Turhaa hösläystä: ei ole luettu perusartikkelia

Risto Koivula, 6/13/2021 6:43:06 AM, 405524

Lainaus: Teppolainen, 12.06.2021 23:16:42, 405522


https://www.sciencedirect.com/science/article/pii/S1873506120304165

Stem Cell Research

Volume 50, January 2021, 102115
Stem Cell Research

Furin cleavage sites naturally occur in coronaviruses

Author links open overlay panel
Yiran Wu, a
Suwen Zhao, ab

a
iHuman Institute, ShanghaiTech University, Shanghai 201210, China

b
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China

Received 30 September 2020, Revised 2 December 2020, Accepted 4 December 2020, Available online 9 December 2020.

Abstract

The spike protein is a focused target of COVID-19, a pandemic caused by SARS-CoV-2. A 12-nt insertion at S1/S2 in the spike coding sequence yields a furin cleavage site, which raised controversy views on origin of the virus. Here we analyzed the phylogenetic relationships of coronavirus spike proteins and mapped furin recognition motif on the tree. Furin cleavage sites occurred independently for multiple times in the evolution of the coronavirus family, supporting the natural occurring hypothesis of SARS-CoV-2.



1. Introduction

The coronavirus disease 2019 (COVID-19) pandemic, caused by coronavirus SARS-CoV-2, is still rapidly spreading. Scientists all around the world are studying the infection process of the virus and looking for therapeutic solutions to prevent and cure the disease. Spike protein is one of most important targets in COVID-19 research, not only in mechanism study but also in vaccine development and therapeutic antibody design (Premkumar et al., 2020; Abraham, 2020).

The spike protein forms a homotrimer (Fig. 1A) and protrudes outside the membrane of the virion (Li, 2016; Wrapp et al., 2020; Walls et al., 2020). It binds to angiotensin-converting enzyme 2 (ACE2) on human cell sur-face, undergoes conformational changes, and mediates the fusion of the virion to human cells (Li et al., 2003; Yan et al., 2020; Lan et al., 2020; Wang et al., 2020; Benton et al., 2020).

The spike protein consists of two subunits: the N-terminal S1 subunit, containing the receptor binding domain that interacts with ACE2 and induces conformational change; while the C-terminal S2 subunit, containing the fusion peptide that is responsible for fusing the membranes of virion and human cells (Li, 2016). To function properly, the spike protein needs to be cleaved by host proteases to separate the two sub-units (Simmons et al., 2013).

Study of SARS-CoV (caused the severe acute respiratory syndrome in 2002–2003 and belonged to the same species as SARS-CoV-2) showed that cleavage at S1/S2 enhances fuso-genicity of spike protein (Bosch et al., 2003).

Another cleavage site producing S2′(fusion peptide and the C-terminal region of S2) was also identified (Fig. 1B) (Belouzard et al., 2009). The S2′ site is not far from the S1/S2 site, and cleavage either or both them can yield the separation of two subunits of spike. For coronaviruses, multiple proteases were reported responsible for the cleavage, including TMPRSS2 (Hoffmann et al., 2020; Glowacka et al., 2011); cathepsin CTSL (Bosch et al., 2008; Ou et al., 2020), and trypsin (Belouzard et al., 2009; Bertram et al., 2011). The spike protein of the Middle East respiratory syndrome corona-virus (MERS-CoV) was discovered to be more effectively cleaved by another protease, furin (Millet and Whittaker, 2014). In MERS-CoV, both S1/S2 and S2′sites have sequence RXXR, the furin recognition motif.

In human body, furin is ubiquitously expressed (Braun and Sauter, 2019). This may explain the highly lethal nature and high rate of multiple system failure caused by MERS (Millet and Whittaker, 2014). Surprisingly, SARS-CoV-2 has the furin recognition motif at S1/S2, causing by a 12-nucleotide insertion not presented even in its closest relatives (Walls et al., 2020; Coutard et al., 2020).

This stimulates a conspiracy that this furin site can only be manual work, thus SARS-CoV- 2 must be created in a laboratory.

Here, we analyzed the sequences of coronaviruses and found furin sites occurred independently for multiple times during evolution.

This exhibits natural occurrence of furin cleavage site in SARS-CoV-2 spike protein is highly possible.

Thus, the insertion of furin cleavage site into SARS-CoV-2 spike protein is not necessarily a result of manual work.


