Genetic Diversity and Detection of Respiratory Viruses Excluding SARS-CoV-2 during the COVID-19 Pandemic in Gabon, 2020-2021.

Acute respiratory infections are a major global burden in resource-limited countries, including countries in Africa. Although COVID-19 has been well studied since the pandemic emerged in Gabon, Central Africa, less attention has been paid to other respiratory viral diseases, and very little data are available. Herein, we provide the first data on the genetic diversity and detection of 18 major respiratory viruses in Gabon during the COVID-19 pandemic. Of 582 nasopharyngeal swab specimens collected from March 2020 to July 2021, which were SARS-CoV-2 negative, 156 were positive (26%) for the following viruses: enterovirus (20.3%), human rhinovirus (HRV) (4.6%), human coronavirus OC43 (1.2%), human adenovirus (0.9%), human metapneumovirus (hMPV) (0.5%), influenza A virus (IAV) (0.3%), and human parainfluenza viruses (0.5%). To determine the genetic diversity and transmission route of the viruses, phylogenetic analyses were performed using genome sequences of the detected viruses. The IAV strain detected in this study was genetically similar to strains isolated in the USA, whereas the hMPV strain belonging to the A2b subtype formed a cluster with Kenyan strains. This study provides the first complete genomic sequences of HRV, IAV, and hMPV detected in Gabon, and provides insight into the circulation of respiratory viruses in the country.

Molecular basis of N-glycan recognition by pradimicin a and its potential as a SARS-CoV-2 entry inhibitor.

Virus entry inhibitors are emerging as an attractive class of therapeutics for the suppression of viral transmission. Naturally occurring pradimicin A (PRM-A) has received particular attention as the first-in-class entry inhibitor that targets N-glycans present on viral surface. Despite the uniqueness of its glycan-targeted antiviral activity, there is still limited knowledge regarding how PRM-A binds to viral N-glycans. Therefore, in this study, we performed binding analysis of PRM-A with synthetic oligosaccharides that reflect the structural motifs characteristic of viral N-glycans. Binding assays and molecular modeling collectively suggest that PRM-A preferentially binds to branched oligomannose motifs of N-glycans via simultaneous recognition of two mannose residues at the non-reducing ends. We also demonstrated, for the first time, that PRM-A can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Significantly, the anti-SARS-CoV-2 effect of PRM-A is attenuated in the presence of the synthetic branched oligomannose, suggesting that the inhibition of SARS-CoV-2 infection is due to the interaction of PRM-A with the branched oligomannose-containing N-glycans. These data provide essential information needed to understand the antiviral mechanism of PRM-A and suggest that PRM-A could serve as a candidate SARS-CoV-2 entry inhibitor targeting N-glycans.

Distribution of Aedes mosquito species along the rural-urban gradient in Lambaréné and its surrounding.

BACKGROUND: Aedes albopictus and Aedes aegypti are known for their potential as vectors of dengue (DENV) and chikungunya (CHIKV) viruses. However, entomological surveys are mostly carried out during epidemics. In Gabon where outbreaks of both viruses have occurred, there is no vector control program targeting these arboviruses. Therefore, we assessed the presence of Aedes species along a rural-urban gradient in Lambaréné (Gabon) and its surroundings and determined ecological factors associated to their presence. METHODS: An entomological survey was conducted in Lambaréné and its surrounding rural areas. Mosquitoes were collected with aspirators around human dwellings, and ecological and environmental data were collected from each study area. Morphological identification keys were used to identify Aedes species. RNA was extracted from pools of female mosquitoes and amplified by RT-qPCR to detect the presence of DENV and CHIKV. RESULTS: Overall, the most common vector collected was Aedes albopictus (97%, 4236/4367 specimens), followed by Aedes aegypti (3%, 131/4367). Albopictus vectors was more abundant in the rural area (Wilcoxon signed-rank test, Z = 627, P = 0.043) than in the urban area. In the urban area, a higher number of mosquitoes (45%) were recorded in the economic zone (zone 3) than in the historical and administrative zones (zone 1 and 2). In the rural area, the proportions of species numbers were significantly higher along the south rural transect (92%) compared to the north rural transect (Wilcoxon signed-rank test, Z = 43, P ˂ 0.016). We also noted a high abundance of vectors in environments characterized by monocultures of Hevea brasiliensis (Hevea) and Manihot esculenta (cassava) (Kruskal-Wallis H-test, H = 25.7, df = 2, P < 0.001). Finally, no mosquito pools were positive for either DENV or CHIKV. CONCLUSION: Aedes albopictus was the dominant vector across the study sites due to its high invasiveness capacity. This presence re-affirms the potential for local transmission of both DENV and CHIKV, as indicated previously by serological surveys conducted in our study area, even though no transmission was detected during the current study. These findings underscore the need for regular arbovirus surveillance in the study region, with the aim of supporting vector control efforts in the event of outbreaks.

