Khosta-2: New bat virus can infect human cells and is resistant to both the monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2

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A recently discovered virus in a Russian bat that is similar to SARS-CoV-2, the virus behind COVID-19, is likely capable of infecting humans and, if it were to spillover, is resistant to current vaccines.

A team lead by researchers in Washington State University’s Paul G. Allen School for Global Health found spike proteins from the bat virus, named Khosta-2, can infect human cells and is resistant to both the monoclonal antibodies and serum from individuals vaccinated for SARS-CoV-2. Both Khosta-2 and SARS- CoV-2 belong to the same sub-category of coronaviruses known as sarbecoviruses.

“Our research further demonstrates that sarbecoviruses circulating in wildlife outside of Asia—even in places like western Russia where the Khosta-2 virus was found—also pose a threat to global health and ongoing vaccine campaigns against SARS-CoV-2,” said Michael Letko, WSU virologist and corresponding author of the study published in the journal PLoS Pathogens.

Letko said the discovery of Khosta-2 highlights the need to develop universal vaccines to protect against sarbecoviruses in general, rather than just against known variants of SARS-CoV-2.

“Right now, there are groups trying to come up with a vaccine that doesn’t just protect against the next variant of SARS-2 but actually protects us against the sarbecoviruses in general,” Letko said. “Unfortunately, many of our current vaccines are designed to specific viruses we know infect human cells or those that seem to pose the biggest risk to infect us. But that is a list that’s everchanging. We need to broaden the design of these vaccines to protect against all sarbecoviruses.”

While hundreds of sarbecoviruses have been discovered in recent years, predominantly in bats in Asia, the majority are not capable of infecting human cells. The Khosta-1 and Khosta-2 viruses were discovered in Russian bats in late 2020, and it initially appeared they were not a threat to humans.

“Genetically, these weird Russian viruses looked like some of the others that had been discovered elsewhere around the world, but because they did not look like SARS-CoV-2, no one thought they were really anything to get too excited about,” Letko said. “But when we looked at them more, we were really surprised to find they could infect human cells. That changes a little bit of our understanding of these viruses, where they come from and what regions are concerning.”

Letko teamed with a pair of WSU faculty members, first author viral ecologist Stephanie Seifert and viral immunologist Bonnie Gunn, to study the two newly discovered viruses. They determined Khosta-1 posed low risk to humans, but Khosta-2 demonstrated some troubling traits.

The team found that like SARS-CoV-2, Khosta-2 can use its spike protein to infect cells by attaching to a receptor protein, called angiotensin converting enzyme 2 (ACE2), found throughout human cells. They next set out to determine if current vaccines protect against the new virus.

Using serum derived from human populations vaccinated for COVID-19, the team saw that Khosta-2 was not neutralized by current vaccines. They also tested serum from people who were infected with the omicron variant, but the antibodies, too, were ineffective.

Fortunately, Letko said the new virus is lacking some of the genes believed to be involved in pathogenesis in humans. There is a risk, however, of Khosta-2 recombining with a second virus like SARS-CoV-2.

“When you see SARS-2 has this ability to spill back from humans and into wildlife, and then there are other viruses like Khosta-2 waiting in those animals with these properties we really don’t want them to have, it sets up this scenario where you keep rolling the dice until they combine to make a potentially riskier virus,” Letko said.

In addition to Letko, Seifert and Gunn, co-authors on this study include Shuangyi Bai and Stephen Fawcett of WSU as well as Elizabeth Norton, Kevin Zwezdaryk and James Robinson of Tulane University.


Horseshoe bats (Rhinolophidae: Rhinolophus) are considered a main natural reservoir and source of zoonotic coronaviruses (CoV), which caused epidemic outbreaks of severe acute respiratory syndrome (SARS) and the COVID-19 pandemic in 2002 and 2019, respectively [1,2]. These viruses, designated SARS-CoV and SARS-CoV-2, together with related viruses found in bats and other animals (SARS-like coronaviruses or SARS-CoV-like viruses), belong to the subgenus Sarbecovirus of the genus Betacoronavirus of the family Coronaviridae [3]. Horseshoe bats are widely distributed in Asia, Europe, and North Africa.

