Generation of single-round infectious rotavirus with a mutation in the intermediate capsid protein VP6.

Rotavirus causes severe diarrhea in infants. Although live attenuated rotavirus vaccines are available, vaccine-derived infections have been reported, which warrants development of next-generation rotavirus vaccines. A single-round infectious virus is a promising vaccine platform; however, this platform has not been studied extensively in the context of rotavirus. Here, we aimed to develop a single-round infectious rotavirus by impairing the function of the viral intermediate capsid protein VP6. Recombinant rotaviruses harboring mutations in VP6 were rescued using a reverse genetics system. Mutations were targeted at VP6 residues involved in virion assembly. Although the VP6-mutated rotavirus expressed viral proteins, it did not produce progeny virions in wild-type cells; however, the virus did produce progeny virions in VP6-expressing cells. This indicates that the VP6-mutated rotavirus is a single-round infectious rotavirus. Insertion of a foreign gene, and replacement of the VP7 gene segment with that of human rotavirus clinical isolates, was successful. No infectious virions were detected in mice infected with the single-round infectious rotavirus. Immunizing mice with the single-round infectious rotavirus induced neutralizing antibody titers as high as those induced by wild-type rotavirus. Taken together, the data suggest that this single-round infectious rotavirus has potential as a safe and effective rotavirus vaccine. This system is also applicable for generation of safe and orally administrable viral vectors.IMPORTANCERotavirus, a leading cause of acute gastroenteritis in infants, causes an annual estimated 128,500 infant deaths worldwide. Although live attenuated rotavirus vaccines are available, they are replicable and may cause vaccine-derived infections. Thus, development of safe and effective rotavirus vaccine is important. In this study, we report the development of a single-round infectious rotavirus that can replicate only in cells expressing viral VP6 protein. We demonstrated that (1) the single-round infectious rotavirus did not replicate in wild-type cells or in mice; (2) insertion of foreign genes and replacement of the outer capsid gene were possible; and (3) it was as immunogenic as the wild-type virus. Thus, the mutated virus shows promise as a next-generation rotavirus vaccine. The system is also applicable to orally administrable viral vectors, facilitating development of vaccines against other enteric pathogens.

Characterization of Sialic Acid-Independent Simian Rotavirus Mutants in Viral Infection and Pathogenesis.

Rotaviruses (RVs) are nonenveloped viruses that cause gastroenteritis in infants and young children. Sialic acid is an initial receptor, especially for animal RVs, including rhesus RV. Sialic acid binds to the VP8* subunit, a part of the outer capsid protein VP4 of RV. Although interactions between virus and glycan receptors influence tissue and host tropism and viral pathogenicity, research has long been limited to biochemical and structural studies due to the unavailability of an RV reverse genetics system. Here, we examined the importance of sialic acid in RV infections using recombinant RVs harboring mutations in sialic acid-binding sites in VP4 via a simian RV strain SA11-based reverse genetics system. RV VP4 mutants that could not bind to sialic acid had replicated to decreased viral titer in MA104 cells. Wild-type virus infectivity was reduced, while that of VP4 mutants was not affected in sialic acid-deficient cells. Unexpectedly, in vivo experiments demonstrated that VP4 mutants suppressed mouse pups' weight gain and exacerbated diarrhea symptoms compared to wild-type viruses. Intestinal contents enhanced VP4 mutants' infectivity. Thus, possibly via interactions with other unknown receptors and/or intestinal contents, VP4 mutants are more likely than wild-type viruses to proliferate in the murine intestine, causing diarrhea and weight loss. These results suggest that RVs binding sialic acid notably affect viral infection in vitro and viral pathogenesis in vivo. IMPORTANCE Various studies have been conducted on the binding of VP8* and glycans, and the direct interaction between purified VP8* and glycans has been investigated by crystalline structure analyses. Here, we used a reverse genetics system to generate rotaviruses (RVs) with various VP4 mutants. The generated mutant strains clarified the importance of glycan binding in vitro and in vivo. Moreover, even when VP4 mutants could not bind to sialic acid, they were able to bind to an unknown receptor. As RVs evolve, pathogenicity can also be modified by easily altering the glycans to which VP4 binds.

