Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236.

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics.

IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

Determination of the factors responsible for the tropism of SARS-CoV-2-related bat coronaviruses to Rhinolophus bat ACE2.

IMPORTANCE: The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level.

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5.

The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.

Virological characterization of the 2022 outbreak-causing monkeypox virus using human keratinocytes and colon organoids.

The outbreak-causing monkeypox virus of 2022 (2022 MPXV) is classified as a clade IIb strain and phylogenetically distinct from prior endemic MPXV strains (clades I or IIa), suggesting that its virological properties may also differ. Here, we used human keratinocytes and induced pluripotent stem cell-derived colon organoids to examine the efficiency of viral growth in these cells and the MPXV infection-mediated host responses. MPXV replication was much more productive in keratinocytes than in colon organoids. We observed that MPXV infections, regardless of strain, caused cellular dysfunction and mitochondrial damage in keratinocytes. Notably, a significant increase in the expression of hypoxia-related genes was observed specifically in 2022 MPXV-infected keratinocytes. Our comparison of virological features between 2022 MPXV and prior endemic MPXV strains revealed signaling pathways potentially involved with the cellular damages caused by MPXV infections and highlights host vulnerabilities that could be utilized as protective therapeutic strategies against human mpox in the future.

Structural Insight into the Resistance of the SARS-CoV-2 Omicron BA.4 and BA.5 Variants to Cilgavimab.

We have recently revealed that the new SARS-CoV-2 Omicron sublineages BA.4 and BA.5 exhibit increased resistance to cilgavimab, a therapeutic monoclonal antibody, and the resistance to cilgavimab is attributed to the spike L452R substitution. However, it remains unclear how the spike L452R substitution renders resistance to cilgavimab. Here, we demonstrated that the increased resistance to cilgavimab of the spike L452R is possibly caused by the steric hindrance between cilgavimab and its binding interface on the spike. Our results suggest the importance of developing therapeutic antibodies that target SARS-CoV-2 variants harboring the spike L452R substitution.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.

The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

Virological characteristics of the SARS-CoV-2 Omicron BA.2 subvariants, including BA.4 and BA.5.

After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.

Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike.

Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant.

The emergence of the Omicron variant of SARS-CoV-2 is an urgent global health concern1. In this study, our statistical modelling suggests that Omicron has spread more rapidly than the Delta variant in several countries including South Africa. Cell culture experiments showed Omicron to be less fusogenic than Delta and than an ancestral strain of SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into two subunits, which facilitates cell-cell fusion2,3, the Omicron S protein was less efficiently cleaved compared to the S proteins of Delta and ancestral SARS-CoV-2. Furthermore, in a hamster model, Omicron showed decreased lung infectivity and was less pathogenic compared to Delta and ancestral SARS-CoV-2. Our multiscale investigations reveal the virological characteristics of Omicron, including rapid growth in the human population, lower fusogenicity and attenuated pathogenicity.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

The SARS-CoV-2 Lambda variant exhibits enhanced infectivity and immune resistance.

SARS-CoV-2 Lambda, a variant of interest, has spread in some South American countries; however, its virological features and evolutionary traits remain unclear. In this study, we use pseudoviruses and reveal that the spike protein of the Lambda variant is more infectious than that of other variants due to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino acid deletion in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies and further augments antibody-mediated enhancement of infection. Although this mutation generates a nascent N-linked glycosylation site, the additional N-linked glycan is dispensable for the virological property conferred by this mutation. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the RSYLTPGD246-253N mutation is closely associated with the substantial spread of the Lambda variant in South America.

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.

SARS-CoV-2 B.1.617 Mutations L452R and E484Q Are Not Synergistic for Antibody Evasion.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine-elicited neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity.

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.

Comprehensive Investigation on the Interplay between Feline APOBEC3Z3 Proteins and Feline Immunodeficiency Virus Vif Proteins.

As the hosts of lentiviruses, almost 40 species of felids (family Felidae) are distributed around the world, and more than 20 feline species test positive for feline immunodeficiency virus (FIV), a lineage of lentiviruses. These observations suggest that FIVs globally infected a variety of feline species through multiple cross-species transmission events during a million-year history. Cellular restriction factors potentially inhibit lentiviral replication and limit cross-species lentiviral transmission, and cellular APOBEC3 deaminases are known as a potent restriction factor. In contrast, lentiviruses have evolutionary-acquired viral infectivity factor (Vif) to neutralize the APOBEC3-mediated antiviral effect. Because the APOBEC3-Vif interaction is strictly specific for viruses and their hosts, a comprehensive investigation focusing on Vif-APOBEC3 interplay can provide clues that will elucidate the roles of this virus-host interplay on cross-species transmission of lentiviruses. Here, we performed a comprehensive investigation with 144 patterns of a round robin test using 18 feline APOBEC3Z3 genes, an antiviral APOBEC3 gene in felid, and 8 FIV Vifs and derived a matrix showing the interplay between feline APOBEC3Z3 and FIV Vif. We particularly focused on the interplay between the APOBEC3Z3 of three felids (domestic cat, ocelot, and Asian golden cat) and an FIV Vif (strain Petaluma), and revealed that residues 65 and 66 of the APOBEC3Z3 protein of multiple felids are responsible for the counteraction triggered by FIV Petaluma Vif. Altogether, our findings can be a clue to elucidate not only the scenarios of the cross-species transmissions of FIVs in felids but also the evolutionary interaction between mammals and lentiviruses. IMPORTANCE Most of the emergences of new virus infections originate from the cross-species transmission of viruses. The fact that some virus infections are strictly specific for the host species indicates that certain "species barriers" in the hosts restrict cross-species jump of viruses, while viruses have evolutionary acquired their own "arms" to overcome/antagonize/neutralize these hurdles. Therefore, understanding of the molecular mechanism leading to successful cross-species viral transmission is crucial for considering the menus of the emergence of novel pathogenic viruses. In the field of retrovirology, APOBEC3-Vif interaction is a well-studied example of the battles between hosts and viruses. Here, we determined the sequences of 11 novel feline APOBEC3Z3 genes and demonstrated that all 18 different feline APOBEC3Z3 proteins tested exhibit anti-feline immunodeficiency virus (FIV) activity. Our comprehensive investigation focusing on the interplay between feline APOBEC3 and FIV Vif can be a clue to elucidate the scenarios of the cross-species transmissions of FIVs in felids.