In East Asia (in particular, China), SARS-CoV-like viruses circulate in multiple rhinolophid species; however, the Chinese rufous (R. sinicus) and the greater (R. ferrumequinum), intermediate (R. affinis), Malayan (R. malayanus), the least (R. pusillus), and king (R. rex) horseshoe bats seem to be of major importance [4]. In Europe, SARS-CoV-like viruses were found in the greater, the lesser (R. hipposideros), the Mediterranean (R. euryale), Mehely’s (R. mehelyi), and Blasius’ (R. blasii) horseshoe bats [5,6,7,8].

The prevalence of SARS-like coronaviruses among bats in different caves/colonies can vary from 0% to 60% [4,7,9,10]. In Russia, three species of horseshoe bats (the greater, lesser, and Mediterranean) are common in the southern regions, lying below about 44° north latitude, mostly including North Caucasus and Crimea. In the present work, we hypothesized that SARS-like coronaviruses circulate in the region in local populations of horseshoe bats.

To test this hypothesis, we examined the colonies of bats located in the southern macroslope of the Greater Caucasus on the northern coast of the Black Sea in Russia. Using metagenomic analysis, we found and genetically described two new SARS-like coronaviruses in feces and oral swabs of the greater and lesser horseshoe bats. Further PCR analysis showed a high degree of infection of bats with discovered viruses in some locations.

Genetic and Phylogenetic Analysis

The genomic organization of Khosta-1 and Khosta-2 was similar to that of other SARS-like coronaviruses (Figure 2). Approximately two-thirds of the genome of coronaviruses is occupied by ORF1a and ORF1b genes, which encode the proteins of the replicative complex and are translated as the ORF1ab polyprotein due to ribosomal shifting. The remainder of the genome contains genes of structural proteins (S, E, M, and N), which form a virion, as well as several nonstructural proteins (ORF3, ORF6, ORF7, ORF8, ORF9, and ORFX), the presence and structure of which vary in different viruses [3]. The genome organization of Khosta-1 and Khosta-2 had the greatest similarity to the BtCoV/BM48-31/2008 and BtKY72 viruses—two SARS-like coronaviruses found in horseshoe bats in Bulgaria and Kenya in 2008 and 2007, respectively [8,15]. The common peculiarity of Khosta-1, Khosta-2, BtCoV/BM48-31/2008, and BtKY72 is the absence of the ORF8 gene, which is common in bat SARS-like coronaviruses from East and Southeast Asia.

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Figure 2

Simplot analysis of Khosta-1 and Khosta-2 with SARS-CoV, SARS-CoV-2, and related viruses. RaTG13, HKU3, and Rs672 were used as representatives of bat SARS-CoV-like viruses from Asia. (A) Khosta-1 was used as a query sequence, and SARS-CoV, RaTG13, HKU3, Rs672, and SARS-CoV-2 were used as reference sequences. (B) Khosta-2 was used as a query sequence, and SARS-CoV, RaTG13, HKU3, Rs672, and SARS-CoV-2 were used as reference sequences. The sequences of the ORF8 gene, which is absent in Khosta-1 and Khosta-2, were removed from alignment before analysis. (C) Khosta-1 was used as a query sequence, and Khosta-2, BM48-31/BGR/2008, and BtKy72 viruses were used as reference sequences. (D) Khosta-2 was used as a query sequence, and Khosta-1, BM48-31/BGR/2008, and BtKY72 were used as reference sequences. The analysis was performed using the Kimura (two-parameter) model, with a window size of 1000 bases and a step size of 100 bases.

