Новые вирусы названы BANAL-52, BANAL-103 и BANAL-236.
Характерно, что у 10 видов мышей из нескольких колоний было выделено 25 альфа- и бета- коронавирусов, 5 из которых относятся к сарбековирусам (среди которых 3 новых)- или роду, куда входят и SARS-CoV-2 (а так же САРС и МЕРС).
Самое опасное, с точки зрения вирусологов, то, что эти коронавирусы ну хоть сейчас готовы связываться с человеческими АСЕ2 рецепторами, и заражения в культуре клеток человека очень напоминают поведения и динамику "уханьского" варианта. Потому что в природе, оказывается, вполне себе существуют подобные патогены, "уже отобранные", готовые перекинуться к людям- только дай им шанс.
Важно, что у некоторых мышей было выделено не один коронавирус, а коронавирусы из разных родов- альфа и бета, одновременно, так что внутри мышей может происходить (в теории) и рекомбинация, позволяющая вирусам получать преимущества друг от друга. Хотя новые штаммы и очень похожи на тот, что вызывает ковид, а шипик практически идентичный- есть важное отличие- у них нет сайта подрезания фурина, который считается важным для инфекционности у людей.
Ученые считают, что путем рекомбинации кусок генома мог попасть от альфа коронавируса к бета, и, вполне может быть, что в какой-то другой популяции летучих мышей, есть вирус, который еще ближе эволюционно к SARS-CoV-2, чем свежеобнаруженные BANALы.
Китайские вирусологи продолжают искать родственников SARS-CoV-2 среди своих мышей, однако, в большом исследовании, с 2016 по 2021 год, с забором образцов от 13 тыс мышей в разных областях страны и популяциях, ничего ближе RaTG13 выловить не удалось, и все, притом, довольно далеко от сарбековирусов.
Так что пока, не смотря на лаосские находки, история попадания ковида к людям - не прояснилась.
Many sarbecoviruses circulate in Rhinolophus colonies living in caves in China and probably also in neighboring countries further south (Laos, Myanmar, Thailand, and Vietnam)25–27. During the course of a prospective study in Northern Laos, we have identified in 10 bat species 25 different coronaviruses belonging to the Alphacoronavirus and Betacoronavirus genera. We then focused our study on the five sarbecoviruses for which we obtained full-length sequences. Among these, three (BANAL-52, -103, and -236) were considered to be close to SARS-CoV-2 because of the similarity in amino acids of one of the S domains (S1-NTD, S1-RBD, or S2) with the homologous domain of SARS-CoV-2. The similarity plot analysis revealed that the evolution history of SARS-CoV-2 is more complex than expected and that R. affinis RaTG13, isolated in Yunnan in 2013, is no longer considered the proximal ancestor of SARS-CoV-2: strains close to R. pusillus RpYN06, R. malayanus RmYN02, and Rhinolophus sp. PrC31 isolated in China in 2018-2019, along with R. malayanus BANAL-52, R. pusillus BANAL-103, and R. marshalli BANAL-236 isolated in Laos in 2020, contributed to the appearance of SARS-CoV-2 in different regions of the genome. No closer viral genome has yet been identified as a possible contributor, and pangolin coronaviruses appear as more distantly related than bat coronaviruses.
Because genomic regions subject to recombination are likely contributing to host-virus interactions and adaptation following spillover events, we compared SARS-CoV-2 strains from the two lineages identified at the very onset of the COVID-19 outbreak19 to these novel bat sarbecoviruses and to pangolin strains within the SARS-CoV-2 clade. We thus identified potential recombination sites, allowing for the reconstruction of the phylogenetic history of early isolated SARS-CoV-2 strains between homologous regions defined by recombination points. The interaction of the SARS-CoV-2 spike with hACE2 is a key event in cell infection. The spike is divided into two subunits, S1 and S2: S1 contains an RBD that specifically binds to hACE2, whereas the S2 subunit contains the fusion peptide. Regarding the spike, we identified a breakpoint at the beginning of the SARS-CoV-2 RBD, resulting in a downstream fragment composed of the RBD, the furin cleavage site, and ending in the N-terminal region of S2. Despite the absence of the furin site in these novel bat sarbecoviruses, phylogenetic reconstruction of this fragment, key for the virus tropism and host spectrum, revealed that Laotian R. malayanus BANAL-52, R. pusillus BANAL-103, and R. marshalli BANAL-236 coronaviruses are the closest ancestors of SARS-CoV-2 known to date. Identification of strains of animal origin with a furin cleavage site may require additional sampling. As seen by others, ORF8 was highly divergent between SARS-CoV-2 related genomes. ORF8 from strains BANAL-52, -103, -236, like that of RaTG13, were closer to SARS-CoV-2 than to pangolin strains. ORF8 encodes a protein that has been proposed to participate in immune evasion28. It is noteworthy that ORF8 was deleted in many human SARS-CoV-2 strains that appeared after March 202029, which is reminiscent of the deletions identified during the 2003 SARS epidemic30. Therefore, ORF8 can be a marker of SARS-CoV-2 adaptation to humans and its presence in bat strains is consistent with bats acting as a natural reservoir of early strains of SARS-CoV-2.
