 expression and adherence (data not shown) suggests that the THP-1 cells have differentiated into a macrophage-like cell. Since incubating blood monocytes with VLPs for 24 h was sufficient time to allow significant EBOV GP-mediated VLP entry (Fig. 3B), we tested whether a 24-h incubation with VLPs, in the absence of differentiation stimuli, would rescue EBOV GP-mediated entry into THP-1 cells and included primary monocytes as a positive control (Fig. 4D and andE).E). As previously seen (Fig. 3B), VSV G VLPs entered THP-1 cells, whereas the increased VLP incubation time of 24 h did not rescue EBOV GP-mediated entry (Fig. 4E). However, the concomitant addition of PMA and EBOV GP VLPs allowed EBOV GP-mediated entry. VLPs pseudotyped with mutant GP-F159A were previously demonstrated to be entry defective (53, 70). Therefore, GP-F159A VLPs were used in entry assays to exclude the possibility that differentiated monocytes (Fig. 4D) or THP-1 cells (Fig. 4E) might indiscriminately take up any GP-expressing VLPs. The GP-F159A VLPs showed no entry, demonstrating that neither the extended time of VLP-cell incubation nor PMA treatment could render cells permissive for this mutant. Taken together, the data demonstrate that an increased VLP-cell incubation time in blood monocytes but not in THP-1 cells eventually allows EBOV GP entry. THP-1 cells, which do not undergo spontaneous differentiation, allow EBOV GP mediated entry only in the presence of PMA. Therefore, entry into monocytes correlates with their differentiation.
Fig 4
THP-1 cells become permissive for EBOV entry after PMA-mediated differentiation. (A) Schematic representation depicts the experimental protocol used for experiment shown in panel B. (B) Purified Mock, EBOV GP, or VSV G VLPs were used to test entry permissiveness ...

EBOV transcription is delayed in monocytes compared to DCs.

Previous studies have shown that EBOV productively infects monocytes. We sought to assess the entry kinetics of replication-competent EBOV into undifferentiated monocytes, DCs, undifferentiated THP-1 cells, and differentiated PMA-treated THP-1 cells. As a surrogate measure for viral entry, we profiled EBOV-infected cells for the onset of viral transcription, using quantitative reverse transcription-PCR (RT-PCR) (Fig. 5). Viral transcription was assessed by measuring viral nucleoprotein (NP) mRNA levels at 0, 6, 12, 24, and 48 h after EBOV infection at an MOI of 1. Consistent with the entry assay data, the appearance of EBOV NP mRNA in monocytes was significantly delayed in comparison to infected DCs (Fig. 5A and andB),B), demonstrating that EBOV infection, like EBOV VLP entry, is delayed in undifferentiated monocytes compared to differentiated cells. Furthermore, NP expression was apparent only in differentiated THP-1 cells and not in untreated THP-1 cells, further supporting the view that differentiation is required for early events in EBOV infection (Fig. 5C and andDD).
Fig 5
Kinetics of EBOV NP mRNA expression and EBOV GP VLPGFP uptake in monocytes. EBOV was used to infect—at an MOI of 1—DCs (A), monocytes (B), and THP-1 cells (C) or THP-1 cells grown in medium supplemented with PMA (D). RNA was harvested ...

EBOV GP VLPGFP uptake in monocytes and DCs.