Khosta-1 and Khosta-2 showed 76–78.2% nt identities with SARS-CoV, SARS-CoV-2, and related viruses found in China according to a full-length genome comparison. The full-length genome of Khosta-1 was most similar to that of the European strain BtCoV/BM48-31/2008 (89.5% nt identity) and had a lower level of similarity to BtKY72 (81.7% nt). The Khosta-2 genome, by contrast, had a near-identical similarity to BtCoV/BM48-31/2008 and BtKY72 (both 79.8% nt), as well as strains isolated in East and Southeast Asia. The genome sequence similarity of Khosta-1, Khosta-2, and other sarbecoviruses was analyzed using the Simplot software (Figure 2). In general, the genetic similarity of Khosta-1 and Khosta-2 with Eastern strains did not exceed 85% nt identity in the most conserved part of the coronavirus genome, i.e., the ORF1b gene, with a decrease to 20–30% in variable regions (Figure 2A,B). The analysis showed the highest degree of similarity of Khosta-1 to BtCoV/BM48-31/2008 in the ORF1ab and N genes, as well as a decrease in the similarity in the S-ORF7b region (Figure 2C,D).

Pairwise alignments of the deduced proteins of Khosta-1 and Khosta-2 virus with those of other SARS-like coronaviruses also showed the highest identity with BtCoV/BM48-31/2008 and BtKY72 (Table 2). Khosta-1 was most closely related to BtCoV/BM48-31/2008, with 92.5% aa and 99% aa identity in the conservative ORF1a and ORF1b proteins, respectively. The similarity of Khosta-1 to SARS-CoV and related viruses from China was, on average, 81.5% aa identity in the ORF1a protein and 96% aa identity in the ORF1b protein. A comparison of Khosta-1 with SARS-CoV-2 viruses revealed 77.5% and 94.2% aa identity in the ORF1a and ORF1b proteins, respectively. Despite the high similarity of Khosta-1 and BtCoV/BM48-31/2008 in the ORF1a and ORF1b proteins, the structural proteins S, E, and M of Khosta-1 were more similar to those of the Kenyan virus BtKY72. Khosta-1 and BtKY72 shared 89.1%, 98.7%, and 97.29% aa identity for the S, E, and M proteins, whereas these values for Khosta-1 and BtCoV/BM48-31/2008 were 84.37%, 89.47%, and 95%, respectively. The N protein of Khosta-1 was more similar to that of BtCoV/BM48-31/2008 (96.64% aa identity) than BtKY72 (92.6% aa identity).

Table 2

Identity (%) of deduced amino-acid sequences of proteins of Khosta-1 and Khosta-2 viruses with certain representatives of the Sarbecovirus subgenus (lineage B of betacoronaviruses).

ProteinVirusesAmino-Acid Identity (%)
Bat SARS-CoV-like BGR/2008
(Bulgaria, 2008)
Bat SARS-CoV-like BtKY72
(Kenya, 2007)
Bat SARS-CoV-like (China, 2005–2016) *Civet SARS-CoV-like SZ3
(China, 2003)
SARS-CoV Urbani (2003)Bat SARS-CoV-2-like RaTG13
(China, 2013)
Pangolin SARS-CoV-2-like (China, 2017)SARS-CoV-2
Wuhan-Hu-1
(2019)
Khosta-1 vs. Khosta-2
ORF1aKhosta-192.9584.681.53–81.681.6781.5377.277.8977.3282
Khosta-281.180.979.4–79.679.579.476.377.176.45
ORF1bKhosta-199.0796.395.82–96.396.1596.1594.2294.2294.2194.75
Khosta-294.793.794.9–95.1795.0294.993.4493.4793.5
SKhosta-184.3789.1175.5–76.275.775.773.072.472.2282
Khosta-279.5479.773.03–73.973.273.072.571.7472.54
S RBDKhosta-181.390.077.1–78.578.076.774.074.272.280
Khosta-274.980.064.3–75.475.375.967.568.569.0
ORF3Khosta-185.9886.766.8–72.370.870.865.166.264.781.8
Khosta-277.982.2267.9–69.3467.1567.564.565.863.27
EKhosta-189.4798.787.0878793.4293.4293.4294.7
Khosta-288.1694.7490.790.790.789.589.589.5
MKhosta-195.097.2991.86–92.3192.3191.8688.2487.7388.1391
Khosta-290.990.588.7–89.690.589.687.387.2787.0
ORF6Khosta-168.2563.049.21–52.3849.2149.2150.8250.8250.8258.73
Khosta-258.158.144.4–47.646.0346.0346.746.746.7
ORF7aKhosta-169.770.658–59.761.3461.3458.559.3258.573.5
Khosta-263.2570.3458.2–59.2660.060.060.058.359.13
ORF7bKhosta-186.0581.471.871.871.861.571.874.470.7
Khosta-271.473.164.264.264.264.264.264.2
NKhosta-196.6492.688.36–88.989.189.187.987.687.491.85
Khosta-291.1390.2185.75–86.7386.586.585.586.485.24

