{"id":16354,"date":"2022-10-08T09:01:02","date_gmt":"2022-10-08T07:01:02","guid":{"rendered":"http:\/\/www.infokuryr.cz\/n\/?p=16354"},"modified":"2022-10-08T09:01:02","modified_gmt":"2022-10-08T07:01:02","slug":"spacha-valek-s-kubkem-sebevrazdu-ustni-vody-jsou-ucinne-proti-koronaviru","status":"publish","type":"post","link":"https:\/\/www.infokuryr.cz\/n\/2022\/10\/08\/spacha-valek-s-kubkem-sebevrazdu-ustni-vody-jsou-ucinne-proti-koronaviru\/","title":{"rendered":"Sp\u00e1ch\u00e1 V\u00e1lek s Kubkem sebevra\u017edu? \u00dastn\u00ed vody jsou \u00fa\u010dinn\u00e9 proti koronaviru!"},"content":{"rendered":"
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V\u011bdeck\u00e9 zpr\u00e1vy: Cetylpyridiniumchlorid v \u00fastn\u00ed vod\u011b zab\u00edj\u00ed koronavirus. V\u011bdci z Hokkaidsk\u00e9 univerzity prok\u00e1zali, \u017ee \u00fastn\u00ed vody mohou zab\u00edjet viry SARS-CoV-2, \u010d\u00edm\u017e sni\u017euj\u00ed virovou z\u00e1t\u011b\u017e.<\/strong><\/span><\/p>\n<\/div>\n

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Uv\u00e1d\u00ed to \u010dl\u00e1nek\u00a0publikovan\u00fd<\/a>\u00a0v \u010dasopise Scientific Reports.<\/p>\n

V\u011bdeck\u00e9 zpr\u00e1vy<\/i><\/a>\u00a0hlasitost<\/span>\u00a012<\/b>\u00a0, \u010c\u00edslo \u010dl\u00e1nku:\u00a0\u00a014050<\/span>\u00a0(\u00a02022<\/span>\u00a0)<\/p>\n

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V\u010dera dezinformace, dnes informace<\/strong><\/span><\/p>\n<\/div>\n
Pandemii zkr\u00e1tka poraz\u00ed sp\u00ed\u0161 o\u010dkov\u00e1n\u00ed a vytrval\u00e1 pr\u00e1ce v\u011bdc\u016f ne\u017e tipy a triky z internetu,\u201c upozor\u0148uje Irena Storov\u00e1, \u0159editelka St\u00e1tn\u00edho \u00fastavu pro kontrolu l\u00e9\u010div. A dal\u0161\u00ed expert na v\u0161e:\u00a0 Kubka roz\u010d\u00edlilo, kdy\u017e zn\u00e1m\u00fd ortoped \u00fastn\u00ed vodu jako prevenci proti covidu. Vlastimil Vor\u00e1\u010dek dostal ot\u00e1zku, zda st\u00e1le vn\u00edm\u00e1 klokt\u00e1n\u00ed \u00fastn\u00ed vody jako prevenci proti onemocn\u011bn\u00ed COVID-19.\u201eAno, virov\u00e1 n\u00e1lo\u017e se t\u00edm d\u00e1 zredukovat. Nejde o to, \u017ee se t\u00edm vyhoj\u00edte, ale sn\u00ed\u017e\u00edte pravd\u011bpodobnost, \u017ee se ten virus tolik rozb\u011bhne,\u201c odv\u011btil Vor\u00e1\u010dek. A kdo potrest\u00e1 Kubka za jeho zlo\u010diny, kter\u00e9 se prok\u00e1zaly?<\/p>\n

Milan Kubek tvrd\u011b ohradil: \u201eJsem r\u00e1d, \u017ee je pan doktor ortoped, a v\u011b\u0159\u00edm, \u017ee dobr\u00fd. Proto\u017ee tohle jsou naprost\u00e9 nesmysly a je smutn\u00e9, kdy\u017e\u00a0takov\u00fdm bizarn\u00edm doporu\u010den\u00edm lid\u00e9 podl\u00e9haj\u00ed. Pak to hlup\u00e1ci \u0161\u00ed\u0159\u00ed d\u00e1l<\/strong>. \u0158ad\u011b lid\u00ed tyhle p\u0159\u00ed\u0161ern\u00e9 rady po\u0161kodily zdrav\u00ed. Doporu\u010duji lidem ignorovat to.\u201c<\/p>\n<\/div>\n<\/div>\n

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Tak\u017ee my jsme dle toho zlo\u010dince Kubka hlup\u00e1ci?<\/strong><\/h2>\n
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Scientific Reports is an online multidisciplinary, open access journal from the publishers of Nature. P\u0159elo\u017eeno z angli\u010dtiny- Scientific Reports je online recenzovan\u00fd v\u011bdeck\u00fd mega \u010dasopis s otev\u0159en\u00fdm p\u0159\u00edstupem vyd\u00e1van\u00fd Nature Portfolio, pokr\u00fdvaj\u00edc\u00ed v\u0161echny oblasti p\u0159\u00edrodn\u00edch v\u011bd. \u010casopis byl zah\u00e1jen v roce 2011. \u010casopis ozn\u00e1mil, \u017ee jejich c\u00edlem je posuzovat pouze v\u011bdeckou validitu p\u0159edlo\u017een\u00e9 pr\u00e1ce. Dnes je nejv\u011bt\u0161\u00ed na sv\u011bt\u011b.<\/em><\/div>\n<\/div>\n

Abstraktn\u00ed<\/h2>\n
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Cetylpyridiniumchlorid (CPC), kvart\u00e9rn\u00ed amoniov\u00e1 slou\u010denina, kter\u00e1 je p\u0159\u00edtomna v \u00fastn\u00ed vod\u011b, je \u00fa\u010dinn\u00e1 proti bakteri\u00edm, pl\u00edsn\u00edm a obalen\u00fdm vir\u016fm. Tato studie byla provedena s c\u00edlem prozkoumat antivirov\u00fd \u00fa\u010dinek CPC na SARS-CoV-2. Existuje n\u011bkolik zpr\u00e1v o \u00fa\u010dinku CPC proti SARS-CoV-2 divok\u00e9ho typu p\u0159i n\u00edzk\u00fdch koncentrac\u00edch, jako je 0,001 %\u20130,005 % (10\u201350 \u00b5g\/ml). Je zaj\u00edmav\u00e9, \u017ee jsme zjistili, \u017ee n\u00edzk\u00e9 koncentrace CPC potla\u010dily infek\u010dnost lidsk\u00fdch izolovan\u00fdch kmen\u016f SARS-CoV-2 (Wuhan, Alpha, Beta a Gamma) dokonce i ve slin\u00e1ch. D\u00e1le jsme pomoc\u00ed anal\u00fdzy hustoty sachar\u00f3zy a elektronov\u00e9ho mikroskopu prok\u00e1zali, \u017ee CPC vykazuje anti-SARS-CoV-2 \u00fa\u010dinky bez naru\u0161en\u00ed virov\u00e9ho obalu. Z\u00e1v\u011brem lze \u0159\u00edci, \u017ee tato studie poskytla experiment\u00e1ln\u00ed d\u016fkaz, \u017ee CPC m\u016f\u017ee inhibovat infekci SARS-CoV-2 i p\u0159i ni\u017e\u0161\u00edch koncentrac\u00edch.<\/p>\n<\/div>\n<\/div>\n<\/section>\n

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\u00davod<\/h2>\n
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Podle ned\u00e1vn\u00fdch informac\u00ed z centra zdroj\u016f pro koronaviry, Johns Hopkins University of Medicine\u00a0https:\/\/coronavirus.jhu.edu\/map.html.<\/a>“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 1″>1<\/sup>\u00a0, je COVID-19 zodpov\u011bdn\u00fd za v\u00edce ne\u017e 420 milion\u016f p\u0159\u00edpad\u016f a p\u0159ibli\u017en\u011b 6 milion\u016f \u00famrt\u00ed po cel\u00e9m sv\u011bt\u011b.<\/p>\n

