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Hakirali emailove od Faucia. (ludi ero opet falio sve 100%)

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Post by mutava baštarda 6/6/2021, 11:59


_________________
“Ako uradimo dobar posao s novim vakcinama, zdravstvenom službom, planiranim roditeljstvom (abortusima), možemo smanjiti broj stanovnika na zemlji za 10% – 15%."
                                                Bill - bi Gates
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mutava baštarda

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Post by T. 6/6/2021, 15:26

Najavio pandemiju još 2017. godine ...
T.
T.

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Post by Gnječ 6/6/2021, 16:11

situacija je frkovita.

nađoh recept za izradu sintetičkog SARS CoV ( možda 2? ) virusa ali je tu umiksan i HIV virus:

ovo ne dokzuje da je tako sintetiziran i SARS CoV2 ali dokazuje da su kinezi već 2004. imali tehnologiju i know-how kako sintetizirati i napraviti zaraznijim ili napraviti zaraznim za ljude bilo koji virus koji prije nije bio zarazan za čovjeka. uz to moguća je i rekombinacija više različitih virusa u potpuno novi prirodi nepoznat virus. ovdje su koristili virus HIV (AIDS) sa virusom SARS na kojem su sintetizirali ljudski ACE2 protein.

koga zanima neka čita

to je objavljeno još daleke 2004. godine


Highly infectious SARS-CoV pseudotyped virus reveals the cell tropism and its correlation with receptor expression

Published online 2004 Aug 1. doi: 10.1016/j.bbrc.2004.07.060

Abstract

Studies of SARS coronavirus (SARS-CoV)—the causative agent of severe acute respiratory syndrome (SARS)—have been hampered by its high transmission rate and the pathogenicity of this virus. To permit analysis of the host range and entry mechanism of SARS-CoV, we incorporated the humanized SARS-CoV spike (S) glycoprotein into HIV particles to generate a highly infectious SARS-CoV pseudotyped virus. The infection on Vero E6—a permissive cell line to SARS-CoV—could be neutralized by sera from convalescent SARS patients, and the entry was a pH-dependent process. With these highly infectious SARS-CoV pseudotypes, several cell lines derived from various tissues were revealed as susceptible to SARS-CoV, which were highly corresponding to the expression pattern of virus’s receptor angiotensin-converting enzyme 2 (ACE2). In addition, we also demonstrated angiotensin 1 converting enzyme (ACE)—the homologue of ACE2 could not function as a receptor for SARS-CoV.

In this report, we expressed SARS spike protein from a synthetic codon-optimized gene, which could dramatically increase S protein expression compared with native S gene. The S protein could incorporate into HIV cores to generate pseudotyped virus with high infectivity on Vero E6 cells. The infection could be neutralized by sera from convalescent SARS patients, and the entry process was pH-dependent. With these SARS-CoV pseudotypes, we analyzed the cell tropism of SARS-CoV and its correlation with its receptor ACE2 expression [11], [12]. We found that various tissue-derived cell lines were susceptible to the pseudotype infection, and the infectivity was correlated with the ACE2 expression. We also demonstrated that ACE—the homologue of ACE2 could not function as a receptor for SARS-CoV.

Materials and methods

Synthetic S gene and plasmid constructions. The cDNA of the SARS-CoV (strain BJ01, GenBank Accession No. AY278488) was a gift from Dr. Li Ruan (China CDC, Beijing). The SARS-CoV S gene was amplified by PCR using the following primers: 5′-CGGGATCCAACGAACATGTTTATTTTCTTATTATTTC-3′ and 5′-CGGAATTCGTTTATGTGTAATGTAATTTGACACCC-3′. The fragment was cloned into pcDNA3.1 (+) vector to generate the plasmid pS. In order to create a codon optimized S gene, 60 overlapping primers were synthesized and assembled by overlapping PCR. Pyrobest DNA polymerase (Takara, Japan) was used in all PCRs. In the first forward primer, tissue plasminogen activator (Tpa) signal sequence (MDAMKRGLCCVLLLCGAVFVSA) was introduced to replace the original signal peptide of the S gene (MFIFLLFLTLTSG). The chimeric wild-type S with Tpa was named TS, the synthetic S gene with Tpa was named TSh. Sequences were determined by sequencing and cloned into a pcDNA3.1 (+) vector to generate pTS and pTSh.