* Strains of bat SARS-CoV-like viruses from China included HKU3 (2005; DQ022305), Rs672 (2006; FJ588686), RsSHC014 (2011; KC881005), WIV1 (2012; KF367457), Rs3367 (2012; KC881006), WIV16 (2013; KT444582), and YN2018B (2016; MK211376).

By contrast, Khosta-2 did not exhibit such an increased similarity with some groups of sarbecoviruses and had 79–81% aa identity with SARS-CoV viruses and 76–77% aa identity with SARS-CoV-2 and related viruses in the ORF1a protein. The ORF1b protein of Khosta-2 had 93.5–95% aa identities with all the other bat SARS-like coronaviruses. A comparison of the proteins of Khosta-1 and Khosta-2 showed that these viruses differ from each other at about the same level at which Khosta-2 differs from other bat SARS-like coronaviruses (Table 2).

Phylogenetic Analysis

A phylogenetic analysis based on the nucleotide sequences of the conserved RdRp gene showed that Khosta-1, Khosta-2, BtCoV/BM48-31/2008, and BtKY72 form a monophyletic lineage located outside the SARS-CoV and SARS-CoV-2 lineages of the Sarbecovirus subgenus (Figure 3A). A separate cluster of this group of viruses was also formed in the phylogenetic tree for the S gene (Figure 3B) and the N gene (Figure 3C). The topology of the trees confirmed a probable recombination event in the evolutionary history of Khosta-1. In the RdRp and N trees, Khosta-1 was grouped together with BtCoV/BM48-31/2008, whereas, in the S gene tree, it was grouped together with BtKY72.

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Figure 3

Phylogenetic trees inferred using maximum likelihood method according to an analysis of nucleotide sequences of the RdRp gene (2766 nt) (A), nucleotide sequences of the S gene (3822 nt (SARS-CoV-2 numbering)) (B), and nucleotide sequences of N gene (1257 nt) (C) of certain sarbecoviruses. The percentage of trees in which the associated taxa clustered together is shown next to the branches (values higher 50% are shown). SARS-CoV and SARS-CoV-2 are marked by black circles; Khosta-1 and Khosta-2, described in the present work, are marked by red circles. The trees were inferred using GTR + G + I model with 1000 bootstrap replicates using the MEGAX.

Discussion

Increasing pieces of evidence from multiple studies do suggest an immediate ancestors of SARS-CoV and SARS-CoV-2 most likely originated from viruses circulated in different species of horseshoe bats [2,19,20,21,22,23]. To date, bat viruses closest related to SARS-CoV-2 have been found in R. affinis (strain RaTG13), R. malayanus (strain RmYN02), and R. pusillus (strain RpYN06) collected in Yunnan province of China [19,23,24]. Other strains, more distant or exhibited high sequence identity to SARS-CoV-2 in certain regions of the genome, were found in bats in Zhejiang province of China (strains ZXC21 and ZC45 from R. pusillus), Thailand (strain RacCS203 from R. acuminatus), Cambodia (strains RshSTT182 and RshSTT200 from R. shameli), Laos (strains BANAL-52, -103, -236 from R. malayanusR. pusillus, and R. marshalli, respectively), and Japan (strain Rc0319 from R. cornutus) [25,26,27,28,29]. Genetic and phylogenetic analysis carried out in these and other studies shows that the genome of sarbecoviruses is subject to frequent recombinations and genome of SARS-CoV-2, similar to related viruses, probably has a complex mosaic origin. Horseshoe bats are widespread and, presumably, SARS-like coronaviruses circulate across the regions of their distribution, including Asia, Europe, and North Africa. However, little information exists on the genetic diversity of bat SARS-like coronaviruses in regions outside East and Southeast Asia. We described here two novel SARS-like coronaviruses circulating in horseshoe bats in the southern region of Russia. Khosta-1 and Khosta-2 viruses are closely related to viruses recently described in Bulgaria (strain BtCoV/BM48-31/2008) and Kenya (strain BtKY72) [8,15]. Together, they form a separate “western” (as they are found to the west of regions home to horseshoe bats) phylogenetic lineage of bat SARS-like coronaviruses. A feature of these viruses is the absence of the ORF8 gene, which is common in SARS-CoV, SARS-CoV-2, and most bat SARS-like coronaviruses of eastern lineages.