SARS-CoV-2 byl p\u016fvodn\u011b hl\u00e1\u0161en ve Wuhanu v \u010c\u00edn\u011b\u00a0https:\/\/doi.org\/10.1056\/NEJMoa2001017<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 2″>2<\/sup>\u00a0a byly hl\u00e1\u0161eny tak\u00e9 n\u011bkter\u00e9 zaj\u00edmav\u00e9 varianty a varianty vzbuzuj\u00edc\u00ed obavy (VOC)\u00a0https:\/\/www.who.int\/en\/activities\/tracking-SARS-CoV-2-variants\/<\/a>\u00a0.“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 3″>3<\/sup>\u00a0. Krom\u011b toho se ob\u00e1v\u00e1, \u017ee n\u011bkter\u00e9 varianty jako Delta a Omicron by mohly m\u00edt schopnost vyhnout se imunit\u011b vyvolan\u00e9 vakc\u00ednou\u00a0https:\/\/doi.org\/10.1056\/NEJMoa2109072<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“>4 ,\u00a0https:\/\/doi.org\/10.1016\/s0140-6736<\/a>(21)02758-6 (2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“>5 ,\u00a0https:\/\/doi.org\/10.1056\/nejmoa2108891<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 6″>6<\/sup>\u00a0. V\u011bdci se proto ob\u00e1vaj\u00ed, \u017ee pandemie SARS-CoV-2 m\u016f\u017ee pokra\u010dovat i po zv\u00fd\u0161en\u00ed proo\u010dkovanosti.<\/p>\n

Bylo hl\u00e1\u0161eno, \u017ee SARS-CoV-2 infikuje epiteli\u00e1ln\u00ed bu\u0148ky sliznice dutiny \u00fastn\u00ed a slinn\u00fdch \u017el\u00e1z, kter\u00e9 exprimuj\u00ed vstupn\u00ed faktory viru, angiotensin-konvertuj\u00edc\u00ed enzym 2 (ACE2) a \u010dleny transmembr\u00e1nov\u00e9 prote\u00e1zy serin (TMPRSS)\u00a0https:\/\/doi.org\/10.1038\/s41591-021-01296-8<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 7″>7<\/sup>\u00a0. T\u00edmto zp\u016fsobem tedy hraje dutina \u00fastn\u00ed z\u00e1sadn\u00ed roli p\u0159i infekci a p\u0159enosu SARS-CoV-2. P\u0159esto\u017ee p\u0159\u00edznakem COVID-19 souvisej\u00edc\u00edm s \u00fastn\u00ed dutinou je dysgeuzie a stomatitida\u00a0https:\/\/doi.org\/10.1053\/j.gastro.2020.05.003<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 8″>8 ,\u00a0https:\/\/doi.org\/10.1111\/dth.14578<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 9″>9<\/sup>\u00a0, mnoho lid\u00ed infikovan\u00fdch SARS-CoV-2 m\u016f\u017ee b\u00fdt asymptomatick\u00fdch, co\u017e vede k jeho p\u0159enosu na dal\u0161\u00ed lidi.<\/p>\n

SARS-CoV-2 se m\u016f\u017ee replikovat v \u00fastn\u00ed dutin\u011b a uvol\u0148ovat se do slin\u00a0https:\/\/doi.org\/10.1038\/s41591-021-01296-8<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 7″>7<\/sup>\u00a0. SARS-CoV-2 se nav\u00edc m\u016f\u017ee replikovat v respira\u010dn\u00edm epitelu\u00a0https:\/\/doi.org\/10.1128\/JVI.00957-20<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 10″>10<\/sup>\u00a0a m\u016f\u017ee se p\u0159en\u00e1\u0161et do dutiny \u00fastn\u00ed ka\u0161lem. P\u0159enos SARS-CoV-2 kap\u00e9nkami a\/nebo aerosolem zp\u016fsobuje jeho infekci a replikaci v plicn\u00edch alveol\u00e1rn\u00edch epiteli\u00e1ln\u00edch bu\u0148k\u00e1ch, co\u017e vede k alveol\u00e1rn\u00edmu po\u0161kozen\u00ed\u00a0https:\/\/doi.org\/10.1038\/s41379-020-0595-z<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 11″>11<\/sup>\u00a0. D\u00e1le se uv\u00e1d\u00ed, \u017ee k p\u0159enosu SARS-CoV-2 doch\u00e1z\u00ed kap\u00e9nkami z exspira\u010dn\u00edch \u010dinnost\u00ed, jako je mluven\u00ed, ka\u0161l\u00e1n\u00ed a k\u00fdch\u00e1n\u00ed\u00a0https:\/\/doi.org\/10.1038\/s41591-020-1092-0<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 12″>12 ,\u00a0https:\/\/doi.org\/10.1126\/science.abd9149<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 13″>13<\/sup>\u00a0. Je zaj\u00edmav\u00e9, \u017ee lid\u00e9 infikovan\u00ed SARS-CoV-2 se mohou st\u00e1t zdrojem p\u0159enosu i b\u011bhem asymptomatick\u00e9 inkuba\u010dn\u00ed doby viru\u00a0https:\/\/doi.org\/10.7326\/M20-0504<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 14″>14<\/sup>. Proto mus\u00edme prozkoumat strategii profylaxe proti COVID-19. D\u00e1le byl hl\u00e1\u0161en vztah mezi aspirac\u00ed kapi\u010dek ze slin obsahuj\u00edc\u00edch SARS-CoV-2 a zhor\u0161en\u00edm COVID-19\u00a0https:\/\/doi.org\/10.1038\/s41586-020-2196-x<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 15″>15<\/sup>\u00a0. Proto je pro prevenci p\u0159enosu SARS-CoV-2 d\u016fle\u017eit\u00e1 \u00fastn\u00ed p\u00e9\u010de.<\/p>\n