2. Results 2.1.

Phylogenetic tree of spike protein sequences reveals major groups of coronaviruses Coronaviruses are members of the subfamily Orthocoronavinae (also named Coronavinae) in the family Coronavidae.

There are four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacor-onavirus.

The first two genera only infect mammals, while the last two primarily infect birds but also mammals (Cui et al., 2019).

We collected sequences of coronavirus spike proteins from the InterPro database (Mitchell et al., 2019) and performed phylogenetic analysis. The phylogenetic tree (Fig. 2A) generally matches reported relationship of coronaviruses (Cui et al., 2019): three genera (Betacoronavirus; Gam-macoronavirus, and Deltacoronavirus) each forms a single clade, while only Alphacoronavirus has a small group left outside (see Fig. 5 for representative sequences). The left-out Alphacoronavirus group contains Rhinolophus bat coronavirus HKU2 that was reported to be closely related to two typical Alphacoronavirus, human coronavirus NL63 and human coronavirus 229E, in a phylogenetic analysis based on the RNA- dependent RNA polymerase coding region (Cui et al., 2019).

We aligned the whole genomes of the three viruses and found Rhinolophus bat coronavirus HKU2 is quite unique in its spike protein-coding region (Fig. S1). Such result explains why this clade was separated from other Alphacoronavirus in our phylogenetic analysis. Furthermore, the five subgenera of Betacoronavirus were nicely displayed as monophyletic (Fig. 2B): Sarbecovirus (e.g. SARS-CoV-2 and SARS-CoV), Merbecovirus (e.g. MERS-CoV), Embecovirus (e.g. human coronavirus OC43 and human coronavirus HKU1, both causing common cold), and two small subgenera Hibecovirus and Nobecovirus. Hibecovirus and Nobecovirus are only discovered in bats so far, and our phylogenetic analysis shows they are related to Sarbecovirus.

2.2. Furin cleavage site at spike S1/S2 also occurs in a virus close to Sarbecovirus We mapped the furin recognition motif RXXR at both S1/S2 and S2′positions in phylogenetic trees of spike protein sequences (Figs. 3–5 and S2, S3).

In the Sarbecovirus +Hibecovirus +Nobecovirus clade (Fig. 3), furin cleavage sites at either position occur only in limited ranges. Strains of SARS-CoV-2 (we also added sequences from the GISAID database) have furin cleavage sites at spike S1/S2. Moreover, SARS-CoV- 2 is the only virus in subgenus Sarbecovirus having this feature, while even its closest relatives, bat coronavirus RaTG13 (sequence identity 97.7%) and pangolin coronaviruses (92.9%–90.7%), do not have furin site. However, in Hibecovirus, the sister clade of Sarbecovirus, a Hippo-sideros bat coronavirus collected in 2013 at Zhejiang Province in China has furin site at S1/S2. Interestingly, the other member in Hibecovirus lacks such site, similar to the situation of SARS-CoV-2 and its close rel-atives. The SARS-CoV-2 strains and Hipposideros bat coronavirus (Zhe-jiang 2013) are not sister groups, in agreement with the distinct sequence patterns of their furin cleavage sites at spike S1/S2 (Fig. 6A). Besides, two strains of bat coronavirus HKU9, belonging to Nobecovirus, are the only members in the Sarbecovirus +Hibecovirus +Nobecovirus clade having furin cleavage site at spike S2′.

2.3.Furin cleavage sites are common in Betacoronavirus Our mapping results showed that the furin recognition motif is more common in Merbecovirus and Embecovirus (Figs. 4 and S2, S3).

In Merbecovirus, furin sites at spike S1/S2 occur in three clades: MERS-CoV strains, the bat coronavirus HKU5 strains, and coronavirus Neoromicia/PML-PHE1/RSA/2011 with its relatives (Figs. 4A and S2). Besides, MERS-CoV and bat coronavirus HKU5 are the only clades in Merbecovirus having furin cleavage site at S2′. In Embecovirus, furin recognition motif at spike S1/S2 is universal: All strains but a few exceptions have furin cleavage sites at spike S1/S2 (Figs. 4B and S3). Interestingly, the Longquan Aa mouse coronavirus (Wang et al., 2015) loses this furin site, while its close relatives (e.g. China Rattus coronavirus HKU24, sequence identity 96.0%) maintains the furin cleavage site. This provides an example of naturally occurred sequence variation at spike S1/S2 among closely related coronaviruses. Besides, for spike S2′, only several single strains have furin recognition motif (Fig. S3).