Development of Quantitative Real-Time PCR and Loop-Mediated Isothermal Amplification Assays for the Surveillance and Diagnosis of Herpes B Virus Infection.

Herpes B virus (BV) is a zoonotic virus which can be transmitted from macaques to humans, which is often associated with high mortality rates. Because macaques often exhibit asymptomatic infections, individuals who come into contact with these animals face unexpected risks of BV infections. A serological test is widely performed to investigate BV infections. However, the assay's sensitivity and specificity appeared to be inadequate, and it does not necessarily indicate ongoing viral shedding. Here, we developed LAMP and qPCR assays aiming to detect BVs with a high sensitivity and specificity in various macaque species and validated them using oral swab samples collected from 97 wild cynomolgus macaques living in Thailand. Our LAMP and qPCR assays detected more than 50 and 10 copies of the target sequences per reaction, respectively. The LAMP assay could detect BV within 25 min, indicating its advantages for the rapid detection of BV. Collectively, our findings indicated that both assays developed in this study exhibit advantages and usefulness for BV surveillance and the diagnosis of BV infections in macaques. Furthermore, for the first time, we determined the partial genome sequences of BVs detected in cynomolgus macaques in Thailand. Phylogenetic analysis revealed the species-specific evolution of BV within macaques.

Genetic Diversity of Hepatitis B and C Viruses Revealed by Continuous Surveillance from 2015 to 2021 in Gabon, Central Africa.

Viral hepatitis remains one of the largest public health concerns worldwide. Especially in Central Africa, information on hepatitis virus infections has been limited, although the prevalence in this region has been reported to be higher than the global average. To reveal the current status of hepatitis B and C virus (HBV and HCV) infections and the genetic diversity of the viruses, we conducted longitudinal surveillance in Gabon. We detected 22 HBV and 9 HCV infections in 2047 patients with febrile illness. Genetic analyses of HBV identified subgenotype A1 for the first time in Gabon and an insertion generating a frameshift to create an X-preC/C fusion protein. We also revealed that most of the detected HCVs belonged to the "Gabon-specific" HCV subtype 4e (HCV-4e), and the entire nucleotide sequence of the HCV-4e polyprotein was determined to establish the first reference sequence. The HCV-4e strains possessed resistance-associated substitutions similar to those of other HCV-4 strains, indicating that the use of direct-acting antiviral therapy may be complex. These results provide a better understanding of the current situation of hepatitis B and C virus infections in Central Africa and will help public health organizations develop effective countermeasures to eliminate chronic viral hepatitis in this region.

Characterization of novel, severely immunodeficient PrkdcΔex57/Δex57 mice.

