Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations1. Using the SARS-CoV reverse genetics system2, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV.
Ricordiamo che si parla del 2015.
Ok, hanno creato in laboratorio un virus-chimera con gli spikes di un particolare strain del coronavirus dei pipistrelli utilizzando un punto di partenza della SARS adattato ai topi, per ovvie esigenze di laboratorio..
Il risultato è che il risultante virus è in grado di attaccare le cellule delle vie aeree umane e hanno ottenuto IN VITRO qualcosa di equivalente a degli strains epidemici della SARS.
Fin qui nulla di particolare. Posso tranquillamente credere alla ricerca medica preventiva di una possibile epidemia.
Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis.
Il virus non solo si è dimostrato in grado di attaccare gli umani ma anche i topi.
Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein.
Successivamente hanno cercato di curare i topi con terapie precedentemente usate per la SARS e i risultati sono stati negativi, il coronavirus del pipistrello una volta modificato non è più curabile con i disponibili (allora) medicinali usabili per la SARS.
Ok, al momento sembra che i ricercatori stessero dicendo "Signori, se il coronavirus che abbiamo usato (ce ne sono molti, in questo caso era il SHC014-CoV) si combina con la struttura della SARS, attacca tanto i topi come gli uomini (così come tutti coloro con un ACE2 equivalente) e non è più attaccabile dai normali medicinali".
Questo mi avrebbe preoccupato non poco, onestamente. La conclusione loro è "Signori, come l'abbiamo fatto in laboratorio non possiamo escludere succeda in natura".
Segue poi il capitolo MAIN, sempre nell'articolo, che è interessantissimo e finalmente spiega cosa stessero cercando di fare in quegli esperimenti.
Il capitolo è molto lungo :
Therefore, to examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs, we built a chimeric virus encoding a novel, zoonotic CoV spike protein—from the RsSHC014-CoV sequence that was isolated from Chinese horseshoe bats1—in the context of the SARS-CoV mouse-adapted backbone. The hybrid virus allowed us to evaluate the ability of the novel spike protein to cause disease independently of other necessary adaptive mutations in its natural backbone.
Questo è la stessa cosa detta più sopra, dicono di nuovo cosa stavano cercando di fare mischiando la SARS con la sequenza RsSHC014-CoV e valutando se gli spikes DA SOLI fossero in grado di causare infezioni ANCHE SENZA ulteriori mutazioni necessarie di fondo.
The sequences of SHC014 and the related RsWIV1-CoV show that these CoVs are the closest relatives to the epidemic SARS-CoV strains (Fig. 1a,b); however, there are important differences in the 14 residues that bind human ACE2, the receptor for SARS-CoV, including the five that are critical for host range: Y442, L472, N479, T487 and Y491 (ref. 7). In WIV1, three of these residues vary from the epidemic SARS-CoV Urbani strain, but they were not expected to alter binding to ACE2 (Supplementary Fig. 1a,b and Supplementary Table 1). This fact is confirmed by both pseudotyping experiments that measured the ability of lentiviruses encoding WIV1 spike proteins to enter cells expressing human ACE2 (Supplementary Fig. 1) and by in vitro replication assays of WIV1-CoV (ref. 1). In contrast, 7 of 14 ACE2-interaction residues in SHC014 are different from those in SARS-CoV, including all five residues critical for host range (Supplementary Fig. 1c and Supplementary Table 1). These changes, coupled with the failure of pseudotyped lentiviruses expressing the SHC014 spike to enter cells (Supplementary Fig. 1d), suggested that the SHC014 spike is unable to bind human ACE2. However, similar changes in related SARS-CoV strains had been reported to allow ACE2 binding7,8,
Come già detto sopra, il SHC014 non era in grado di attaccare gli ACE2 umani, mentre alcuni SARS-CoV sì. Stanno ripetendo l'intro spiegandola meglio.
Therefore, we synthesized the SHC014 spike in the context of the replication-competent, mouse-adapted SARS-CoV backbone (we hereafter refer to the chimeric CoV as SHC014-MA15) to maximize the opportunity for pathogenesis and vaccine studies in mice
E rieccoci al succo del concetto, e cioè il creare un ibrido SARS-CoV/SHC014 per studiare le infezioni che causa nei topi e massimizzare lo studio di un vaccino.