Cell lines. 293T cells were used as packaging cell lines for preparing the pseudotyped virus. Huh7 was a gift from Dr. Stanley M. Lemon (The University of Texas Medical Branch, USA). Human cell lines (Bel7402, T84, Colo320, SW480, SPC-A1, A549, Glc-82, and Hep-2), a monkey cell line (Vero E6), and a mouse cell line (CHO) were obtained from Wuhan Institute of Virology (Wuhan, China). To establish the ACE or ACE2 expressing cell lines, the cDNA of human ACE (gift from Dr. Pierre Corvol) or ACE2 was transduced into NIH/3T3 or HeLa cells by retroviral vector pMX, and the expression of ACE or ACE2 was detected by FACS using anti-ACE or anti-ACE2 polyclonal antibody (R&D systems, Minneapolis).

FACS analysis of S protein expression. S expression constructs were transfected into 293T by the calcium phosphate precipitation method. The cells were harvested 48 h after transfection and incubated with the sera for 1 h. These cells were then incubated with FITC-conjugated goat anti-human IgG (Sigma, St. Louis) for 30 min. After 3 washes, the cells were subjected to analysis using a Moflo cytometer (DAKO Cytomation, Denmark).

Western blot. Viral pellets and lysates of the transfected 293T cells were then subjected to 8% SDS–PAGE, which was followed by transfer to a nitrocellulose membrane (Amersham–Pharmacia, Germany) and incubated with sera (1:200 dilution) from rabbits immunized with the N-terminal domain (14–670) of the S protein; and an alkaline phosphatase-labeled goat anti-rabbit IgG (Santa Cruz Biotechnology, California) was used as the secondary antibody.

Pseudotyped virus infection assays. The SARS-CoV pseudotyped virus HIV/SARS was produced by a similar method which was described previously [8]. Briefly, 10 μg pNL4.3.Luc. R−E−pro− [13] and 10 μg pTSh were co-transfected into 293T cells in 10 cm dishes. Supernatants were harvested 48 h later and used in infection assays. The pseudotyped virus was purified by ultracentrifugation through a 20% sucrose cushion at 50,000g for 90 min, resuspended in 100 μl PBS, and normalized by p24 ELISA using a Vironostika HIV-1 Antigen MicroELISA Kit (Biomerieux bv, Boxtel, The Netherlands). The supernatant containing 5 ng pseudotyped virus (p24) was used to infect cells in 24-well plates (4–8 × 104 cells/well). The cells were lysed at 48 h post-infection. Twenty microliters of lysate was tested for luciferase activity by the addition of 50 μl of luciferase substrate and measured for 10 s in a Wallac Multilabel 1450 Counter (Perkin–Elmer, Singapore). For neutralization tests, diluted SARS patient’s sera were mixed with equal volumes of pseudotyped virus supernatants. After incubation at 37 °C for 30 min, 100 μl mixtures were added to Vero E6 cells in 96-well plates. The cells were lysed at 48 h post-infection and tested for luciferase activity as described above.

Cell fusion assay. COS-7 cells were transduced with S gene (TS or TSh) by retroviral vector pBabe-puro and selected in culture medium containing 5 μg/ml puromycin for one week. The puromycin resistant cells were checked for S protein expression and named TS-COS and TSh-COS, respectively. These cells were mixed with Vero E6 cells at a ratio of 1:1 and co-cultured for 8 h, and the syncytium was examined under microscope.