\u00dastn\u00ed voda se zam\u011b\u0159uje na prevenci mikrobiomov\u00e9 infekce\u00a0https:\/\/doi.org\/10.1007\/s40121-018-0200-7<\/a>\u00a0(2018).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 16″>16<\/sup>\u00a0. Krom\u011b toho bylo ned\u00e1vno hl\u00e1\u0161eno, \u017ee n\u011bkolik slo\u017eek \u00fastn\u00ed vody sni\u017euje viriony SARS-CoV-2 v \u00fastn\u00ed dutin\u011b\u00a017<\/a>\u00a0,\u00a018<\/a><\/sup>\u00a0. Cetylpyridiniumchlorid (CPC) je \u0161iroce pou\u017e\u00edv\u00e1n jako jedna z baktericidn\u00edch slo\u017eek \u00fastn\u00edch vod, tablet, sprej\u016f a kapek. CPC m\u016f\u017ee naru\u0161it lipidovou membr\u00e1nu prost\u0159ednictv\u00edm fyzik\u00e1ln\u011b-chemick\u00fdch interakc\u00ed. Ji\u017e bylo hl\u00e1\u0161eno, \u017ee CPC m\u00e1 baktericidn\u00ed \u00fa\u010dinky a tak\u00e9 antivirov\u00e9 \u00fa\u010dinky proti viru ch\u0159ipky\u00a0https:\/\/doi.org\/10.20411\/pai.v2i2.200<\/a>\u00a0(2017).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 19″>19<\/sup>\u00a0a koronavir\u016fm\u00a0https:\/\/doi.org\/10.1128\/JVI.00023-19<\/a>\u00a0(2019).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“>20 ,\u00a021<\/a>\u00a0,\u00a0https:\/\/doi.org\/10.1080\/20002297.2022.2030094<\/a>\u00a0(2022).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 22″>22<\/sup>. Ve srovn\u00e1n\u00ed s jin\u00fdmi slo\u017ekami \u00fastn\u00edch vod, v\u010detn\u011b povidonu, jodu a chlorhexidinu (CHX); CPC je bez chuti a z\u00e1pachu, a proto je vhodn\u00fd pro pou\u017eit\u00ed v p\u0159\u00edpravc\u00edch pro p\u00e9\u010di o \u00fastn\u00ed dutinu. K dne\u0161n\u00edmu dni existuje jen m\u00e1lo zpr\u00e1v popisuj\u00edc\u00edch virucidn\u00ed aktivitu CPC proti SARS-CoV-2. Seneviratne a kol. uvedli, \u017ee CPC sn\u00ed\u017eila virovou z\u00e1t\u011b\u017e SARS-CoV-2 ve slin\u00e1ch \u010dty\u0159 pacient\u016f s COVID-19\u00a0https:\/\/doi.org\/10.1007\/s15010-020-01563-9<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 23″>23<\/sup>\u00a0ve srovn\u00e1n\u00ed s kontroln\u00ed vodou, ale virov\u00e1 infek\u010dnost ve slin\u00e1ch nebyla pops\u00e1na. Ned\u00e1vn\u00e1 zpr\u00e1va uk\u00e1zala \u00fa\u010dinek CPC v mnohem ni\u017e\u0161\u00ed koncentraci ne\u017e CPC v komer\u010dn\u011b dostupn\u00fdch \u00fastn\u00edch vod\u00e1ch proti pseudoviru\u00a0https:\/\/doi.org\/10.1177\/00220345211029269<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 24″>24<\/sup>. Neexistuje v\u0161ak \u017e\u00e1dn\u00e1 zpr\u00e1va o \u00fa\u010dinku CPC p\u0159i n\u00edzk\u00fdch koncentrac\u00edch, jako je 0,001 %\u20130,005 % (10\u201350 \u00b5g\/ml) proti divok\u00e9mu typu SARS-CoV-2 ve slin\u00e1ch. V Japonsku je koncentrace CPC v komer\u010dn\u011b dostupn\u00fdch \u00fastn\u00edch vod\u00e1ch t\u00e9m\u011b\u0159 30\u201350 \u00b5g\/ml, co\u017e je mnohem m\u00e9n\u011b ne\u017e v \u00fastn\u00edch vod\u00e1ch pou\u017e\u00edvan\u00fdch v p\u0159edchoz\u00edch zpr\u00e1v\u00e1ch\u00a0https:\/\/doi.org\/10.1177\/00220345211029269<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 24″>24 ,\u00a0https:\/\/doi.org\/10.1016\/j.ajoms.2021.02.002<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 25″>25<\/sup>\u00a0. Proto jsme zkoumali antivirov\u00e9 \u00fa\u010dinky CPC na SARS-CoV-2 p\u0159i n\u00edzk\u00fdch koncentrac\u00edch. Krom\u011b toho jsme tak\u00e9 zkoumali mechanismus anti-SARS-CoV-2 aktivity CPC anal\u00fdzou hustoty sachar\u00f3zy a pozorov\u00e1n\u00edm elektronov\u00fdm mikroskopem.<\/p>\n<\/div>\n<\/div>\n<\/section>\n

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V\u00fdsledek<\/h2>\n
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CPC potla\u010dila infek\u010dnost SARS-CoV-2<\/h3>\n

Zkoumali jsme kmeny SARS-CoV-2, v\u010detn\u011b Wuhan, Alpha, Beta a Gamma, kter\u00e9 pat\u0159\u00ed k VOC. Test plak\u016f prok\u00e1zal, \u017ee CPC v\u00fdznamn\u011b potla\u010dila infek\u010dnost v\u0161ech vy\u0161et\u0159ovan\u00fdch SARS-CoV-2 p\u0159\u00edmo v z\u00e1vislosti na d\u00e1vce (obr.\u00a0\u00a01<\/a>\u00a0a\u2013d,\u00a0S1<\/a>\u00a0). L\u00e9\u010dba CPC (50 \u03bcg\/ml) zcela inaktivovala kmen SARS-CoV-2 Wuhan podobn\u011b jako Triton X-100 (1 %) (obr.\u00a0\u00a01e<\/a>\u00a0). Komer\u010dn\u00ed \u00fastn\u00ed voda (SP-T l\u00e9ka\u0159sk\u00e9 kloktadlo: SP-T) obsahuj\u00edc\u00ed stejnou koncentraci CPC vykazovala lep\u0161\u00ed antivirov\u00fd \u00fa\u010dinek ne\u017e roztok CPC bez ru\u0161iv\u00fdch slo\u017eek. Titr viru SARS-CoV-2 l\u00e9\u010den\u00e9ho SP-T byl pod hranic\u00ed detekce 2,0 \u00d7 10\u00a03<\/sup>\u00a0PFU\/ml (obr.\u00a0S2<\/a>). Tyto v\u00fdsledky uk\u00e1zaly, \u017ee ni\u017e\u0161\u00ed koncentrace CPC (10\u201340 \u03bcg\/ml) ne\u017e koncentrace komer\u010dn\u011b dostupn\u00e9 \u00fastn\u00ed vody (50 \u03bcg\/ml) vykazovaly anti-SARS-CoV-2 \u00fa\u010dinky u mnoha kmen\u016f, v\u010detn\u011b VOC.<\/p>\n

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Obr\u00e1zek 1<\/b><\/figcaption>
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\"Obr\u00e1zek<\/picture><\/a><\/div>\n
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Antivirov\u00e1 \u00fa\u010dinnost CPC proti SARS-CoV-2 pomoc\u00ed plakov\u00e9ho testu s pou\u017eit\u00edm bun\u011bk Vero E6 exprimuj\u00edc\u00edch gen TMPRSS2 (VeroE6\/TMPRSS2). Byly spo\u010d\u00edt\u00e1ny titry viru a titr viru SARS-CoV-2 Wuhan (\u00a0a<\/b>\u00a0), Alpha (\u00a0b<\/b>\u00a0), Beta (\u00a0c<\/b>\u00a0) a Gamma (\u00a0d<\/b>\u00a0) kmen\u016f o\u0161et\u0159en\u00fdch CPC (0\u201340 \u03bcg\/ml) p\u0159i pokojov\u00e9 teplot\u011b po dobu 30 min byly kvantifikov\u00e1ny a reprezentov\u00e1ny jako PFU\/ml. Test plak\u016f byl tak\u00e9 proveden v p\u0159\u00edtomnosti PBS, CPC (50 ug\/ml) nebo Triton X-100 (1 %) po dobu 10 minut. Pot\u00e9 byly vzorky filtrov\u00e1ny na kolon\u00e1ch PD-10, aby se odstranila \u010dinidla (\u00a0e<\/b>\u00a0). Statistick\u00e1 anal\u00fdza byla provedena pomoc\u00ed jednosm\u011brn\u00e9 anal\u00fdzy rozptylu. (*\u00a0p<\/i>\u00a0\u2009< 0,05).<\/p>\n<\/div>\n<\/div>\n