To assess the kinetics of EBOV GP VLP uptake into monocytes, we used fluorescently tagged VLPs that were generated by coexpression of a chimeric GFP-VP40 protein and GP, leading to the formation of EBOV GP VLPGFP (51). Monocytes were treated with equivalent amounts of EBOV GP VLPGFPs, washed gently, and incubated for 0, 1.5, and 4 h to allow the uptake of particles. Using confocal microscopy, VLP uptake was assessed by visualizing the location of EBOV GP VLPGFPs relative to the monocyte surface membrane. At 0 and 1.5 h postinfection, there was a clear association of fluorescently labeled cell membranes (red) with fluorescent particles (green) (Fig. 5E). The VLPs remained associated with the cell periphery until 4 h, when significant numbers of the EBOV GP VLPGFPs had relocalized toward the interior of the monocytes (Fig. 4E). Specifically, at 0, 1.5, and 4 h postinfection, 0, 5, and 48% of EBOV GP VLPGFPs, respectively, were in the cell interior regions of monocytes (Fig. 5F). These data, which have been reproduced, demonstrate that EBOV GP VLPGFPs readily associate with monocytes before entry occurs.

Monocytes secrete limited levels of cytokines upon EBOV infection.

Cytokines produced by APCs are proposed to contribute to EBOV pathogenesis (18). To test for the presence and levels of several inflammatory cytokines, a multiplex assay was performed on supernatants harvested 3 days after EBOV infection of monocytes, macrophages, and DCs. A representative panel of secreted cytokines (IL-12, IL-6, IL-8, MIP-1α, MCP-1, RANTES, IFN-α, IP-10 and G-CSF) is presented in Fig. 6. Consistent with previous studies, macrophages in general secreted comparably more inflammatory cytokines, but monocytes did secrete some inflammatory cytokines, albeit at lower levels. Therefore, the differentiation status and the type of APC affects both the efficiency/kinetics by which EBOV infection is established and the inflammatory cytokine response.
Fig 6
Secreted cytokines induced by EBOV infection of PBMCs, monocytes, macrophages, DCs. PBMCs, monocytes, monocyte-derived macrophages, or DCs were mock infected or infected with EBOV at MOI of 5 (the cell type is identified with a label under the first of ...

Expression levels of host factors critical for entry change during monocyte differentiation and EBOV infection.

Since significant changes occur in the monocyte transcriptome during differentiation (71), we tested the hypothesis that the levels of host factors implicated in EBOV entry also change during monocyte differentiation. The relative mRNA levels of select host entry factors (Table 2) were compared by quantitative RT-PCR between undifferentiated monocytes and DCs. Of the mRNAs examined, only the mRNA levels of cathepsin B, NPC1, IFITM1, and IFITM3 showed a >10-fold difference between monocytes and DCs. Therefore, these specific mRNAs were chosen for further investigation. The kinetics of mRNA expression of these host factors was determined in cultured monocytes at 0, 3, 6, 12, 24, and 48 h after plating. For comparison, the 120-h time point represents the relative mRNA levels in DCs. We also tested for the expression of cathepsin B, NPC1, IFITM1, IFITM2, and IFITM3 mRNA levels after monocyte infection with EBOV at an MOI of 1 (Fig. 7). Comparison of undifferentiated monocytes with or without EBOV infection revealed several striking findings. Although IFITM entry restriction factor expression in cultured monocytes dramatically decreases over time to the levels measured in DCs, their expression initially increases during the first few hours after plating. However, upon EBOV infection of monocytes, IFITM expression immediately decreases. On the other hand, these same infected cells demonstrate an upregulation of factors required for entry, namely, NPC1 and cathepsin B. These data demonstrate that the levels of host entry factors not only change during monocyte differentiation, but EBOV infection also hastens the downregulation of entry restriction factors, while at the same time upregulating host factors that are required for entry.
Table 2
RelativemRNA levels of EBOV entry factors expressed by monocytes and DCs

Stable expression of NPC1 from THP-1 cells partially rescues EBOV entry.