2.4.Furin cleavage sites also occur in other genera of coronavirus

Our mapping results showed furin cleavage sites are widely present in the whole coronavirus family (Fig. 5). For spike S1/S2, furin recog-nition motif is universal in Gammacoronavirus, and also occurs in two clades of Alphacorovanvirus: feline coronavirus and relatives, and


Chevrier’s field mouse coronavirus. For spike S2′, furin recognition motif occurs in several independent clades, covering all the three genera. Notably, in the two human coronaviruses in Alphacoronavirus causing common cold, HCoV NL63 has furin cleavage site at spike S2′, while the HCoV 229E (protein sequence identity 63.8%) lacks such feature.

2.5. Furin cleavages sites occurred independently for six times in Betacoronavirus

The alignment of linking regions of spike S1 and S2 domains in representative Betacoronavirus (Fig. 6A) shows this region is less conserved than the neighboring folded S1 and S2 segments. Within a subgenus the sequences are well aligned, but among subgenera the similarity is low. The furin cleavage site of SARS-CoV-2 spike S1/S2 is formed by a insertion of PRRA in comparison to other Sarbecovirus including close relative RaTG13, showing it occurred very recently and independently. Similarly, Hipposideros bat coronavirus (Zhejiang 2013) in Hibecovirus has furin site of independent origin, though the occurring time is hard to decide for in this subgenera only two sequences were published. Merbecovirus and Embecovirus both have multiple coronavirus species with furin cleavage sites at spike S1/S2, but their situations are different: In Merbecovirus, furin cleavage sites prevail in three non-sister clades (Figs. 4A and 6A).

Moreover, the positions of furin recognition motifs in the linking regions are unique to each clade, as exhibited in alignments of both protein sequences (Fig. 6A) and nucleotide sequences (Fig. S4A). These indicated for of the three clades in Merbecovirus, furin cleavage sites have an independent origin. In Embecovirus, to the contrast, all the furin cleavage sites are variations based on a 5-residue region with consensus sequence RRXRR. The region is well aligned in both protein and nucleotide sequences (Figs. 6A and S4B). This suggested the furin cleavage sites of Embecovirus share a common ancestor. In addition, in Alphacoronavirus and Gammacoronavirus, S1/S2 cleavage sites reside at a different loop comparing to the site in Beta-coronavirus (Fig. 6B), therefore furin cleavage sites at spike S1/S2 in these two genera occurred independently from those in Betacoronavirus in evolution.

3. Discussion Furin cleavage is critical to many viral diseases, including HIV, Ebola, and influenza H5 and H7 (Becker et al., 2012). Furin is a ubiq-uitously expressed protease. In human body, it has a wider distribution range than the major protease responsible for cleaving spike, TMPRSS2 (Fig. S5). Therefore, coronaviruses with spike containing furin cleavage site may have advantage in spreading. Deletions of furin cleavage site in SARS-CoV-2 attenuates replication on respiratory cells (Johnson et al., 2020) and pathogenesis in hamster (Johnson et al., 2020; Lau et al., 2020). Furin inhibitors suppress virus production and cytopathic effects in kidney cells (Cheng et al., 2020). Natural polymorphisms losing furin recognition motif in SARS-CoV-2 spike S1/S2 are observed, but very rare (Xing et al., 2020). Variations in this region are more common in viruses cultured in vitro than viruses isolated from clinical samples, suggesting this cleavage site is under selection pressure in human body (Lau et al., 2020; Liu et al., 2020). Our analysis exhibits furin cleavage sites at spike S1/S2 occurred independently for several times in coronavirus. Consequently, natural occurring of the site in SARS-CoV-2 is highly possible. This is further supported by other observed natural variations at the linking region of S1 and S2: A natural insertion in SARS-CoV spike though not related to furin recognition motif was reported (Zhou et al., 2020). In Embecovirus; Longquan Aa mouse coronavirus (Wang et al., 2015) has a frameshift mutation led to the loss of furin recognition motif (Fig. S4B); Some strains of murine coronavirus lose furin recognition motif through substitution mutations (Fig. S3), e.g. in MHV-2 (Yamada et al., 1997) urther study of losing the furin cleavage site in Embecovirus would help to interpret the S1/S2 cleavage of Betacoronaviruses. Besides, indepen-dent occurrences of furin cleavage sites in surface glycoproteins are not unique to coronavirus: for the hemagglutinin of influenza, only H5 and H7 have furin cleavage sites (Bottcher-Friebertshauser et al., 2013); and these subtypes are distant in phylogenetic tree (Fig. S6). 4. Conclusion Furin cleavage sites in spike proteins naturally occurred indepen-dently for multiple times in coronaviruses. Such feature of SARS-CoV-2 spike protein is not necessarily a product of manual intervention, though our observation does not rule out the lab-engineered scenario.