Obr\u00e1zek v pln\u00e9 velikosti<\/a><\/div>\n<\/figure>\n<\/div>\n

D\u00e1le jsme hodnotili \u00fa\u010dinek CPC na bun\u011b\u010dn\u00fd vstup SARS-CoV-2. Bu\u0148ky VeroE6\/TMPRSS2 byly infikov\u00e1ny kmenem SARS-CoV-2 Wuhan o\u0161et\u0159en\u00fdm CPC p\u0159i multiplicit\u011b infekce (MOI) 0,01. \u00darove\u0148 exprese virov\u00e9 RNA v bu\u0148k\u00e1ch byla v\u00fdznamn\u011b sn\u00ed\u017eena pomoc\u00ed CPC zp\u016fsobem z\u00e1visl\u00fdm na d\u00e1vce 24 hodin po infekci (obr.\u00a0\u00a02<\/a>\u00a0). Po\u010det kopi\u00ed virov\u00e9 RNA byl sn\u00ed\u017een na p\u0159ibli\u017en\u011b jednu t\u0159icetinu pomoc\u00ed CPC p\u0159i koncentraci 15 \u03bcg\/ml ve srovn\u00e1n\u00ed s kontrolou. Tato data uk\u00e1zala, \u017ee mno\u017estv\u00ed infek\u010dn\u00edch virion\u016f bylo sn\u00ed\u017eeno CPC p\u0159ed vstupem do bun\u011bk. V\u0161echny experimenty byly provedeny s pou\u017eit\u00edm CPC v koncentraci, kter\u00e1 nezp\u016fsobila cytotoxicitu (obr.\u00a0S3<\/a>\u00a0).<\/p>\n

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Obr\u00e1zek 2<\/b><\/figcaption>
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\"obr\u00e1zek<\/picture><\/a><\/div>\n
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Antivirov\u00e1 \u00fa\u010dinnost CPC proti SARS-CoV-2 pomoc\u00ed qRT-PCR. Bu\u0148ky VeroE6\/TMPRSS2 byly nao\u010dkov\u00e1ny kmenem SARS-CoV-2 Wuhan p\u0159i multiplicit\u011b infekce (MOI) 0,01 po sm\u00edch\u00e1n\u00ed stejn\u00e9ho mno\u017estv\u00ed CPC. 24 hodin po infekci byly relativn\u00ed hladiny virov\u00e9 N proteinov\u00e9 RNA vyhodnoceny kvantitativn\u011b pomoc\u00ed qRT-PCR. (*\u00a0p<\/i>\u00a0\u2009< 0,05).<\/p>\n<\/div>\n<\/div>\n

Obr\u00e1zek v pln\u00e9 velikosti<\/a><\/div>\n<\/figure>\n<\/div>\n

CPC m\u00e1 antivirovou aktivitu proti SARS-CoV-2 i ve slin\u00e1ch<\/span><\/h3>\n

Abychom zjistili, zda je CPC \u00fa\u010dinn\u00e1 na SARS-CoV-2 ve slin\u00e1ch, kter\u00e9 obsahuj\u00ed mnoho protein\u016f a jsou vysoce visk\u00f3zn\u00ed, m\u011b\u0159ili jsme infek\u010dnost kmene SARS-CoV-2 Wuhan pomoc\u00ed plakov\u00e9ho testu po inkubaci s CPC ve slin\u00e1ch odebran\u00fdch od zdrav\u00fdch dobrovoln\u00edk\u016f. Plaque test prok\u00e1zal inhibi\u010dn\u00ed \u00fa\u010dinek CPC (25\u201340 \u03bcg\/ml) proti SARS-CoV-2 ve slin\u00e1ch v\u00fdznamn\u011b v z\u00e1vislosti na d\u00e1vce (obr.\u00a0\u00a03<\/a>\u00a0).<\/p>\n

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Obr\u00e1zek 3<\/b><\/figcaption>
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\"obr\u00e1zek<\/picture><\/a><\/div>\n
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Antivirov\u00e1 \u00fa\u010dinnost CPC proti SARS-CoV-2 se slinami pomoc\u00ed plakov\u00e9ho testu s pou\u017eit\u00edm bun\u011bk Vero E6 exprimuj\u00edc\u00edch gen TMPRSS2 (VeroE6\/TMPRSS2). Kmen SARS-CoV-2 Wuhan byl p\u0159id\u00e1n do slin a sm\u00edch\u00e1n se stejn\u00fdm mno\u017estv\u00edm CPC. (*\u00a0p<\/i>\u00a0\u2009< 0,05).<\/p>\n<\/div>\n<\/div>\n

Obr\u00e1zek v pln\u00e9 velikosti<\/a><\/div>\n<\/figure>\n<\/div>\n

Anal\u00fdza hustoty sachar\u00f3zy a anal\u00fdza transmisn\u00ed elektronov\u00e9 mikroskopie SARS-CoV-2 o\u0161et\u0159en\u00e9ho CPC<\/span><\/h3>\n

Abychom analyzovali mechanismus CPC na infek\u010dnost SARS-CoV-2, provedli jsme anal\u00fdzu hustoty sachar\u00f3zy virion\u016f SARS-CoV-2 o\u0161et\u0159en\u00fdch bu\u010f 1 \u00d7 fosf\u00e1tem pufrovan\u00fdm fyziologick\u00fdm roztokem (PBS), CPC (50 \u03bcg\/ml) nebo Tritonem X- 100 (1 %) po dobu 10 minut p\u0159i pokojov\u00e9 teplot\u011b (obr.\u00a0\u00a04<\/a>A). SARS-CoV-2 S a N protein vykazoval specifickou distribuci v cel\u00e9m gradientu. Posun p\u00e1su byl pozorov\u00e1n u virion\u016f o\u0161et\u0159en\u00fdch Tritonem X-100; nicm\u00e9n\u011b frakce byly jin\u00e9 ne\u017e frakce o\u0161et\u0159en\u00e9 PBS nebo CPC. Jin\u00fdmi slovy, PBS a CPC nemus\u00ed m\u00edt \u017e\u00e1dn\u00fd vliv na strukturu virion\u016f SARS-CoV-2, zat\u00edmco Triton X-100 strukturu zm\u011bnil. D\u00e1le byla morfologie virion\u016f analyzov\u00e1na transmisn\u00ed elektronovou mikroskopi\u00ed (TEM). Elektronov\u00e1 mikroskopick\u00e1 anal\u00fdza odhalila, \u017ee struktura sf\u00e9rick\u00fdch \u010d\u00e1stic SARS-CoV-2 o\u0161et\u0159en\u00e9ho PBS z\u016fstala nezm\u011bn\u011bna. Tak\u00e9 jsme zjistili, \u017ee v\u011bt\u0161ina virov\u00fdch \u010d\u00e1stic o\u0161et\u0159en\u00fdch 10 ug\/ml CPC z\u016fstala nezm\u011bn\u011bna, zat\u00edmco n\u011bkter\u00e9 se rozpadly s 50 ug\/ml CPC. Naproti tomu t\u00e9m\u011b\u0159 v\u0161echny virov\u00e9 \u010d\u00e1stice o\u0161et\u0159en\u00e9 250 ug\/ml CPC byly jasn\u011b rozru\u0161eny (jako 1% Triton X-100). Mno\u017estv\u00ed 50 ug\/ml bylo pova\u017eov\u00e1no za koncentraci, p\u0159i kter\u00e9 se v\u0161echny virov\u00e9 \u010d\u00e1stice nerozbily. Tento v\u00fdsledek je v souladu s daty anal\u00fdzy hustoty sachar\u00f3zy (obr.\u00a04b<\/a>\u00a0).<\/p>\n