Genetic engineering strategy for generating a stable dsRNA virus vector using a virus-like codon-modified transgene.

IMPORTANCE: The stabilities of transgenes in RNA virus vectors differ between the genes of interest, but the molecular mechanisms determining genetic stability remain unknown. This study demonstrated that the stability of a transgene was affected by the nucleotide composition, and altering the codon usage of transgenes to resemble that of the viral genome significantly increased transgene stability in double-stranded RNA virus vectors. The virus-like codon modification strategy enabled generation of stable rotavirus and mammalian orthoreovirus vectors, which could be developed as machinery for gene delivery to the intestines and/or respiratory organs. This technology has further potential to be expanded to other RNA viruses.

N-Glycosylation of Rotavirus NSP4 Protein Affects Viral Replication and Pathogenesis.

Rotavirus (RV), the most common cause of gastroenteritis in children, carries a high economic and health burden worldwide. RV encodes six structural proteins and six nonstructural proteins (NSPs) that play different roles in viral replication. NSP4, a multifunctional protein involved in various viral replication processes, has two conserved N-glycosylation sites; however, the role of glycans remains elusive. Here, we used recombinant viruses generated by a reverse genetics system to determine the role of NSP4 N-glycosylation during viral replication and pathogenesis. The growth rate of recombinant viruses that lost one glycosylation site was as high as that of the wild-type virus. However, a recombinant virus that lost both glycosylation sites (glycosylation-defective virus) showed attenuated replication in cultured cell lines. Specifically, replications of glycosylation-defective virus in MA104 and HT29 cells were 10- and 100,000-fold lower, respectively, than that of the wild-type, suggesting that N-glycosylation of NSP4 plays a critical role in RV replication. The glycosylation-defective virus showed NSP4 mislocalization, delay of cytosolic Ca2+ elevation, and less viroplasm formation in MA104 cells; however, these impairments were not observed in HT29 cells. Further analysis revealed that assembly of glycosylation-defective virus was severely impaired in HT29 cells but not in MA104 cells, suggesting that RV replication mechanism is highly cell type dependent. In vivo mouse experiments also showed that the glycosylation-defective virus was less pathogenic than the wild-type virus. Taken together, the data suggest that N-glycosylation of NSP4 plays a vital role in viral replication and pathogenicity. IMPORTANCE Rotavirus is the main cause of gastroenteritis in young children and infants worldwide, contributing to 128,500 deaths each year. Here, we used a reverse genetics approach to examine the role of NSP4 N-glycosylation. An N-glycosylation-defective virus showed attenuated and cell-type-dependent replication in vitro. In addition, mice infected with the N-glycosylation-defective virus had less severe diarrhea than mice infected with the wild type. These results suggest that N-glycosylation affects viral replication and pathogenesis. Considering the reduced pathogenicity in vivo and the high propagation rate in MA104 cells, this glycosylation-defective virus could be an ideal live attenuated vaccine candidate.

Genotypic Analysis of Transmitted and Acquired HIV Drug Resistance in People Living with HIV/AIDS in Surabaya, Indonesia, from 2018 to 2019.