Severely immunodeficient mice are useful for understanding the pathogenesis of certain tumors and for developing therapeutic agents for such tumors. In addition, engraftment of these mice with human hematopoietic cells can yield information that helps us understand the in vivo molecular mechanisms underlying actual human viral infections. In our present research, we discovered a novel, severely immunodeficient strain of mice having a mutation in exon 57 of the Prkdc gene (PrkdcΔex57/Δex57) in an inbred colony of B10.S/SgSlc mice. Those PrkdcΔex57/Δex57 mice showed thymic hypoplasia and lack of mature T cells and B cells in peripheral lymphoid tissues, resulting in very low levels of production of serum immunoglobulins. In addition, those mice were highly susceptible to influenza viruses due to the lack of acquired immune cells. On the other hand, since they had sufficient numbers of NK cells, they rejected tumor transplants, similarly to Prkdc+/+ mice. Next, we generated Foxn1nu/nuPrkdcΔex57/Δex57Il2rg-/- (NPG) mice on the BALB/cSlc background, which lack all lymphocytes such as T cells, B cells and innate lymphoid cells, including NK cells. As expected, these mice were able to undergo engraftment of human tumor cell lines. These findings suggest that PrkdcΔex57/Δex57 mice will be useful as a novel model of immunodeficiency, while NPG mice will be useful for xenografting of various malignancies.

Comparison of genome replication fidelity between SARS-CoV-2 and influenza A virus in cell culture.

Since the emergence of COVID-19, several SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) variants have emerged and spread widely. These variants are produced through replication errors of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Seasonal epidemics of influenza are also known to occur because of new variants of influenza A virus (IAV), which are generated by the introduction of mutations by viral RdRp with low fidelity. Variants with different antigenicities appear because of mutations in envelope glycoproteins. In this study, we calculated and compared the mutation rates in genome replication of IAV and SARS-CoV-2. Average mutation rates per passage were 9.01 × 10-5 and 3.76 × 10-6 substitutions/site for IAV and SARS-CoV-2, respectively. The mutation rate of SARS-CoV-2 was 23.9-fold lower than that of IAV because of the proofreading activity of the SARS-CoV-2 RdRp complex. Our data could be useful in establishing effective countermeasures against COVID-19.

The NSs protein of severe fever with thrombocytopenia syndrome virus differentially inhibits the type 1 interferon response among animal species.

Severe fever with thrombocytopenia syndrome virus (SFTSV), which has been reported in China, Korea, Japan, Vietnam, and Taiwan, is a causative agent of severe fever thrombocytopenia syndrome. This virus has a high mortality and induces thrombocytopenia and leukocytopenia in humans, cats, and aged ferrets, whereas immunocompetent adult mice infected with SFTSV never show symptoms. Anti-SFTSV antibodies have been detected in several animals-including goats, sheep, cattle, and pigs. However, there are no reports of severe fever thrombocytopenia syndrome in these animals. Previous studies have reported that the nonstructural protein NSs of SFTSV inhibits the type I interferon (IFN-I) response through the sequestration of human signal transducer and activator of transcription (STAT) proteins. In this study, comparative analysis of the function of NSs as IFN antagonists in human, cat, dog, ferret, mouse, and pig cells revealed a correlation between pathogenicity of SFTSV and the function of NSs in each animal. Furthermore, we found that the inhibition of IFN-I signaling and phosphorylation of STAT1 and STAT2 by NSs depended on the binding ability of NSs to STAT1 and STAT2. Our results imply that the function of NSs in antagonizing STAT2 determines the species-specific pathogenicity of SFTSV.

Calcium Influx Regulates the Replication of Several Negative-Strand RNA Viruses Including Severe Fever with Thrombocytopenia Syndrome Virus.