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Obr\u00e1zek 4<\/b><\/figcaption>
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\"obr\u00e1zek<\/picture><\/a><\/div>\n
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Anal\u00fdza hustoty sachar\u00f3zy a TEM anal\u00fdza \u010d\u00e1stic SARS-CoV-2. (\u00a0a<\/b>\u00a0) Anal\u00fdza hustoty sachar\u00f3zy sestavy kapsidy v p\u0159\u00edtomnosti 1 x PBS, CPC (50 \u03bcg\/ml) a Triton X-100 (1 %). Kmen SARS-CoV-2 Wuhan byl o\u0161et\u0159en popsan\u00fdmi \u010dinidly po dobu 10 minut a o\u0161et\u0159en\u00e9 viriony byly aplikov\u00e1ny na ultracentrifugaci s gradientem hustoty. Ka\u017ed\u00e1 frakce byla aplikov\u00e1na na SDS-PAGE a analyzov\u00e1na Western blotem s protil\u00e1tkami proti S proteinu a N proteinu. (\u00a0b<\/b>\u00a0) Elektronov\u00e9 mikrofotografie virion\u016f SARS-CoV-2 po o\u0161et\u0159en\u00ed \u010dinidly. Kmen SARS-CoV-2 Wuhan byl o\u0161et\u0159en 1 x PBS, CPC (10, 50, 250 \u03bcg\/ml) a Triton X-100 (1 %) po dobu 10 minut p\u0159i teplot\u011b m\u00edstnosti. Ka\u017ed\u00e1 stupnice p\u0159edstavuje 50 nm.<\/p>\n<\/div>\n<\/div>\n

Obr\u00e1zek v pln\u00e9 velikosti<\/a><\/div>\n<\/figure>\n<\/div>\n<\/div>\n<\/div>\n<\/section>\n
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Diskuse<\/h2>\n
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V tomto v\u00fdzkumu jsme prok\u00e1zali, \u017ee CPC p\u0159i n\u00edzk\u00e9 koncentraci 50 \u03bcg\/ml nebo m\u00e9n\u011b potla\u010duje infek\u010dnost kmene SARS-CoV-2 Wuhan a VOC, v\u010detn\u011b kmen\u016f Alpha, Beta a Gamma. CPC prok\u00e1zal virucidn\u00ed \u00fa\u010dinky proti SARS-CoV-2 i ve slin\u00e1ch. Zjistili jsme tedy, \u017ee n\u00edzk\u00e9 koncentrace CPC vykazovaly dostate\u010dnou antivirovou aktivitu proti SARS-CoV-2. Proto\u017ee CPC naru\u0161uje lipidov\u00e9 dvojvrstvy, vysok\u00e9 koncentrace CPC mohou vykazovat cytotoxick\u00e9 \u00fa\u010dinky. Proto lze CPC aplikovat ve formulaci, kter\u00e1 m\u016f\u017ee uplat\u0148ovat sv\u016fj \u00fa\u010dinek po dlouhou dobu p\u0159i n\u00edzk\u00fdch koncentrac\u00edch. Proto\u017ee prototypov\u00e1 formulace \u00fastn\u00ed vody m\u016f\u017ee obsahovat dal\u0161\u00ed interferuj\u00edc\u00ed slo\u017eky, kter\u00e9 potla\u010duj\u00ed n\u00edzk\u00e9 hladiny CPC pou\u017eit\u00e9 v experimentech, testovali jsme komer\u010dn\u00ed \u00fastn\u00ed vodu se stejnou koncentrac\u00ed CPC,S2<\/a>\u00a0). D\u00e1le bychom mohli navrhnout, \u017ee antivirov\u00fd \u00fa\u010dinek nemus\u00ed b\u00fdt zp\u016fsoben destrukc\u00ed lipidov\u00e9 membr\u00e1ny, ale denaturac\u00ed proteinu SARS-CoV-2.<\/p>\n

Bylo hl\u00e1\u0161eno, \u017ee CPC je \u00fa\u010dinn\u00fd proti SARS-CoV-2 Alfa varianta\u00a0https:\/\/doi.org\/10.1177\/00220345211029269<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 24″>24<\/sup>\u00a0. Na\u0161e studie odhalila, \u017ee CPC zm\u00edr\u0148uje infek\u010dnost dal\u0161\u00edch dvou variant (kmeny Beta a Gamma) p\u0159i n\u00edzk\u00fdch koncentrac\u00edch. L\u00e9\u010dba CPC po dobu 1 hodiny neuk\u00e1zala cytotoxicitu v\u016f\u010di VeroE6\/TMPRSS2 a\u017e do 40 \u03bcg\/ml (obr.\u00a0S3<\/a>\u00a0). Nav\u00edc v p\u0159edchoz\u00edch studi\u00edch byla pou\u017eita \u00fastn\u00ed voda obsahuj\u00edc\u00ed vy\u0161\u0161\u00ed koncentraci CPC (500\u2013750 \u03bcg\/\u00a0ml\u00a0<\/sup>https:\/\/doi.org\/10.1177\/00220345211029269<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 24″>)\u00a0https:\/\/doi.org\/10.1016\/j.ajoms.2021.02.002<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 25″>24,25<\/sup>\u00a0. P\u0159i t\u011bchto koncentrac\u00edch se m\u00e1 za to, \u017ee lipidov\u00e1 membr\u00e1na bun\u011bk m\u016f\u017ee b\u00fdt po\u0161kozena.<\/p>\n

Doba trv\u00e1n\u00ed antivirov\u00e9ho \u00fa\u010dinku \u00fastn\u00ed vody obsahuj\u00edc\u00ed CPC nen\u00ed dosud jasn\u00e1; na\u0161e v\u00fdsledky v\u0161ak nazna\u010duj\u00ed, \u017ee k prok\u00e1z\u00e1n\u00ed antivirov\u00e9ho \u00fa\u010dinku sta\u010d\u00ed ni\u017e\u0161\u00ed koncentrace. Omezen\u00ed tohoto experimentu spo\u010d\u00edv\u00e1 v tom, \u017ee kdy\u017e se CPC pou\u017eije jako \u00fastn\u00ed voda, p\u0159edpokl\u00e1d\u00e1 se, \u017ee trv\u00e1n\u00ed \u00fa\u010dinku je do minuty. Nicm\u00e9n\u011b s ohledem na slo\u017eitost experiment\u00e1ln\u00ed techniky a mo\u017enost chyb v trv\u00e1n\u00ed \u00fa\u010dinku mezi vzorky byla minim\u00e1ln\u00ed doba p\u016fsoben\u00ed v na\u0161\u00ed studii stanovena na 10 minut. V tomto ohledu Anderson a kol. objasnili \u00fa\u010dinek CPC v dob\u011b p\u016fsoben\u00ed 30 s pomoc\u00ed metody k neutralizaci CPC\u00a0https:\/\/doi.org\/10.1099\/jmm.0.001508<\/a>\u00a0(2022).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 26″>26<\/sup>\u00a0. Je tedy zaru\u010deno vyv\u00edjet nov\u00e9 produkty, jako jsou tablety, kapky a n\u00e1plasti, kter\u00e9 mohou uvol\u0148ovat CPC v bezpe\u010dn\u00e9 koncentraci a mohou b\u00fdt uchov\u00e1ny v \u00fastn\u00ed dutin\u011b co nejd\u00e9le.<\/p>\n

Uk\u00e1zali jsme, \u017ee CPC potla\u010dila infek\u010dnost SARS-CoV-2 ve visk\u00f3zn\u00edch slin\u00e1ch, kter\u00e9 obsahuj\u00ed r\u016fzn\u00e9 proteiny. Zd\u00e1 se v\u0161ak, \u017ee \u00fa\u010dinek je ve slin\u00e1ch ve srovn\u00e1n\u00ed s PBS oslaben. To m\u016f\u017ee b\u00fdt zp\u016fsobeno p\u0159\u00edtomnost\u00ed negativn\u011b nabit\u00fdch protein\u016f slin, sn\u00ed\u017eenou \u00fa\u010dinnost\u00ed dif\u00faze v d\u016fsledku viskozity a tvorbou micel\u00a0https:\/\/doi.org\/10.1016\/j.ejpb.2015.01.007<\/a>\u00a0(2015).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 27″>27<\/sup>\u00a0. Nicm\u00e9n\u011b i n\u00edzk\u00e9 koncentrace CPC prok\u00e1zaly dostate\u010dn\u00e9 potla\u010den\u00ed infek\u010dnosti SARS-CoV-2. Supresivn\u00ed \u00fa\u010dinky n\u00edzk\u00fdch koncentrac\u00ed CPC na infek\u010dnost SARS-CoV-2 ve slin\u00e1ch skute\u010dn\u00fdch pacient\u016f s COVID-19 zb\u00fdv\u00e1 objasnit.<\/p>\n