Structural and functional biology studies have identified the RBD domain that mediates the interaction with hACE2, as well as the major amino acids that are involved24. The host range is dependent on this interaction31,32. We show that the spike of SARS-CoV-2 is a mosaic of sequences close to the following bat viruses: S1-NTD (BANAL-52 and RaTG13), S1-RBD (BANAL-52, -103, and -236), and S2 (BANAL-52, -236, -103, and RaTG13). Notably, the RBDs (BANAL-52, -103, and -236) are closer to SARS-CoV-2 than that of any other bat strain described so far. Overall, one (H498Q (strains BANAL-103 and -52)) or two (K493Q and H498Q (strain BANAL-236)) amino acids interacting with hACE2 are substituted in these strains in comparison to SARS-CoV-2. These mutations did not destabilize the BANAL-236 / hACE2 interface, as shown by the BLI experiments (Fig. 3A).
Our results contribute to understanding the origin of SARS-CoV-2: they show that sequences very close to those of the early strains of SARS-CoV-2 responsible for the pandemic exist in nature and are found in several Rhinolophus bat species. The RBDs of the viruses found in our study are closer to that of SARS-CoV-2 than to the RaTG13 RBD, the virus identified in R. affinis from the Mojiang mineshaft where pneumonia cases with clinical characteristics strikingly similar to COVID-196 were recorded in 201233,34. We found here sarbecoviruses with RBDs closest to that of SARS-CoV-2 in three different bat species, R. marshalli, R. malayanus, and R. pusillus. Our results therefore support the hypothesis that SARS-CoV-2 could originally result from a recombination of sequences pre-existing in Rhinolophus bats living in the extensive limestone cave systems of South-East Asia and South China35, which provides ideal conditions for interspecies interactions among Rhinolophus bats. They are restricted to limestone caves for their roosting sites and forage in the vicinity of these caves, and many species have been found foraging in the same cave areas, including R. malayanus and R. pusillus36. In addition, the distribution of R. marshalli, R. malayanus, and R. pusillus overlaps in the Indo-Chinese subregion, which means they may share caves as roost sites and foraging habitats37.
The pangolin has been suspected as being an intermediate host of SARS-CoV-2. The pangolin CoV-2017 and -2019 genomes have a high overall protein identity with SARS-CoV-2 and RaTG13 (up to 100% for certain proteins). In particular, RBD was highly conserved between pangolin-CoV-2019 and SARS-CoV-2. On the other hand, the amino-acid sequence identity of S1-NTD is only 63.1% identical between pangolin-CoV-2017 and SARS-CoV-27. With the novel viruses here described, the pool of sequences found in Rhinolopus spp. allows the reconstitution of a genome sufficiently close to that of SARS-CoV-2 without the need to hypothesize recombination or natural selection for increased RBD affinity for hACE2 in an intermediate host before spillover38, nor natural selection in humans following spillover39. However, we found no furin site in any of these viruses on sequences determined from original fecal swab samples, devoid of any bias associated with counterselection of the furin site by amplification in Vero cells16. Lack of furin cleavage may be explained by insufficient sampling in bats, or by acquisition of the furin cleavage site through passages of the virus in an alternate host or during an early poorly symptomatic unreported circulation in humans. Finally, where these intergenomic recombinations arose and the epidemiological link with the first human cases remains to be established.
As expected from the high affinity for ACE2 of the S ectodomain of the BANAL-236, pseudoviruses expressing it were able to enter efficiently human cells expressing hACE2 using an ACE2-dependent pathway. Entry was blocked by a serum neutralizing SARS-CoV-2. The RaTG13 strain, the closest to SARS-CoV-2 known before, had never been isolated. In contrast, preliminary studies show that BANAL-236 replicated in primate VeroE6 cells with a small plaque phenotype compared to SARS-CoV-2.
Further analysis may indicate more clearly whether post-entry steps also shape infectivity.To conclude, our results pinpoint the presence of new bat sarbecoviruses that seem to have the same potential for infecting humans as early strains of SARS-CoV-2. People working in caves, such as guano collectors, or certain ascetic religious communities who spend time in or very close to caves, as well as tourists who visit the caves, are particularly at risk of being exposed. Further investigations are needed to assess if such exposed populations have been infected by one of these viruses, if these infections are associated with symptoms, and whether they could confer protection against subsequent SARS-CoV-2 infections. In this context, it is noteworthy that SARS-CoV-2 with the furin site deleted replicates in hamsters and in transgenic mice expressing hACE2, but leads to ablated disease while protecting from rechallenge with wild-type SARS-CoV-216.