RT-PCR. The total RNA of target cells was isolated with a Trizol reagent (Life Technologies, Rockville, MD). The expression of ACE2 was analyzed with the following primers: forward 5′-GCACTCACGATTGTTGGGACT-3′; reverse 5′-ATTAGCCACTCGCACATCCTC-3′. The expression of mouse ACE2 was analyzed with the following primers: forward 5′-GCATTGACAATTGTTGGAACA-3′; reverse 5′-ATCACTCACTCGTACATCTTC-3′. The expression of human ACE was analyzed with the following primers: forward 5′-CGGCTCAATGGCTATGTAGATGC-3′; reverse 5′-TCCCTCAAGGCCACAGGTAAGTC-3′.

Treatment with NH 4 Cl. Huh7 cells in 24-well plates were pretreated for 1 h with serum-free DMEM containing NH4Cl at various concentrations (0–30 mM), at 37 °C. HIV/SARS, HIV/AMLV or HIV/VSV-G pseudoviruses were added onto Huh7 cells in the presence of NH4Cl at various concentrations. After 12 h incubation, the supernatant was replaced with DMEM containing 10% FBS. The luciferase activity was determined at 48 h post-infection as described above.

Results and discussion

The expression of the S protein using a codon-optimized S gene.

The spike (S) glycoprotein of the coronavirus is the major envelope protein that plays a key role in viral entry. It not only binds to the cellular receptor but also initiates membrane fusion [14], [15], [16], [17]. To develop the pseudotyped virus system for studying SARS-CoV entry, we initially transfected pTS containing the native S gene into 293T cells, but found that the expression of the S protein in transfected cells was too weak to be detected either by FACS or Western blot (Figs. 1 A and B). The poor expression from the native S gene might be due to codon bias, since the native S gene had a codon usage that differed substantially from those of human genes (Fig. 1C).

We therefore synthesized a humanized S gene (TSh) in which native codons were replaced with the degenerate codons used most frequently in human genes. When the pTSh was transfected into 293T cells, the S protein could be easily detected by FACS analysis (Fig. 1A). Western blot analysis of lysates from the pTSh transfected 293T cells showed two major protein bands that reacted with anti-S sera: a 180 kDa protein corresponding to the previously described size for the mature S protein on SARS-CoV virions [18] and a 130 kDa protein consistent with the calculated molecular weight of the nonglycosylated precursors of the S protein (Fig. 1B).

Infectious SARS-CoV pseudotyped virus can be assembled in vitro


We then produced the pseudotyped virus HIV/SARS by co-transfecting 293T cells with pTSh and pNL4.3.Luc.R−E−pro− [8]. As controls, pTS or pVSV-G was co-transfected with pNL4.3.Luc.R−E−pro− into 293T cells. The supernatant was collected and the virus particles were concentrated by ultra centrifugation and then normalized by p24 ELISA. These viral particles were subjected to immunoblots using rabbit polyclonal antisera raised against the S protein. The results showed that a 180 kDa band of the mature S protein was detected in virus particles generated from pTSh, indicating that the S glycoprotein expressed in 293T cells could incorporate into pseudotyped particles (Fig. 2 A). The absence of detectable S protein in virus particles generated from pTS further confirmed that the codon optimization of native S gene could improve the expression of S glycoprotein.

o test if the pseudotyped virus was infectious and displayed the same host range as SARS-CoV, we infected Vero E6, MDCK, and NIH/3T3 cells with HIV/SARS. Our data showed that Vero E6 and MDCK were susceptible to HIV/SARS infection (Fig. 2B). This is consistent with the infection pattern of the wild-type SARS-CoV [3], [19]. To determine whether the infectivity of HIV/SARS can be mediated by S protein and was SARS-CoV specific, we incubated the pseudotyped virus with sera from convalescent SARS patients before adding them onto Vero E6 cells for infection. We showed that convalescent SARS patients’ sera had a high neutralizing activity on SARS-CoV pseudotypes, while they failed to neutralize pseudotypes with VSV G glycoprotein (Fig. 2C). This result indicated that the entry process of SARS-CoV was mediated by S protein and this mechanism is similar to those of murine hepatitis virus and transmissible gastroenteritis coronavirus [16], [17].