Fig. 2.Phylogenetic tree of coronavirus spike protein sequences. A) Noting genera of coronavirus. B) Subtree of Betacoronavirus, noting subgenera. Y. Wu and S. Zhao



Stem Cell Research 50 (2021) 1021156Fig. 5.Mapping of furin recognition motif on phylogenetic tree of spike protein sequences, the Alphacoronavirus +Gammacoronavirus +Deltacoronavirus clade. Sequences clustered with 95% identity threshold. Y. Wu and S. Zhao




Stem Cell Research 50 (2021) 1021154Fig. 3.

Mapping of furin recognition motif on phylogenetic tree of spike protein sequences, the Sarbecovirus +Hibecovirus +Nobecovirus clade. Sequences in the SARS-CoV-2 clade were clustered with 95% identity threshold; in the SARS-CoV-2 clade were clustered with 99% identity threshold. Y. Wu and S. Zhao

Furiiniryhmän esiintyminen elukoiden koronaviruksissa.

Muokannut: Risto Koivula, 6/13/2021 7:48:33 AM


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                    ERKON HÖRHÖLÄ BIDENIN SUUHUN: "KORONAN EI OLE HAVAITTU VOIVAN TARTTUA ELÄIMILTÄ IHMISILLE"...   [ 405382 , Risto Koivula , 28.05.2021 07:28:01 ]
                        ERKON HÖRHÖLÄ BIDENIN SUUHUN: "KORONAN EI OLE HAVAITTU VOIVAN TARTTUA ELÄIMILTÄ IHMISILLE"...   [ 405384 , PeP , 28.05.2021 09:55:24 ]
                            ERKON HÖRHÖLÄ BIDENIN SUUHUN: "KORONAN EI OLE HAVAITTU VOIVAN TARTTUA ELÄIMILTÄ IHMISILLE"...   [ 405385 , Risto Koivula , 28.05.2021 11:07:45 ]
                                Ainakin koronaviruksen lähimmät sukulaiset lepakoilla tarttuvat ihmisiinkin.   [ 405386 , Risto Koivula , 28.05.2021 11:52:23 ]
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                    Bidenin USA ja Kiina vastuussa WHO:n korruptiosta   [ 405530 , _Wesa_ , 13.06.2021 13:04:09 ]
        Viruksen laboratorioalkuperää pidetään tiedepiireissä epätodennäköisenä   [ 406924 , Otto Mäkelä , 15.09.2021 17:16:50 ]
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            Turhaa hösläystä: ei ole luettu perusartikkelia   [ 405642 , Risto Koivula , 26.06.2021 09:16:04 ]
        USA:n senaatin tiedotustilaisuus kertoo Big Techin sensuurista ja koronaviruksen alkuperästä   [ 405531 , _Wesa_ , 13.06.2021 13:28:35 ]
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                                                            Tautia tuodaan taas bussilasteittain ulkomailta   [ 405661 , _Wesa_ , 27.06.2021 10:32:51 ]
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                                                                            Edes tartuttajien henkilöllisyyksiä ei rajalla tarkistettu ja myös THL panttaa tietoja - Enqvist varoittaa fasismista   [ 405703 , Teppolainen , 28.06.2021 11:11:40 ]
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                                                                                                        Poliisin totalitaristinen toiminta   [ 405725 , Teppolainen , 29.06.2021 18:43:33 ]
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                                                                                                                Poliisin ohjeet ylemmältä taholta myös näitä kahta koskien   [ 405734 , Teppolainen , 30.06.2021 12:50:35 ]
                                                                                                                    Aloituksessasi kysyit juuri näistä kahdesta henkilöstä   [ 405737 , Trepidaatio , 30.06.2021 16:22:30 ]
                                                                                                                        Aloituksessasi kysyit juuri näistä kahdesta henkilöstä   [ 405739 , Teppolainen , 30.06.2021 16:50:08 ]
                                                                                                                            Aloituksessasi kysyit juuri näistä kahdesta henkilöstä   [ 405740 , Trepidaatio , 30.06.2021 16:55:55 ]
                                                                                                                                Laki sallii pakkotestit, mutta ei kuitenkaan salli?   [ 405742 , Teppolainen , 30.06.2021 23:45:37 ]
                                                                                Edes tartuttajien henkilöllisyyksiä ei rajalla tarkistettu ja myös THL panttaa tietoja - Enqvist varoittaa fasismista   [ 405713 , _Wesa_ , 29.06.2021 14:18:35 ]