SARS-CoV and SARS-CoV-2 recognize the host’s angiotensin-converting enzyme 2 (ACE2) as their receptor. Amino acids (442, 487, 479, 487, and 491) crucial for binding of ACE2 are located in the RBM of the S protein [17,30,31,32,33]. These amino acids and their surrounding residues in the RBM of Khosta-1 and Khosta-2, as in most other bat SARS-like coronaviruses, are quite different from those in SARS-CoV and SARS-CoV-2. Most bat SARS-like coronaviruses are unable to bind the ACE2 receptor of humans and, thus, are not infectious toward their cells [34]. However, several strains of bat SARS-like coronaviruses that can use the ACE2 receptor have been recently found in the Chinese rufous (R. sinicus) and the intermediate (R. affinis) horseshoe bats in China [35,36,37,38]. Distantly related to SARS-CoV-2 strains (RsYN04, RmYN05 and RmYN08, all also from China) have been found to bind to the human ACE2 receptor, albeit with very low affinity [23]. Finally, strains with an almost identical to SARS-CoV-2 RBD and a high receptor binding capacity have been found in bats in Laos [28]. These data suggest that the ability to bind a human ACE2 receptor could arise naturally and independently in different phylogenetic lineages of sarbecoviruses.

Since recombination is of great importance in the evolution of coronaviruses, we analyzed possible recombination events in the western lineage of bat SARS-like coronaviruses. Despite the small number of known full-length sequences (only four), we observed evidence of recombination in the evolutionary history of Khosta-1. The alleged recombination event involved the acquisition of structural proteins S, E, and M, as well as nonstructural genes ORF3, ORF6, ORF7a, and ORF7b, from a virus that is closer to the Kenyan isolate BtKY72 than to the European strain BtCoV/BM48-31/2008. Accordingly, we can assume that the genetic diversity of viruses in the region is significantly higher than currently established, and there is a constant exchange of genes across viruses. These findings require further investigation of the diversity of circulating variants, with particular emphasis on the diversity of the S gene.

Using RT-PCR, we showed that 14% of tested horseshoe bats were positive for Khosta-1 virus and 1.75% were positive for Khosta-2 virus. However, most of the Khosta-1-positive samples were found in only one cave (Kolokolnaya cave), where the infection rate of greater horseshoe bats reached 62.5%. This bias, together with the small number of samples from other locations, makes it difficult to accurately estimate the prevalence of Khosta-1 in the region; hence, further research is required. The closest European region where such studies have been carried out is Bulgaria; according to the data obtained by Drexler et al. (2010), SARS-like coronaviruses were detected in 13.3% of greater horseshoe, 15.9% of Blasius’s horseshoe, 30.8% of Mehely’s horseshoe bat, and 32.1% of Mediterranean horseshoe bats [8]. Other studies found 38.8% positivity in lesser horseshoe bats in Slovenia and 37.9% positivity in greater horseshoe bats in France [5,6]. All these data show that the prevalence of SARS-like coronaviruses in horseshoe bats in Europe can vary widely across different species, locations, and possibly the time of year of observation.

In conclusion, we showed that SARS-like coronaviruses circulate in horseshoe bats in the southern region of Russia, and we provided new information on their genomic diversity. The genetic diversity, prevalence, host range, and potential threat to humans of these viruses remain to be determined.

reference link :https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8779456/


More information: An ACE2-dependent Sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines, PLoS Pathogens (2022). DOI: 10.1371/journal.ppat.1010828

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