BACKGROUND: Despite the availability of various effective antiretroviral (ARV) drugs, human immunodeficiency virus (HIV) infection has come with HIV drug resistance (HIVDR), which compromises its effectiveness in reducing HIV-related morbidity, mortality, and transmission. The emergence of transmitted (TDR) and acquired HIVDR (ADR) among antiretroviral therapy (ART)-naïve and experienced individuals have been reported in several Indonesian regions. Therefore, continuous HIVDR surveillance is needed in Indonesia, especially in Surabaya, which is identified as having the highest prevalence of HIV infection in East Java; thus, this study aimed to identify the emergence of TDR and ADR among people living with HIV/acquired immune deficiency syndrome (AIDS) (PLWHA). METHODS: Fifty-eight PLWHA infected with HIV type 1 (HIV-1), comprising 21 and 37 ART-naïve and experienced individuals were enrolled in this study, respectively. Blood samples collected from study participants were subjected to genotypic analysis, mainly towards the pol gene encoding protease (PR gene) and reverse transcriptase (RT gene) of HIV-1. RESULTS: Seventeen PR and 21 RT genes were successfully amplified and sequenced from 29 samples. HIV-1 subtyping revealed CRF01_AE as the most dominant subtype (24/29; 82.76%), followed by subtype B (3/29; 10.34%). Uncommon subtypes, including subtype D and a recombinant containing subtypes B and G genomic fragments, were also identified. TDR for PR inhibitors was not detected; however, TDR and ADR for RT inhibitors were identified in 11.11% and 41.67% of samples, respectively. Two amino acid insertions at position 69 of the RT gene (69ins), a previously never-reported mutation in Indonesia, were identified in this study. CONCLUSION: Both TDR and ADR have emerged among PLWHA residing in Surabaya, East Java, Indonesia. Uncommon drug-resistance mutations and subtypes were identified in this study. These situations might hamper ART efficacy and treatment success. Continuous surveillance of HIVDR is necessary to monitor both TDR and ADR in Indonesia.

The Dominance of CRF01_AE and the Emergence of Drug Resistance Mutations Among Antiretroviral Therapy-Experienced, HIV-1-infected Individuals in Medan, Indonesia.

BACKGROUND: human immunodeficiency virus type 1 (HIV-1) infection is a serious public health threat worldwide. Medan is one example of big cities in Indonesia with a high prevalence of HIV-1 infection; however, quite a limited study had conducted for detecting the circulation of HIV-1 subtypes in Medan. In addition, a serious factor that can implicate the treatment of HIV-1-infected individuals is the emergence of drug resistance mutations. Thus, the information on HIV-1 infection is important to improve the treatment for infected individuals. METHODS: sixty-seven antiretroviral therapy-experienced, HIV-1-infected individuals were recruited for this study. HIV-1 pol genes encoding protease (PR genes) and reverse transcriptase (RT gene), as well as env and gag genes, were amplified from DNA derived from peripheral blood samples. HIV-1 subtyping was conducted to study the dominant HIV-1 subtype circulating in the region. In addition, the emergence of drug resistance mutations was analyzed based on the guidelines published by the International Antiviral Society-United States of America (IAS-USA). RESULTS: the dominant HIV-1 subtype found in Medan was CRF01_AE (77.6%). In addition, another subtype and recombinant viruses such as recombinants between CRF01_AE and subtype B (12.2%), subtype B (4.1%), and CRF02_AG (4.1%) were also found. Drug resistance-associated major mutations were found in 21.6% (8/37) of RT genes and 3.1% (1/32) of PR genes studied. CONCLUSION: our study showed that the dominant subtype found in ART-experienced, HIV-1-infected individuals residing in Medan was CRF01_AE. The emergence of drug resistance mutations in RT and PR genes indicated the importance to monitor the prevalence of drug resistance mutations among HIV-1-infected individuals in Medan.

Clinical Risk Factors for Death Caused by Extended-Spectrum Beta-Lactamase: Producing Bacteria.

OBJECTIVES: Extended-spectrum beta-lactamase (ESBL)-producing bacteria often causes bacteremia, leading serious outcomes. In this study, we conducted a retrospective analysis to identify the risk factors associated with death by bacteremia of ESBL-producing bacteria. METHODS: Patients with bacteremia by ESBL-producing bacteria were retrospectively collected in Kobe University Hospital, Japan, between January 2011 and December 2015. Potential risk factors for death caused by ESBL-bacteremia were analyzed for patients' outcome (recovery or death) by univariate and multivariate analysis. RESULTS: A total of 101 patients (64 male and 37 female) were recruited. The most frequently detected ESBL-producing bacteria were Escherichia coli (91 cases; 90.1%), followed by Klebsiella pneumoniae (8 cases; 7.9%). Most frequently used antibiotics after the detection of bacteremia was meropenem (66.3%; 67/101) followed by cefmetazole (51.5%; 52/101). Univariate analysis showed a significantly positive correlation with mortality in ICU admission (p < 0.001), circulatory diseases (p = 0.022), shock (p = 0.044), and respirator requirement (p = 0.002). Multivariate analysis showed ICU admission remained significant risk factor for mortality (p = 0.0192). CONCLUSIONS: We showed ICU admission was significantly correlated with death from bacteremia by ESBL-producing bacteria. These factors should be monitored to estimate severity of ESBL causing-bacteremia for better patients' outcomes.