                                                                                    Edes tartuttajien henkilöllisyyksiä ei rajalla tarkistettu ja myös THL panttaa tietoja - Enqvist varoittaa fasismista   [ 405715 , Teppolainen , 29.06.2021 14:27:27 ]
                                                                                        Edes tartuttajien henkilöllisyyksiä ei rajalla tarkistettu ja myös THL panttaa tietoja - Enqvist varoittaa fasismista   [ 405729 , _Wesa_ , 29.06.2021 20:36:53 ]
                                                                                Enqvist varoittaa (hyvin) fasismista - mutta ei huomaa itse olevansa NATSI "eurotieteen" "rotumurhapierupeilineurooneineen"...   [ 405735 , Risto Koivula , 30.06.2021 14:49:08 ]
                                                                            Tautia tuodaan taas bussilasteittain ulkomailta   [ 405712 , _Wesa_ , 29.06.2021 14:16:26 ]
                                                                                Tautia tuodaan taas bussilasteittain ulkomailta   [ 405716 , Teppolainen , 29.06.2021 14:33:09 ]
                                                                                    Tautia tuodaan taas bussilasteittain ulkomailta   [ 405728 , _Wesa_ , 29.06.2021 20:28:42 ]
        Totalitaristista hallitusta ei kansanterveys oikeasti kiinnosta   [ 405774 , Teppolainen , 02.07.2021 13:00:56 ]
            Toisen postauksesi linkistä.   [ 405776 , Trepidaatio , 02.07.2021 13:04:45 ]
                Eduskunta ei ole palauttanut lakia voimaan   [ 405777 , Teppolainen , 02.07.2021 13:23:21 ]
                    Toisen postauksesi linkistä:   [ 405778 , Trepidaatio , 02.07.2021 13:31:59 ]
                        Edelleen oleellinen unohtuu   [ 405779 , Teppolainen , 02.07.2021 13:52:43 ]
                            Edelleen oleellinen unohtuu   [ 405781 , Trepidaatio , 02.07.2021 14:12:56 ]
                                Edelleen oleellinen unohtuu   [ 405783 , Teppolainen , 02.07.2021 14:21:04 ]
                                    Virheet korjataan   [ 405784 , Trepidaatio , 02.07.2021 14:31:25 ]
                                        Virheet ja "virheet" korjataan   [ 405787 , Teppolainen , 02.07.2021 14:51:25 ]
            Totalitaristista hallitusta ei kiinnosta maksaa edes terveydenhoidosta vastaavien palkkoja   [ 405943 , Teppolainen , 12.07.2021 20:38:58 ]
                Totalitaristista hallitusta ei kiinnosta maksaa edes terveydenhoidosta vastaavien palkkoja   [ 405947 , _Wesa_ , 12.07.2021 23:27:11 ]
                    Sillä välin Ranska vajoaa totalitarismiin   [ 405949 , Teppolainen , 13.07.2021 01:32:12 ]
                        Sillä välin Ranska vajoaa totalitarismiin   [ 405950 , _Wesa_ , 13.07.2021 02:22:32 ]
                            Sillä välin Ranska vajoaa totalitarismiin   [ 405951 , Teppolainen , 13.07.2021 04:00:41 ]
                                Sillä välin Ranska vajoaa totalitarismiin   [ 405957 , _Wesa_ , 13.07.2021 16:00:05 ]
                                    Sillä välin Ranska vajoaa totalitarismiin   [ 405962 , Teppolainen , 13.07.2021 22:18:40 ]
                                        Sillä välin Ranska vajoaa totalitarismiin   [ 405965 , _Wesa_ , 14.07.2021 11:08:25 ]
                                            Sillä välin Ranska vajoaa totalitarismiin   [ 405970 , Teppolainen , 14.07.2021 15:39:22 ]
                                                Sillä välin Ranska vajoaa totalitarismiin   [ 405975 , _Wesa_ , 15.07.2021 12:24:37 ]
                                                    Sillä välin Ranska vajoaa totalitarismiin   [ 405976 , Teppolainen , 15.07.2021 12:58:14 ]
                                                        Sillä välin Ranska vajoaa totalitarismiin   [ 405977 , _Wesa_ , 15.07.2021 13:11:49 ]
                                                            Sillä välin Ranska vajoaa totalitarismiin   [ 405979 , Teppolainen , 15.07.2021 13:51:42 ]
                                                                Sillä välin Ranska vajoaa totalitarismiin   [ 405983 , _Wesa_ , 15.07.2021 22:05:50 ]
                                                                    Sillä välin Ranska vajoaa totalitarismiin   [ 405985 , Teppolainen , 15.07.2021 22:42:08 ]
                                                                        WHO:n toiminta paljastaa, että kyse ei ole koronasta   [ 405986 , Teppolainen , 16.07.2021 11:04:50 ]