Negative-strand RNA viruses (NSVs) represent one of the most threatening groups of emerging viruses globally. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic emerging virus that was initially reported in 2011 from China. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV. Here, L-type calcium channel blockers obtained from a U.S. Food and Drug Administration (FDA)-approved compound library were identified as effective anti-SFTSV compounds. Manidipine, a representative L-type calcium channel blocker, restricted SFTSV genome replication and exhibited inhibitory effects against other NSVs. The result from the immunofluorescent assay suggested that manidipine inhibited SFTSV N-induced inclusion body formation, which is believed to be important for the virus genome replication. We have shown that calcium possesses at least two different roles in regulating SFTSV genome replication. Inhibition of calcineurin, the activation of which is triggered by calcium influx, using FK506 or cyclosporine was shown to reduce SFTSV production, suggesting the important role of calcium signaling on SFTSV genome replication. In addition, we showed that globular actin, the conversion of which is facilitated by calcium from filamentous actin (actin depolymerization), supports SFTSV genome replication. We also observed an increased survival rate and a reduction of viral load in the spleen in a lethal mouse model of SFTSV infections after manidipine treatment. Overall, these results provide information regarding the importance of calcium for NSV replication and may thereby contribute to the development of broad-scale protective therapies against pathogenic NSVs. IMPORTANCE SFTS is an emerging infectious disease and has a high mortality rate of up to 30%. There are no licensed vaccines or antivirals against SFTS. In this article, L-type calcium channel blockers were identified as anti-SFTSV compounds through an FDA-approved compound library screen. Our results showed the involvement of L-type calcium channel as a common host factor for several different families of NSVs. The formation of an inclusion body, which is induced by SFTSV N, was inhibited by manidipine. Further experiments showed that SFTSV replication required the activation of calcineurin, a downstream effecter of the calcium channel. In addition, we identified that globular actin, the conversion of which is facilitated by calcium from filamentous actin, supports SFTSV genome replication. We also observed an increased survival rate in a lethal mouse model of SFTSV infection after manidipine treatment. These results facilitate both our understanding of the NSV replication mechanism and the development of novel anti-NSV treatment.

The roles of BST-2 in murine B cell development and on virus propagation.

The bone marrow (BM) stromal cell antigen-2 (BST-2), also known as tetherin, CD317, PDCA-1, or HM1.24, is a membrane protein overexpressed in several types of tumors and may act as a promising target for cancer treatment via antibody-dependent cellular cytotoxicity. BST-2 is also expressed in human BM stromal cells (BMSC), which support B cell development. While the activity of BST-2 as an antiviral factor has been demonstrated, the expression patterns and the role of BST-2 on B-cell development and activation have not been investigated, especially in vivo. In this study, Bst2 knockout (Bst2-/- ) mice were generated to assess the role of BST-2 on B cell development and activation. It was observed that BST-2 was not expressed in BMSC or all B cell progenitors even in wild-type mice and does not play a significant role in B cell development. In addition, the loss of BST-2 had no effect on B cell activation. Furthermore and in contrast to the well-known antiviral role of BST-2, infection of vesicular stomatitis Indiana virus to the BM cells collected from the Bst2-/- mice produced less infectious virus compared with that from the WT mice. These results suggest that murine BST-2 is different from human BST-2 in the expression pattern, physiological function, in vivo, and might possess positive role on VSV replication.

Long-term validation of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 from March 2020 to October 2021 in Central Africa, Gabon.

BACKGROUND: Despite the development of several methods for diagnosing COVID-19, long-term validation of such methods remains limited. In the early phase of the COVID-19 pandemic, we developed a rapid and sensitive diagnostic method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology, which is suitable for point-of-care application or for use in resource-limited settings to detect SARS-CoV-2. To assess the applicability of the RT-LAMP assay technique to resource-limited regions, such as rural areas in Africa, and to verify the usability of the method against various SARS-CoV-2 variants, the method was validated using clinical samples collected longitudinally during the pandemic. METHODOLOGY/PRINCIPAL FINDINGS: First, the sensitivity of the RT-LAMP assay for detecting 10 SARS-CoV-2 variants was evaluated using viral RNA samples extracted from cell culture with a portable battery-supported device, resulting in the successful detection of 20-50 copies of the viral genome within 15 min, regardless of the variant. COVID-19 positive samples collected in Gabon between March 2020 and October 2021 were used to evaluate the sensitivity of the assay and to calculate the copy number of the SARS-CoV-2 genome. More than 292 copies of the viral genome were detected with 100% probability within 15 min in almost all tests. CONCLUSIONS: This long-term validation study clearly demonstrated the applicability of the RT-LAMP assay for the clinical diagnosis of COVID-19 in resource-limited settings of Africa, such as rural areas in Gabon. The results show the potential of the assay as a promising COVID-19 diagnostic method, especially in rural and remote regions located far from the official diagnosis facilities in urban or semi-urban areas.