The Emergence of HIV-1 Transmitted Drug Resistance Mutations Among Antiretroviral Therapy-naive Individuals in Buleleng, Bali, Indonesia.

BACKGROUND: the global scale-up of antiretroviral therapy (ART) is the primary factor contributing to the decline in deaths from acquired immune deficiency syndrome (AIDS)-related illnesses. However, the emergence of transmitted drug resistance (TDR) compromises the effects of ART in treatment-naïve individuals, which may hinder treatment success. The present study aimed to identify the presence of TDR among treatment-naive individuals in Buleleng, Bali, which is currently ranked sixth among Indonesian provinces with the highest cumulative human immunodeficiency virus type 1 (HIV-1) infection cases. METHODS: thirty-nine ART-naive individuals in Buleleng Regency General Hospital were enrolled in the present study. Blood samples from participants were subjected to a genotypic analysis. RESULTS: 28 protease (PR) and 30 reverse transcriptase (RT) genes were successfully amplified and sequenced from 37 samples. HIV-1 subtyping revealed CRF01_AE as the dominant circulating recombinant form in the region. No TDR for PR inhibitors was detected; however, TDR for RT inhibitors was identified in five out of 30 samples (16.7%). CONCLUSION: these results indicate the emergence of TDR among ART-naive individuals in Buleleng, Bali. This issue warrants serious consideration because TDR may hamper treatment success and reduce ART efficacy among newly diagnosed individuals. Continuous surveillance with a larger sample size is necessary to monitor TDR among ART-naive individuals.

Sero- and Molecular Epidemiology of HIV-1 in Papua Province, Indonesia.

BACKGROUND: human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) cause serious health problems and affect the Indonesian economy. Papua province has the highest prevalence of HIV infection in the country; however, epidemiological data are limited. Therefore, in order to reveal the current situation of HIV/AIDS in Papua province, sero- and molecular epidemiological studies of HIV were conducted. METHODS: serological tests were conducted on 157 healthy individuals from the general population residing in Paniai, Papua. In addition, a molecular epidemiological study was then conducted on HIV type 1 (HIV-1) genes derived from infected individuals. Peripheral blood samples from HIV-1-positive individuals and 15 additionally enrolled, previously confirmed HIV-1-positive individuals were subjected to a genotypic analysis. RESULTS: serological tests revealed that 2 out of 157 (1.27%) healthy individuals were HIV-positive. In addition, HIV-1 subtyping revealed that subtype B and CRF01_AE were the major subtype and circulating recombinant form (CRF) of HIV-1 prevalent in the region, while subtype A1 and a recombinant form including viral gene fragments of CRF01_AE and subtype B was also detected. In addition, HIV drug resistance-associated major mutations were detected in the reverse transcriptase gene derived from infected individual on antiretroviral therapy. CONCLUSION: these results provide important information for clearer understanding on the current situation of HIV/AIDS in Papua province in Indonesia.

Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

HIV-1 transmitted drug resistance mutations among antiretroviral therapy-Naïve individuals in Surabaya, Indonesia.