P\u0159edpokl\u00e1d\u00e1 se, \u017ee mechanismus \u00fa\u010dinku, kter\u00fd potla\u010duje infek\u010dnost SARS-CoV-2, je zp\u016fsoben destrukc\u00ed virov\u00e9ho obalu. Tato studie uk\u00e1zala, \u017ee CPC inaktivuje SARS-CoV-2 bez naru\u0161en\u00ed virov\u00e9 \u010d\u00e1stice p\u0159i koncentraci a trv\u00e1n\u00ed experiment\u016f. Bylo hl\u00e1\u0161eno, \u017ee vysok\u00e1 koncentrace (250\u2013500 \u03bcg\/ml) CPC naru\u0161uje obal SARS-CoV-2\u00a0https:\/\/doi.org\/10.1080\/20002297.2022.2030094<\/a>\u00a0(2022).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 22″>22 ,\u00a0https:\/\/doi.org\/10.3390\/covid1020037<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 28″>28<\/sup>. Stupe\u0148 morfologick\u00e9ho rozpadu z\u00e1visl\u00fd na koncentraci CPC se tedy li\u0161\u00ed. Jin\u00fdmi slovy, \u010d\u00e1stice jsou zma\u010dkan\u00e9, ale ne rozbit\u00e9, v koncentraci, kter\u00e1 je st\u00e1le dostate\u010dn\u00e1 k deaktivaci viru. Tyto v\u00fdsledky jsou v souladu s v\u00fdsledky anal\u00fdzy gradientu hustoty sachar\u00f3zy. Nav\u00edc, z v\u00fd\u0161e uveden\u00fdch d\u016fvod\u016f, antivirov\u00fd mechanismus CPC uk\u00e1zan\u00fd na\u0161\u00ed studi\u00ed podporuje zji\u0161t\u011bn\u00ed, \u017ee CPC interferuje hlavn\u011b s lipidovou\u00a0https:\/\/doi.org\/10.1128\/AAC.00576-20<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 29″>membr\u00e1nou29<\/sup>\u00a0. Obr\u00e1zek\u00a0\u00a04b<\/a>\u00a0ukazuje virov\u00e9 \u010d\u00e1stice r\u016fzn\u00fdch velikost\u00ed. V p\u0159edchoz\u00edch zpr\u00e1v\u00e1ch se pr\u016fm\u011br SARS-CoV-2 m\u011bnil od p\u0159ibli\u017en\u011b 60 do 140 nm, co\u017e je v souladu se sou\u010dasn\u00fdmi v\u00fdsledky\u00a0https:\/\/doi.org\/10.1056\/NEJMoa2001017<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 2″>2 ,\u00a0https:\/\/doi.org\/10.1016\/s0140-6736<\/a>(21)02758-6 (2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Reference 5″>5 ,\u00a0https:\/\/doi.org\/10.1016\/S0140-6736<\/a>(20)31185-5 (2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 30″>30<\/sup>. Podrobn\u00fd mechanismus inaktivace SARS-CoV-2 ni\u017e\u0161\u00ed koncentrac\u00ed CPC je v\u0161ak st\u00e1le nejasn\u00fd. Na\u0161e v\u00fdsledky nazna\u010duj\u00ed, \u017ee na vstupu se m\u016f\u017ee pod\u00edlet denatura\u010dn\u00ed \u00fa\u010dinek proteinu S a zd\u00e1 se, \u017ee hraje kl\u00ed\u010dovou roli.<\/p>\n

P\u0159edpokl\u00e1d\u00e1 se, \u017ee vstup vir\u016f do organismu prob\u00edh\u00e1 p\u0159es dutinu \u00fastn\u00ed a nosn\u00ed\u00a0https:\/\/doi.org\/10.1183\/13993003.01948-2020<\/a>\u00a0(2020).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 31″>dutinu31<\/sup>\u00a0. Aplikac\u00ed nosn\u00edch sprej\u016f obsahuj\u00edc\u00edch CPC a sn\u00ed\u017een\u00edm mno\u017estv\u00ed viru v nosn\u00ed dutin\u011b m\u016f\u017ee v\u00e9st ke kontrole infekce COVID-19. K dne\u0161n\u00edmu dni v\u0161ak neexistuje \u017e\u00e1dn\u00e1 zpr\u00e1va ukazuj\u00edc\u00ed pou\u017eit\u00ed nosn\u00edch sprej\u016f CPC a jejich preventivn\u00ed \u00fa\u010dinek je st\u00e1le nezn\u00e1m\u00fd.<\/p>\n

V sou\u010dasn\u00e9 dob\u011b prov\u00e1d\u00edme klinickou studii zkoumaj\u00edc\u00ed \u00fa\u010dinek CPC u pacient\u016f s COVID-19, kter\u00e1 se zab\u00fdv\u00e1 vlivem CPC na virovou z\u00e1t\u011b\u017e SARS-CoV-2 ve slin\u00e1ch pacient\u016f. N\u00edzk\u00e1 virov\u00e1 z\u00e1t\u011b\u017e ve slin\u00e1ch m\u016f\u017ee m\u00edt za n\u00e1sledek n\u00edzkou rychlost p\u0159enosu a men\u0161\u00ed progresi stavu onemocn\u011bn\u00ed.<\/p>\n

Pou\u017eit\u00ed produkt\u016f obsahuj\u00edc\u00edch CPC m\u016f\u017ee v\u00e9st ke sn\u00ed\u017een\u00ed po\u010dtu nov\u011b infikovan\u00fdch pacient\u016f s covidem. Nav\u00edc m\u016f\u017ee b\u00fdt prost\u0159edkem preventivn\u00edch opat\u0159en\u00ed ve \u0161patn\u011b proo\u010dkovan\u00fdch zem\u00edch. P\u0159edpokl\u00e1d\u00e1me, \u017ee CPC bude pou\u017e\u00edv\u00e1n jako jeden z n\u00e1stroj\u016f k prevenci vzniku a infekce SARS-CoV-2.<\/p>\n<\/div>\n<\/div>\n<\/section>\n

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Materi\u00e1ly a metody<\/h2>\n
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Bun\u011b\u010dn\u00e1 kultura<\/h3>\n

Bu\u0148ky Vero E6 (ATCC, Manassas, VA, USA) byly udr\u017eov\u00e1ny v Dulbeccov\u011b modifikovan\u00e9m Eagleov\u011b m\u00e9diu (DMEM) dopln\u011bn\u00e9m 10% fet\u00e1ln\u00edm bovinn\u00edm s\u00e9rem (FBS) (v\/v) a inkubov\u00e1ny p\u0159i 37 \u00b0C s 5% CO2. Pro tuto studii byly tak\u00e9 pou\u017eity bu\u0148ky Vero E6 stabiln\u011b exprimuj\u00edc\u00ed lidsk\u00e9 TMPRSS2 (VeroE6\/TMPRSS2)\u00a0https:\/\/doi.org\/10.1371\/journal.ppat.1009233<\/a>\u00a0(2021).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 32″>32 .<\/sup><\/p>\n