Therefore, we synthesized the SHC014 spike in the context of the replication-competent, mouse-adapted SARS-CoV backbone (we hereafter refer to the chimeric CoV as SHC014-MA15) to maximize the opportunity for pathogenesis and vaccine studies in mice (Supplementary Fig. 2a). Despite predictions from both structure-based modeling and pseudotyping experiments, SHC014-MA15 was viable and replicated to high titers in Vero cells (Supplementary Fig. 2b). Similarly to SARS, SHC014-MA15 also required a functional ACE2 molecule for entry and could use human, civet and bat ACE2 orthologs (Supplementary Fig. 2c,d). To test the ability of the SHC014 spike to mediate infection of the human airway, we examined the sensitivity of the human epithelial airway cell line Calu-3 2B4 (ref. 9) to infection and found robust SHC014-MA15 replication, comparable to that of SARS-CoV Urbani (Fig. 1c). To extend these findings, primary human airway epithelial (HAE) cultures were infected and showed robust replication of both viruses (Fig. 1d). Together, the data confirm the ability of viruses with the SHC014 spike to infect human airway cells and underscore the potential threat of cross-species transmission of SHC014-CoV.
E rieccoci al risultato : il nuovo virus SHC014-MA15 creato è perfettamente in grado di adattarsi all'ACE2 umano e dei topi. Per capire come si adattasse alle cellule umane, hanno utilizzato "primary human airway epithelial (HAE) cultures", il che vuol dire che hanno utilizzato colture di specifiche cellule umane delle vie aeree per vedere come le cellule reagivano al virus.
Animals infected with SARS-MA15 experienced rapid weight loss and lethality by 4 d post infection (d.p.i.); in contrast, SHC014-MA15 infection produced substantial weight loss (10%) but no lethality in mice.
We next analyzed infection in more susceptible, aged (12-month-old) animals. SARS-MA15–infected animals rapidly lost weight and succumbed to infection (Supplementary Fig. 3a,b). SHC014-MA15 infection induced robust and sustained weight loss, but had minimal lethality.
Questo è interessantissimo, hanno fatto un confronto tra uno strain della SARS e uno del nuovo coronavirusvirus appena creato e hanno visto che la SARS causava una molto più rapida perdita di peso e una mortalità molto alta, mentre il nuovo coronavirus dava una perdita di peso molto più lunga e sostenuta MA sia nei topi più giovani che nei più vecchi una mortalità lievissima o quasi 0.
Nei topi giovani nessuna mortalità, mentre nei topi vecchi minima.
Together, the data indicate that viruses with the SHC014 spike are capable of inducing weight loss in mice in the context of a virulent CoV backbone.
"Se si aggiungono, naturalmente o no, gli spikes del SHC014 ad una struttura coronavirus virulenta, questo causa perdita di peso nei topi."
We examined the effect of these antibodies on viral replication (expressed as percentage inhibition of viral replication) and found that whereas wild-type SARS-CoV Urbani was strongly neutralized by all four antibodies at relatively low antibody concentrations (Fig. 2a–d), neutralization varied for SHC014-MA15.
Come dicevamo sopra, gli antibiotici usati per la SARS funzionavano per la SARS ma avevano risultati molto differenti col nuovo coronavirus.
Fm6, an antibody generated by phage display and escape mutants11,12, achieved only background levels of inhibition of SHC014-MA15 replication (Fig. 2a). Similarly, antibodies 230.15 and 227.14, which were derived from memory B cells of SARS-CoV–infected patients13, also failed to block SHC014-MA15 replication (Fig. 2b,c). For all three antibodies, differences between the SARS and SHC014 spike amino acid sequences corresponded to direct or adjacent residue changes found in SARS-CoV escape mutants (fm6 N479R; 230.15 L443V; 227.14 K390Q/E), which probably explains the absence of the antibodies' neutralizing activity against SHC014. Finally, monoclonal antibody 109.8 was able to achieve 50% neutralization of SHC014-MA15, but only at high concentrations (10 μg/ml) (Fig. 2d). Together, the results demonstrate that broadly neutralizing antibodies against SARS-CoV may only have marginal efficacy against emergent SARS-like CoV strains such as SHC014.
Questo è un elenco dei vari antibiotici che hanno usato, dei quali solo il monoclonal antibody 109.8 ha ottenuto, in alte concentrazioni, una capacità di neutralizzazione di non più del 50%. I risultati sono stati frustranti.
To evaluate the efficacy of existing vaccines against infection with SHC014-MA15, we vaccinated aged mice with double-inactivated whole SARS-CoV (DIV). Previous work showed that DIV could neutralize and protect young mice from challenge with a homologous virus14; however, the vaccine failed to protect aged animals in which augmented immune pathology was also observed, indicating the possibility of the animals being harmed because of the vaccination.