The key role of the S protein in viral entry was further confirmed by cell fusion assay. We transduced pTS or pTSh into COS-7 cells to establish COS-TS and COS-TSh cell lines. When these cells were co-cultured with Vero E6 cells, large amounts of syncytia were observed in Vero E6 plus COS-TSh, but not in Vero E6 plus COS-7 or Vero E6 plus COS-TS (Fig. 2D). The entry mechanism of SARS-CoV pseudotyped virus in our results was similar to those recently demonstrated by Graham Simmons et al. [20], who increased the expression of S protein by utilizing a chicken β-actin promoter and generated HIV(SARS-S) pseudovirions. In our experiment, codon optimization dramatically increased the expression of S protein and led to high titer HIV/SARS production. Moreover, the luciferase reporter gene used in our experiment provided a convenient and high throughput assay.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092805/
Gnječ
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Post by AlfaOmega 7/6/2021, 16:38

PROBLEMATIČNO, U NAJMANJU RUKU: Ključni tekst koji ponovno pokreće teoriju o virusu iz kineskog laboratorija potpisuje utjecajni novinar koji je 2002. prvi pisao o nepostojećem "iračkom oružju za masovno uništenje"


Kinesko ministarstvo vanjskih poslova oštro je osudilo povratak interesa za teoriju o podrijetlu koronavirusa iz (kineskog) laboratorija. Podsjetimo, iz Pekinga još od prošle godine tvrde kako ta teorija ima za cilj vršiti pritisak na Kinu koji se sad opet pojačava.


Glasnogovornik kineskog ministarstva vanjskih poslova, Wang Wenbin, konkretno je izdvojio jednu osobu, Michaela R. Gordona, korespondenta za nacionalnu sigurnost koji radi za Wall Street Journal, ističući kako je Gordon jedan od autora izvještaja u kojem se spekulira o tome da je Covid-19 nastao u laboratoriju.
"Riječ je o istoj osobi koja je prije 19 godina širila lažne informacije o neutemeljenim tvrdnjama da Irak nastoji 'nabaviti nuklearno oružje' što je pak direktno dovelo do Rata u Iraku", rekao je Wenbin referirajući se na američku vojnu invaziju Iraka 2003. godine.


Očito je kako Michael R. Gordon ima popriličan utjecaj jer 3 dana nakon njegovog teksta u WSJ-u američki predsjednik Joe Biden naredio je obavještajnoj zajednici da "udvostruče napore" po pitanju potrage za informacijama kako je ovaj virus nastao uključujući i mogućnost da je nastao u laboratoriju.


Spomenimo pritom kako je Gordon prije WSJ-a radio više od 30 godina kao vojni diplomatski korespondent za The New York Times. Upravo je on prvi pisao o iračkom navodnom programu oružja za masovno uništenje. 8. rujna 2002. objavio je članak, zajedno s Judith Miller, pod naslovom: "SAD kaže kako Hussein sve intenzivnije traga za dijelovima za atomsku bombu". U tom tekstu citiraju se "neimenovani dužnosnici" koji tvrde, odnosno Gordon i Miller tvrde da tvrde, kako se Irak dao u "globalnu potragu za dijelovima atomske bombe".


Nedugo zatim George W. Bush počeo je pripremati planove za invaziju na Irak koristeći se upravo ovim lažnim tvrdnjama. Na kraju nikakvo oružje za masovno uništenje ili programi proizvodnje istog u Iraku nisu pronađeni, ali su zato izginule stotine tisuća ljudi.

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Post by AlfaOmega 7/6/2021, 16:39

E jbg sad, retardinjosi iz čemerike i kad hoće nekog da optuže za nekaj, odmah se sapletu u startu. Čuj angažirali lika koji je već laga za iračko oružje za masovno uništenje.

Zar ni bija niko drugi?

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2015-09-11


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