Re-emergence of dengue, chikungunya, and Zika viruses in 2021 after a 10-year gap in Gabon.

Mosquito-borne viral infections are a major concern in endemic areas, such as Africa. Although outbreaks have been reported throughout Africa, only a few surveillance studies have been conducted in Gabon since the outbreaks of dengue virus (DENV) and chikungunya virus (CHIKV) in 2010. Therefore, the current situation is unknown. This study aimed to investigate the presence of arboviruses, especially DENV (serotypes 1-4), CHIKV, and Zika virus (ZIKV), in Gabon, Central Africa. Between 2020 and 2021, we collected 1060 serum samples from febrile patients and screened them against viruses using reverse transcription-quantitative PCR. We detected two DENV serotypes 1 (DENV-1), one CHIKV, and one ZIKV, and subsequently analyzed the genome sequences. To determine the genetic diversity and transmission route of the viruses, phylogenetic analysis was performed using complete or partial genome sequences. The DENV-1 and CHIKV strains detected in this study were closely related to the previous Gabonese strains, whereas the recent ZIKV strain was genetically different from a strain detected in 2007 in Gabon. This study provides new genomic information on DENV-1, CHIKV, and ZIKV that were detected in Gabon and insight into the circulation of the viruses in the country and their introduction from neighboring African countries.

Identification of novel orthonairoviruses from rodents and shrews in Gabon, Central Africa.

In Africa, several emerging zoonotic viruses have been transmitted from small mammals such as rodents and shrews to humans. Although no clinical cases of small mammal-borne viral diseases have been reported in Central Africa, potential zoonotic viruses have been identified in rodents in the region. Therefore, we hypothesized that there may be unrecognized zoonotic viruses circulating in small mammals in Central Africa. Here, we investigated viruses that have been maintained among wild small mammals in Gabon to understand their potential risks to humans. We identified novel orthonairoviruses in 24.6 % of captured rodents and shrews from their kidney total RNA samples. Phylogenetic analysis revealed that the novel viruses, Lamusara virus (LMSV) and Lamgora virus, were closely related to Erve virus, which was previously identified in shrews of the genus Crocidura and has been suspected to cause neuropathogenic diseases in humans. Moreover, we show that the LMSV ovarian tumour domain protease, one of the virulence determination factors of orthonairoviruses, suppressed interferon signalling in human cells, suggesting the possible human pathogenicity of this virus. Taken together, our study demonstrates the presence of novel orthonairoviruses that may pose unrecognized risks of viral disease transmission in Gabon.

Potential and action mechanism of favipiravir as an antiviral against Junin virus.

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.

5-Aminolevulinic acid antiviral efficacy against SARS-CoV-2 omicron variant in vitro.

The coronavirus disease 2019 (COVID 19) pandemic continues to pose a threat to global health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529) has spread rapidly worldwide and became dominant in many countries. A natural 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) has demonstrated antiviral activity in Wuhan, Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 infections in vitro. In this study, we report antiviral activity of 5-ALA, 5-ALA with SFC led to IC50 of 329 and 765/191, respectively after infection with Omicron variant of SARS-CoV-2 in vitro. Our finding suggests that 5-ALA could be used as antiviral drug candidate to treat Omicron variant infected patients.