NPC1 is essential for EBOV infection, and there was a 100-fold increase in NPC1 mRNA in DCs and macrophages relative to monocytes (Fig. 8A). Western blotting for the levels of NPC1 confirmed that the lower levels of mRNA correspond to lower levels NPC1 protein expression in monocytes and higher levels in DCs and macrophages (Fig. 8B). PMA differentiated THP-1 cells also demonstrated higher levels of NPC1 protein compared to their undifferentiated counterparts (Fig. 8B). To test whether overexpressing NPC1 can rescue EBOV entry into undifferentiated monocytic cells, THP-1 cells were stably transduced with a retrovirus that expresses NPC1 and tested for EBOV GP VLP entry. Overexpressing NPC1 partly rescued EBOV GP-mediated entry (Fig. 8C), suggesting that low levels of NPC1 in monocytes may determine, in part, their resistance to EBOV entry. This experiment was repeated with similar results.
Fig 8
Stable expression of NPC1 in THP-1 cells rescues EBOV GP VLP entry. (A) Relative NPC1 mRNA levels in monocytes, macrophages, and DCs as normalized to GAPDH mRNA. (B) Western blots of the levels of NPC1 expressed in monocytes, DCs, macrophages, THP-1 monocytes, ...

DISCUSSION

This study addresses seemingly contradictory observations that monocytes are productively infected by EBOV but are nonpermissive for EBOV GP-mediated entry (4, 2023). Our initial studies, wherein VLPs were incubated with undifferentiated or differentiated target cells for 3.5 h (Fig. 1A), indicated that in the presence of GM-CSF+IL-4 (a standard protocol used to generate monocyte-derived DCs), monocytes become permissive for entry by 48 h after cytokine addition (Fig. 2A and andB).B). On the other hand, if VLPs were incubated for longer periods of time with cultured monocytes, which spontaneously differentiate, substantial entry was detected as early as 24 h post-VLP addition (Fig. 3B), although overall entry efficiency was consistently lower compared to DCs (Fig. 3B). Studies with infectious EBOV, where we used NP mRNA expression as a surrogate marker for entry (Fig. 5), support this conclusion. These data suggest that monocytes are not incompetent for EBOV entry. Rather, entry is substantially delayed and somewhat less efficient compared to DCs.
Primary monocytes spontaneously differentiate in culture. Therefore, it is problematic to determine whether or not the delay in entry requires the differentiation process. We therefore turned to THP-1 cells, because these cells do not spontaneously differentiate in culture, exhibit a strict resistance to entry as monocytes, and become permissive following differentiation overnight with PMA (Fig. 4B) (22). In these cells, prolonged incubation with EBOV VLPs, in the absence of PMA did not lead to successful entry. However, if prolonged incubation occurred with the coaddition of PMA, then THP-1 cells became entry positive by 24 h postinfection (Fig. 4E). Although we cannot fully exclude the possibility that entry requirements differ in THP-1 cells compared to primary monocytes, these results suggested that differentiation is a requirement for EBOV entry into monocytes.
Significant changes occur in the monocyte transcriptome during differentiation (71), and this leads to altered expression of select cell surface markers. Although no significant differences were seen in the expression levels of such cell surface markers when comparing freshly isolated monocytes and 24-h-cultured monocytes, which are entry permissive (Fig. 3C), monocyte differentiation and entry permissiveness did correlate with a decrease in mRNA levels for entry restriction factors and an upregulation of factors critical for EBOV entry, all of which occurred within hours of monocyte culturing. Therefore, it is likely that monocytes become permissive for entry before the differentiation process is complete. Among the entry-relevant factors that change, IFITM1, IFTIM2, and IFITM3 are among a family of IFN-inducible proteins that when overexpressed restrict the entry of a variety of viruses, including EBOV (47, 48, 7275). We observed a dramatic downregulation of the mRNA for each of these proteins during the differentiation process, and this downregulation was accelerated in EBOV-infected monocytes. Conversely, essential entry factors cathepsin B and NPC1 are dramatically upregulated during monocyte differentiation. In THP-1s, the patterns of NPC1 expression mirror what was seen in primary monocyte to macrophage or DC differentiation, and the expression of NPC1 via a retroviral vector was sufficient to partially rescue entry into undifferentiated THP-1 cells (Fig. 8). Whether NPC1 facilitates entry directly, by acting as a receptor, or by some indirect mechanism remains to be determined. Nonetheless, these data point to NPC1 upregulation as a critical determinant of entry into APCs. Whether coexpression of other entry factors (e.g., cathepsin B) or simultaneous knockdown of restriction factors (IFITMs) would further enhance entry remains to be determined. In addition to their role in EBOV entry, it will be of interest to determine how the regulation of these factors generally affects monocyte and macrophage function.