Viry<\/h3>\n

Kmen SARS-CoV-2 Wuhan (WK-521; EPI_ISL_408667), Alpha (QK002; EPI_ISL_768526), \u200b\u200bBeta (TY8-612; EPI_ISL_1123289) a Gamma (TY7-501; EPI_ISL_8) byly poskytnuty spole\u010dnost\u00ed EPI_ISL_8 \u00dastav infek\u010dn\u00edch nemoc\u00ed, Tokio, Japonsko). Tyto viry byly p\u0159ipraveny pomoc\u00ed bun\u011bk VeroE6\/TMPRSS2. V\u0161echny experimenty s pou\u017eit\u00edm SARS-CoV-2 byly provedeny v za\u0159\u00edzen\u00ed Biosafety Level-3 (BSL-3) Mezin\u00e1rodn\u00edho institutu pro kontrolu zoon\u00f3z (schv\u00e1len\u00e9 \u010d\u00edslo: 19(19), #21002-3), Hokkaido University a dodr\u017eovaly standard provozn\u00ed postupy BSL-3. V\u0161echny experiment\u00e1ln\u00ed n\u00e1vrhy vyu\u017e\u00edvaj\u00edc\u00ed patogeny byly nav\u00edc schv\u00e1leny Graduate School of Dental Medicine, Hokkaido University (schv\u00e1len\u00e9 \u010d\u00edslo: R-2-4-1).<\/p>\n

Reagencie<\/h3>\n

CPC (TCI, Tokio, Japonsko) byl rozpu\u0161t\u011bn v deionizovan\u00e9 destilovan\u00e9 vod\u011b (DDW) a sterilizov\u00e1n filtrem (0,45 um v pr\u016fm\u011bru), (Sartorius, G\u00f6ttingen, N\u011bmecko). Krom\u011b toho byl jako pozitivn\u00ed kontrola pou\u017eit povrchov\u011b aktivn\u00ed \u010dinidlo Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). PBS byl pou\u017eit jako negativn\u00ed kontrola.<\/p>\n

Sliny od zdrav\u00fdch dobrovoln\u00edk\u016f<\/h3>\n

Sliny byly poskytnuty od p\u011bti zdrav\u00fdch neo\u010dkovan\u00fdch dobrovoln\u00edk\u016f. V\u0161echny vzorky slin byly p\u0159ed experimenty stanoveny jako negativn\u00ed na SARS-CoV-2 pomoc\u00ed kvantitativn\u00ed reverzn\u00ed transkrip\u010dn\u00ed polymer\u00e1zov\u00e9 \u0159et\u011bzov\u00e9 reakce (qRT-PCR) a sm\u00edch\u00e1ny v jedn\u00e9 zkumavce. Tento experiment byl schv\u00e1len Institucion\u00e1ln\u00edm etick\u00fdm v\u00fdborem Hokkaidsk\u00e9 univerzity k pou\u017eit\u00ed materi\u00e1l\u016f poch\u00e1zej\u00edc\u00edch z \u010dlov\u011bka. P\u0159ed odb\u011brem slin byl od ka\u017ed\u00e9ho dobrovoln\u00edka z\u00edsk\u00e1n informovan\u00fd souhlas (schv\u00e1len\u00e9 \u010d\u00edslo: 2021-2).<\/p>\n

Test p\u0159e\u017eit\u00ed bun\u011bk<\/h3>\n

Bun\u011b\u010dn\u00e1 \u017eivotaschopnost VeroE6\/TMPRSS2 byla m\u011b\u0159ena testem MTS [3-(4,5-dimethylthylthiazol-2-yl)-5-(3-karboxymethoxyfenyl)-2-(4-sulfofenyl)-2H-tetrazolium] za pou\u017eit\u00ed CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA) v p\u0159\u00edtomnosti r\u016fzn\u00e9 koncentrace CPC (0\u201350 \u00b5g\/ml) po dobu 1 hodiny p\u0159i 37 \u00b0C. Absorbance byla m\u011b\u0159ena pomoc\u00ed GloMax Multiplus Plate Reader\/Luminometer (Promega). T\u0159i nez\u00e1visl\u00e9 experimenty byly provedeny trojmo.<\/p>\n

Plakov\u00fd test<\/h3>\n

Kmeny SARS-CoV-2 byly sm\u00edch\u00e1ny se stejn\u00fdm mno\u017estv\u00edm roztoku CPC (kone\u010dn\u00e1 koncentrace: 0\u201350 \u00b5g\/ml s DMEM obsahuj\u00edc\u00edm 2 % FBS) nebo l\u00e9ka\u0159sk\u00e9ho kloktadla SP-T (Lion Corporation, Tokio, Japonsko), kter\u00e9 bylo na\u0159ed\u011bno. v PBS na koncentraci 50 ug\/ml, podobnou CPC. Sm\u011bs byla inkubov\u00e1na po dobu 30 minut p\u0159i teplot\u011b m\u00edstnosti a z\u0159ed\u011bna na 1\/10 2% FBS DMEM, aby se sn\u00ed\u017eil CPC ve sm\u011bsi. Z\u0159ed\u011bn\u00e1 sm\u011bs byla nao\u010dkov\u00e1na do bun\u011bk VeroE6\/TMPRSS2 a inkubov\u00e1na p\u0159i 37 \u00b0C po dobu 1 hodiny s rotac\u00ed. Po inkubaci byly bu\u0148ky dvakr\u00e1t promyty 1 x PBS, aby se odstranil CPC, a pot\u00e9 byly p\u0159ekryty 2% FBS DMEM obsahuj\u00edc\u00edm 1,2% Bacto Agar (Becton Dickinson, Franklin Lakes, NJ, USA). Po 48 hodin\u00e1ch inkubace p\u0159i 37 \u00b0C byly bu\u0148ky p\u0159es noc fixov\u00e1ny 3,7% pufrovan\u00fdm formaldehydem. Fixovan\u00e9 bu\u0148ky byly obarveny 1% krystalovou violet\u00ed.<\/p>\n

Test vstupu viru<\/h3>\n

Bu\u0148ky VeroE6\/TMPRSS2 byly nasazeny na 24jamkov\u00e9 desti\u010dky v hustot\u011b 1,0 x 105 bun\u011bk\/jamku\u00a0.\u00a0<\/sup>Kmen SARS-CoV-2 Wuhan byl sm\u00edch\u00e1n se stejn\u00fdm mno\u017estv\u00edm CPC (kone\u010dn\u00e1 koncentrace: 0\u201325 \u03bcg\/ml). P\u0159i ka\u017ed\u00e9 koncentraci l\u00e9\u010diva byly jamky infikov\u00e1ny 1,0 x\u00a0103<\/sup>PFU (MOI = 0,01) viru. Sm\u011bsi byly inkubov\u00e1ny po dobu 30 minut p\u0159i teplot\u011b m\u00edstnosti. Po inkubaci byly sm\u011bsi nao\u010dkov\u00e1ny do bun\u011bk VeroE6\/TMPRSS2 a inkubov\u00e1ny p\u0159i 37 \u00b0C po dobu 1 hodiny s rotac\u00ed. Po 1 hodin\u011b absorpce byly bu\u0148ky dvakr\u00e1t promyty 1 x PBS k odstran\u011bn\u00ed CPC a kultivov\u00e1ny v udr\u017eovac\u00edm m\u00e9diu. 24 hodin po infekci (hpi) byly celkov\u00e9 RNA extrahov\u00e1ny z nao\u010dkovan\u00fdch bun\u011bk pomoc\u00ed TRIzol\u2122 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) a celkov\u00e1 RNA byla extrahov\u00e1na pomoc\u00ed RNeasy Mini Kit (QIAGEN, Hilden, N\u011bmecko). Extrahovan\u00e9 RNA byly podrobeny anal\u00fdze qRT-PCR pomoc\u00ed soupravy THUNDERBIRD Probe One-step qRT-PCR Kit (TOYOBO, Osaka, Japonsko). Genom SARS-CoV-2 byl kvantifikov\u00e1n pomoc\u00ed sad primerov\u00fdch sond pro N2 (Takara, Shiga, Japonsko). Jako endogenn\u00ed kontrola byl pou\u017eit \u03b2-aktin prim\u00e1t\u016f jin\u00e9ho ne\u017e lidsk\u00e9ho p\u016fvodu.https:\/\/doi.org\/10.1016\/j.cyto.2005.07.002<\/a>\u00a0(2005).“ data-track=“click“ data-track-action=“reference anchor“ data-track-label=“link“ data-test=“citation-ref“ aria-label=“Odkaz 33″>33<\/sup>\u00a0. Hladiny N genu SARS-CoV-2 byly normalizov\u00e1ny s hladinami mRNA p-\u00a0aktinu34<\/a><\/sup>\u00a0. Krom\u011b toho byly hladiny virov\u00e9 RNA p\u0159i 24 hpi normalizov\u00e1ny s hladinami virov\u00e9 RNA p\u0159i 0 hpi. V\u0161echny testy RT-PCR byly provedeny pomoc\u00ed syst\u00e9mu CFX96 Real-Time PCR System (BioRad, Hercules, CA, USA). T\u0159i nez\u00e1visl\u00e9 experimenty byly provedeny trojmo.<\/p>\n