                                                                            Taistelu syrjintää vastaan on alkanut   [ 405989 , Teppolainen , 16.07.2021 16:31:46 ]
                                                                                Ja Yle levittää taas totalitaristisen hallituksen valheita   [ 405990 , Teppolainen , 16.07.2021 17:06:02 ]
                                                                                    Ja Yle levittää taas totalitaristisen hallituksen valheita   [ 406001 , _Wesa_ , 18.07.2021 13:27:49 ]
                                                                                        Nyt jo Ylekin jälleen myöntää valheellisen hysterian   [ 406027 , Teppolainen , 22.07.2021 11:28:55 ]
                                                                                            Nyt jo Ylekin jälleen myöntää valheellisen hysterian   [ 406029 , _Wesa_ , 22.07.2021 12:33:48 ]
                                                                        Sillä välin Ranska vajoaa totalitarismiin   [ 406002 , _Wesa_ , 18.07.2021 13:40:56 ]
                                                                            Demokratian palauttaminen   [ 406015 , Teppolainen , 20.07.2021 10:23:57 ]
                                                                                Demokratian palauttaminen   [ 406016 , _Wesa_ , 20.07.2021 11:17:07 ]
                                                                                    Demokratian palauttaminen   [ 406021 , Teppolainen , 20.07.2021 20:36:47 ]
                                                                                        Demokratian palauttaminen   [ 406022 , _Wesa_ , 21.07.2021 14:49:15 ]
                                                                                            Demokratian palauttaminen   [ 406023 , Teppolainen , 21.07.2021 15:25:50 ]
                                                                                                Demokratian palauttaminen   [ 406024 , _Wesa_ , 21.07.2021 16:11:13 ]
                                                                                                    Demokratian palauttaminen   [ 406025 , Teppolainen , 21.07.2021 16:19:28 ]
                                                                                                        Demokratian palauttaminen   [ 406026 , _Wesa_ , 21.07.2021 16:59:02 ]
                                                                                                            Italia seuraa fasismin jaloa perinnettä - ja Suomi tulossa perässä   [ 406974 , Teppolainen , 17.09.2021 15:06:39 ]
                                                                                                                Jutussa kerrotaan oikeasti...   [ 406976 , Trepidaatio , 17.09.2021 15:20:56 ]
                                                                                                                    Jutussa kerrotaan oikeasti...   [ 406977 , Teppolainen , 17.09.2021 15:49:32 ]
                                                                                                                        Jutussa kerrotaan oikeasti...   [ 406981 , _Wesa_ , 18.09.2021 20:26:45 ]
                                                                                                                            Jutussa kerrotaan oikeasti...   [ 406982 , Teppolainen , 18.09.2021 23:08:46 ]
                                                                                                                                Rokotuspassi on rokotuspassi on syrjintää - naishuijarit taas vedättämässä suomalaisia   [ 406989 , Teppolainen , 20.09.2021 20:25:40 ]
                                                                                                                                    Yle valehtelee jälleen   [ 406995 , Teppolainen , 21.09.2021 15:03:28 ]
                                                                                                                                        Marinin hörhöoppien mukaan pizzansyönnin rajoittamisella lisätään rokotusten määrää   [ 406996 , Teppolainen , 21.09.2021 17:38:46 ]
                                                                                                        Hallituksen riveistä sataa tunnustuksia Suomen vaalivilpistä   [ 406990 , Teppolainen , 20.09.2021 20:44:36 ]