Our microscopy studies indicate that VLPs attach to undifferentiated monocytes at some frequency and that the VLPs can remain associated with the cells for at least several hours. However, by the 4-h time point when the β-lactamase-based entry assay does not detect significant entry (Fig. 3) and NP mRNA remains undetectable in EBOV-infected monocytes (Fig. 5), a significant percentage of the particles had moved to an intracellular location (Fig. 5). It remains to be determined whether the inward migration of VLPs at 4 h postinfection represents a pathway that leads to productive entry. However, the data suggest that monocytes are capable of supporting a certain level of viral attachment and that VLPs remain monocyte-associated as these cells undergo differentiation and become fully permissive for entry.
Previous studies implicated cell adherence versus nonadherence as a determinant of EBOV entry into THP-1 and 293 cells (22). Because differentiation of primary monocytes is intertwined with the acquisition of adherence, we cannot exclude the possibility that the modulation of entry factors is a direct consequence of adherence. Comparison of the ability of nonadherent and adherent cells, including monocytes and macrophages, to bind to a recombinant EBOV GP receptor binding domain (RBD)-Fc protein also indicated that adherence correlates with the translocation of an RBD binding activity from an intracellular compartment to the cell surface (21). Although our studies did demonstrate stable association of VLPs with freshly isolated, nonadherent primary monocytes, these studies do not directly address the presence of an RBD binding factor on monocytes versus macrophages.
There are multiple potential consequences for the delayed entry of EBOV into monocytes. Monocytes are blood-borne phagocytic mononuclear cells (76) that can capture and present antigen. Under inflammatory conditions, monocytes can also enter tissues in order to differentiate into macrophages or DCs (77, 78). The persistence of EBOV GP VLPs associated with monocytes suggests the possibility that EBOV can hijack monocytes and potentially “hide” from the host immune response within monocytes, using migrating monocytes as a vehicle for dissemination. In this model, completion of the entry process and onset of viral replication might only occur after the monocytes enter tissue and differentiate. In addition, we show that EBOV-infected monocyte cultures secrete limited levels of cytokines (Fig. 6). Further studies are required to determine whether the lack of robust secretion of a subset of cytokines is related to the delayed kinetics of viral entry.
EBOV infection appears to accelerate the transcriptional regulation of entry-relevant host factors compared to uninfected, plated monocytes (Fig. 7). EBOV can trigger signaling in macrophages within 1 h postinfection (79) and EBOV VLPs, which lack viral replication machinery can also activate macrophages as well as DCs. Furthermore, several of these studies have shown that EBOV VLPs stimulate APCs in a GP-dependent manner, illustrating a central role for the attachment protein in an entry process that itself induces signaling (3, 51, 80). Therefore, it will be of interest to identify the signals responsible for EBOV-induced transcriptional regulation. By defining the pathways required for EBOV to differentiate and enter monocytes, it may be possible to devise strategies that will impair the infection of APCs, potentially blunt their cytokine and other responses and thereby influence EBOV pathogenesis.

ACKNOWLEDGMENTS

This study was supported in part by National Institutes of Health grants R01AI059536, R21AI097568, and AI057158 (Northeast Biodefense Center-Lipkin), U.S. Army grant W81XWH-10-1-0683 to C.F.B., and NIH fellowship 5F32AI084453-02 to R.S.S.

Footnotes

Published ahead of print 23 January 2013

Article information

J Virol. Apr 2013; 87(7): 3801–3814.