Anal\u00fdza virucidn\u00ed aktivity CPC proti SARS-CoV-2 ve slin\u00e1ch<\/h3>\n

Kmen SARS-CoV-2 Wuhan byl p\u0159id\u00e1n do slin odebran\u00fdch od zdrav\u00e9ho dobrovoln\u00edka a sm\u00edch\u00e1n se stejn\u00fdm mno\u017estv\u00edm CPC (kone\u010dn\u00e1 koncentrace: 0\u201340 \u03bcg\/ml). Sm\u011bsi slin byly z\u0159ed\u011bny na 1\/100 pro sn\u00ed\u017een\u00ed viskozity a p\u0159efiltrov\u00e1ny p\u0159es 0,45 \u03bcm filtry (Sartorius) k odstran\u011bn\u00ed bakteri\u00ed a pl\u00edsn\u00ed. Plakov\u00fd test byl proveden tak, jak bylo pops\u00e1no d\u0159\u00edve (\u010d\u00e1st \u201e\u00a0Plakov\u00fd test<\/a>\u00a0\u201c). T\u0159i nez\u00e1visl\u00e9 experimenty byly provedeny trojmo.<\/p>\n

Anal\u00fdza gradientu hustoty sachar\u00f3zy<\/h3>\n

Kmen SARS-CoV-2 Wuhan byl o\u0161et\u0159en CPC (50 \u03bcg\/ml) nebo Triton X-100 (1 %) p\u0159i pokojov\u00e9 teplot\u011b po dobu 10 minut. Po inkubaci byly sm\u011bsi naneseny na 10%\u201350% gradienty hustoty sachar\u00f3zy. Po ultracentrifugaci s Optima XE-90 (Beckman Coulter, Brea, CA, USA) po dobu 6 hodin p\u0159i 250 000 \u00d7\u00a0g<\/i>, ka\u017ed\u00fdch 100 \u03bcl gradient\u016f bylo frakcionov\u00e1no do 22 frakc\u00ed a sm\u00edch\u00e1no se 100 \u03bcl vzorkov\u00e9ho pufru SDS-PAGE a va\u0159eno p\u0159i 95 \u00b0C po dobu 5 minut. Po uva\u0159en\u00ed byly analyzov\u00e1ny pomoc\u00ed 10% SDS-PAGE s n\u00e1slednou imunoblotovou anal\u00fdzou s pou\u017eit\u00edm my\u0161\u00ed monoklon\u00e1ln\u00ed protil\u00e1tky proti SARS-CoV-2 S proteinu (GTX632604) nebo kr\u00e1li\u010d\u00edho polyklon\u00e1ln\u00edho N proteinu (GTX135357) (GeneTex, Irvine, CA, USA). Membr\u00e1ny byly po p\u0159enosu na\u0159\u00edznuty p\u0159i 100 kDa a hybridizov\u00e1ny s protil\u00e1tkou proti S proteinu, respektive s protil\u00e1tkou proti N proteinu a podrobeny vizualizaci. Pro zobrazov\u00e1n\u00ed byl pou\u017eit ImageQuant LAS 4000 mini (FUJIFILM Corporation, Tokio, Japonsko) (obr.\u00a0S4<\/a>\u00a0).<\/p>\n

Transmisn\u00ed elektronov\u00e1 mikroskopie<\/h3>\n

Kmen SARS-CoV-2 Wuhan byl o\u0161et\u0159en 1 x PBS, CPC (10, 50 a 250 ug\/ml) a Triton X-100 (1 %) po dobu 10 minut p\u0159i teplot\u011b m\u00edstnosti. Sm\u011bsi byly fixov\u00e1ny 2,5% glutaraldehydem p\u0159i 4 \u00b0C po dobu 24 hodin. Fixovan\u00fd vzorek (5 \u03bcl) byl um\u00edst\u011bn na list Parafilmu. Formvar Film (#10-1009, Okenshoji, Tokio, Japonsko) byl um\u00edst\u011bn na ka\u017edou kapku, aby adsorboval virus po dobu 5 minut. M\u0159\u00ed\u017eky byly promyty DDW a um\u00edst\u011bny na kapku filtrovan\u00e9ho 2,0% roztoku octanu uranylu na dal\u0161\u00ed 1 minutu, vysu\u0161eny na vzduchu a vy\u0161et\u0159eny pomoc\u00ed JEM-1400 TEM (JEOL, Tokyo, Japonsko) p\u0159i 80 kV.<\/p>\n

Statistika<\/h3>\n

Statistick\u00e9 anal\u00fdzy byly provedeny pomoc\u00ed Graphpad Prism v9 (GraphPad Software Inc., San Diego, CA, USA). Data jsou prezentov\u00e1na jako pr\u016fm\u011brn\u00e9 hodnoty \u00b1 SD biologick\u00fdch triplik\u00e1t\u016f. Statistick\u00e1 anal\u00fdza byla provedena pomoc\u00ed jednosm\u011brn\u00e9 anal\u00fdzy rozptylu. Pro v\u0161echny soubory dat byla za v\u00fdznamnou pova\u017eov\u00e1na hodnota\u00a0p men\u0161\u00ed ne\u017e 0,05.<\/i><\/p>\n

Institucion\u00e1ln\u00ed revizn\u00ed komise<\/h3>\n

Studie byla provedena v souladu s Helsinskou deklarac\u00ed a schv\u00e1lena Institucion\u00e1ln\u00edm etick\u00fdm v\u00fdborem Univerzity Hokkaido (schv\u00e1len\u00e9 \u010d\u00edslo: 2021\u20132).<\/p>\n

Informovan\u00fd souhlas<\/h3>\n

Informovan\u00fd souhlas byl z\u00edsk\u00e1n od v\u0161ech subjekt\u016f zapojen\u00fdch do studie.<\/p>\n<\/div>\n<\/div>\n<\/section>\n<\/div>\n

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Dostupnost dat<\/h2>\n
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Soubory dat vytvo\u0159en\u00e9 b\u011bhem a\/nebo analyzovan\u00e9 b\u011bhem aktu\u00e1ln\u00ed studie jsou k dispozici od odpov\u00eddaj\u00edc\u00edho autora na p\u0159im\u011b\u0159enou \u017e\u00e1dost.<\/p>\n<\/div>\n<\/div>\n<\/section>\n<\/div>\n

ZDROJ<\/a><\/p>\n

 <\/p>\n","protected":false},"excerpt":{"rendered":"

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