Notably, DIV vaccination resulted in robust immune pathology (Supplementary Table 4) and eosinophilia (Supplementary Fig. 5d–f). Together, these results confirm that the DIV vaccine would not be protective against infection with SHC014 and could possibly augment disease in the aged vaccinated group.
Un tentativo di vaccinazione DIV addirittura sembra indicare la possibilità che i topi ANZIANI siano danneggiati nel sistema immunitario dal vaccino.
Having established that the SHC014 spike has the ability to mediate infection of human cells and cause disease in mice, we next synthesized a full-length SHC014-CoV infectious clone based on the approach used for SARS-CoV (Fig. 3a)2. Replication in Vero cells revealed no deficit for SHC014-CoV relative to that for SARS-CoV (Fig. 3b); however, SHC014-CoV was significantly (P < 0.01) attenuated in primary HAE cultures at both 24 and 48 h after infection (Fig. 3c). In vivo infection of mice demonstrated no significant weight loss but showed reduced viral replication in lungs of full-length SHC014-CoV infection, as compared to SARS-CoV Urbani (Fig. 3d,e). Together, the results establish the viability of full-length SHC014-CoV, but suggest that further adaptation is required for its replication to be equivalent to that of epidemic SARS-CoV in human respiratory cells and in mice.
Successivamente i ricercatori sintetizzarono un virus completo e dimostrarono che erano necessarie successive mutazioni/adattamenti perchè arrivasse ad avere una capacità di replicamento simile a quella della SARS, che è quello che i passi precedenti indicavano.
Il prossimo passo è il più importante
During the SARS-CoV epidemic, links were quickly established between palm civets and the CoV strains that were detected in humans4. Building on this finding, the common emergence paradigm argues that epidemic SARS-CoV originated as a bat virus, jumped to civets and incorporated changes within the receptor-binding domain (RBD) to improve binding to civet Ace2 (ref. 1
. Subsequent exposure to people in live-animal markets permitted human infection with the civet strain, which, in turn, adapted to become the epidemic strain (Fig. 4a). However, phylogenetic analysis suggests that early human SARS strains appear more closely related to bat strains than to civet strains18. Therefore, a second paradigm argues that direct bat-human transmission initiated SARS-CoV emergence and that palm civets served as a secondary host and reservoir for continued infection (Fig. 4b)19. For both paradigms, spike adaptation in a secondary host is seen as a necessity, with most mutations expected to occur within the RBD, thereby facilitating improved infection.Both theories imply that pools of bat CoVs are limited and that host-range mutations are both random and rare, reducing the likelihood of future emergence events in humans.
Although our study does not invalidate the other emergence routes, it does argue for a third paradigm in which circulating bat CoV pools maintain 'poised' spike proteins that are capable of infecting humans without mutation or adaptation (Fig. 4c). This hypothesis is illustrated by the ability of a chimeric virus containing the SHC014 spike in a SARS-CoV backbone to cause robust infection in both human airway cultures and in mice without RBD adaptation. Coupled with the observation of previously identified pathogenic CoV backbones3,20, our results suggest that the starting materials required for SARS-like emergent strains are currently circulating in animal reservoirs. Notably, although full-length SHC014-CoV probably requires additional backbone adaption to mediate human disease, the documented high-frequency recombination events in CoV families underscores the possibility of future emergence and the need for further preparation.
Questa è la conclusione del rapporto per quanto riguarda la sezione infettività, ipotizzano che le mutazioni tra un host e l'altro sono RARE E CASUALI e che questo riduce la possibilità di future infezioni, MA allo stesso tempo teorizzano che si possano trovare in natura spikes adattabili che possano diventare virulenti e ribadiscono che al momento il virus SHC014 richiede ulteriori modifiche per essere in grado di sviluppare malattie negli umani.
Inoltre suggeriscono immediati provvedimenti per prepararsi per una eventuale mutazione.
Overall, our approach has used metagenomics data to identify a potential threat posed by the circulating bat SARS-like CoV SHC014. Because of the ability of chimeric SHC014 viruses to replicate in human airway cultures, cause pathogenesis in vivo and escape current therapeutics, there is a need for both surveillance and improved therapeutics against circulating SARS-like viruses. Our approach also unlocks the use of metagenomics data to predict viral emergence and to apply this knowledge in preparing to treat future emerging virus infections.
Apparentemente stavano dicendo "Signori, dobbiamo essere pronti perchè se questa cosa si espande in natura siamo nei guai".
Il resto dell'articolo spiega le METODOLOGIE con cui hanno analizzato i topi, le misure di sicurezza prese nel laboratorio.
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