A screen of FDA-approved drugs with minigenome identified tigecycline as an antiviral targeting nucleoprotein of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus and is the causative agent of a viral hemorrhagic disease with a case fatality rate of 30%. However, limited studies have been conducted to explore antiviral compounds specific to CCHFV. In this study, we developed a minigenome system of orthonairoviruses, CCHFV and Hazara virus to analyze viral replication and screened an FDA-approved compound library. The transfection of the minigenome components induced marked increase in luciferase expression, indicating the sufficient replication and translation of reporter RNA. Compound library screening identified 14 candidate compounds that significantly decreased luciferase activity. Some of the compounds also inhibited the replication of the infectious Hazara virus. The mechanism of inhibition by tigecycline was further analyzed, and a decrease in the interaction between the viral N protein and RNA by tigecycline was observed. This work provides a basis for validation using animal models and the design of chemical derivatives with stronger activity in future studies on the development of an antiviral against CCHFV.

Identification of novel chemical compounds targeting filovirus VP40-mediated particle production.

The central role of Ebola virus (EBOV) VP40 in nascent virion assembly and budding from infected host cells makes it an important therapeutic target. The mechanism of dimerization, following oligomerization of VP40 leading to the production of virus-like particles (VLP) has never been investigated for the development of therapeutic candidates against Ebola disease. Molecular dynamics-based computational screening targeted VP40 dimer with 40,000,000 compounds selected 374 compounds. A novel in vitro screening assay selected two compounds, NUSU#1 and NUSU#2. Conventional VLP assays consistently showed that both compounds inhibited EBOV VP40-mediated VLP production. Intriguingly, NUSU#1 inhibited the VP40-mediated VLP production in other ebolavirus species and the Marburg virus, but did not inhibit Lassa virus Z-mediated VLP production. These results strongly suggested that the selected compounds are potential lead drug candidates against Filovirus disease via disruption of VP40-mediated particle production.

Ebola Virus GP Activates Endothelial Cells via Host Cytoskeletal Signaling Factors.

Ebola virus disease (EVD) is a lethal disease caused by the highly pathogenic Ebola virus (EBOV), and its major symptoms in severe cases include vascular leakage and hemorrhage. These symptoms are caused by abnormal activation and disruption of endothelial cells (ECs) whose mediators include EBOV glycoprotein (GP) without the need for viral replication. However, the detailed molecular mechanisms underlying virus-host interactions remain largely unknown. Here, we show that EBOV-like particles (VLPs) formed by GP, VP40, and NP activate ECs in a GP-dependent manner, as demonstrated by the upregulation of intercellular adhesion molecules-1 (ICAM-1) expression. VLPs-mediated ECs activation showed a different kinetic pattern from that of TNF-α-mediated activation and was associated with apoptotic ECs disruption. In contrast to TNF-α, VLPs induced ICAM-1 overexpression at late time points. Furthermore, screening of host cytoskeletal signaling inhibitors revealed that focal adhesion kinase inhibitors were found to be potent inhibitors of ICAM-1 expression mediated by both TNF-α and VLPs. Our results suggest that EBOV GP stimulates ECs to induce endothelial activation and dysfunction with the involvement of host cytoskeletal signaling factors, which represent potential therapeutic targets for EVD.

Unique Evolution of SARS-CoV-2 in the Second Large Cruise Ship Cluster in Japan.

In the initial phase of the novel coronavirus disease (COVID-19) pandemic, a large-scale cluster on the cruise ship Diamond Princess (DP) emerged in Japan. Genetic analysis of the DP strains has provided important information for elucidating the possible transmission process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on a cruise ship. However, genome-based analyses of SARS-CoV-2 detected in large-scale cruise ship clusters other than the DP cluster have rarely been reported. In the present study, whole-genome sequences of 94 SARS-CoV-2 strains detected in the second large cruise ship cluster, which emerged on the Costa Atlantica (CA) in Japan, were characterized to understand the evolution of the virus in a crowded and confined place. Phylogenetic and haplotype network analysis indicated that the CA strains were derived from a common ancestral strain introduced on the CA cruise ship and spread in a superspreading event-like manner, resulting in several mutations that might have affected viral characteristics, including the P681H substitution in the spike protein. Moreover, there were significant genetic distances between CA strains and other strains isolated in different environments, such as cities under lockdown. These results provide new insights into the unique evolution patterns of SARS-CoV-2 in the CA cruise ship cluster.