BACKGROUND: The emergence of transmitted drug resistance (TDR) compromises the effect of antiretroviral therapy (ART), resulting in treatment failure of human immunodeficiency virus (HIV) disease. Although more than a decade has passed since ART was introduced into Indonesia, information on TDR is limited. Here, a genotypic study of TDR among ART-naïve individuals was conducted in Surabaya, Indonesia. METHOD: HIV-1 seropositive participants were recruited from the communities of commercial sex workers and intravenous drug users as well as from the university teaching hospital in Surabaya. Protease (PR) and reverse transcriptase (RT) genes were sequenced in order to conduct HIV-1 subtyping and phylogenetic analysis and to detect TDR. TDR was defined as the presence of at least one surveillance drug resistance mutation on the WHO list or major drug resistance mutations in the International AIDS Society-USA panel. RESULT: Fifty two and 47 of the PR and RT genes, respectively, were successfully sequenced in the 58 samples. HIV-1 subtyping revealed that 86.3% (50/58) of the sequenced samples were classified as CRF01_AE, 8.6% as subtype B, 3.4% as B/CRF01_AE, and 1.7% as A/G/CRF01_AE. TDR of PR inhibitors was not detected in this study. In contrast, TDR of RT inhibitors was detected in 4.3% (2/47) of samples. In addition, minor drug resistance mutations were detected in 98.1% (51/52) and 12.8% (6/47) of PR and RT genes, respectively. CONCLUSION: This study clarified the predominance of the CRF01_AE strain in Surabaya, Indonesia. The prevalence of TDR was below 5%, indicating that the currently available first-line regimen is still effective in Surabaya. However, the prevalence might be underestimated since we detected only major population of HIV-1 in individuals. Therefore, continuous surveillance is required in order to detect the emergence of TDR in the early phase.

A mouse monoclonal antibody against dengue virus type 1 Mochizuki strain targeting envelope protein domain II and displaying strongly neutralizing but not enhancing activity.

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans.

Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE: The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

Spatial-temporal transmission dynamics of HIV-1 CRF01_AE in Indonesia.

Human immunodeficiency virus type 1 (HIV-1) remains a serious health threat in Indonesia. In particular, the CRF01_AE viruses were the predominant HIV-1 strains in various cities in Indonesia. However, information on the dynamic transmission characteristics and spatial-temporal transmission of HIV-1 CRF01_AE in Indonesia is limited. Therefore, the present study examined the spatial-temporal transmission networks and evolutionary characteristics of HIV-1 CRF01_AE in Indonesia. To clarify the epidemiological connection between CRF01_AE outbreaks in Indonesia and the rest of the world, we performed phylogenetic studies on nearly full genomes of CRF01_AE viruses isolated in Indonesia. Our results showed that five epidemic clades, namely, IDN clades 1-5, of CRF01_AE were found in Indonesia. To determine the potential source and mode of transmission of CRF01_AE, we performed Bayesian analysis and built maximum clade credibility trees for each clade. Our study revealed that CRF01_AE viruses were commonly introduced into Indonesia from Southeast Asia, particularly Thailand. The CRF01_AE viruses might have spread through major pandemics in Asian countries, such as China, Vietnam, and Laos, rather than being introduced directly from Africa in the early 1980s. This study has major implications for public health practice and policy development in Indonesia. The contributions of this study include understanding the dynamics of HIV-1 transmission that is important for the implementation of HIV disease control and prevention strategies in Indonesia.

The diversity of shell matrix proteins: genome-wide investigation of the pearl oyster, Pinctada fucata.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2-8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2-8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.

Subtype Distribution and Drug Resistance Patterns Among HIV-1 Strains Prevalent in Makassar, Indonesia.

Human immunodeficiency virus type 1 (HIV-1) is characterized by a large degree of genetic variability because of high rates of recombination and mutation, sizable population sizes, and rapid replication. Therefore, this study investigated HIV-1 subtype distribution and the appearance of drug resistance mutations (DRMs) in viruses that are prevalent in Makassar, South Sulawesi, Indonesia. The HIV-1 pol, env, and gag genes were amplified from 63 infected individuals and sequenced for a subtyping analysis. CRF01_AE was identified as the predominant HIV-1 circulating recombinant form (CRF) in Makassar, South Sulawesi, Indonesia. Subtype B and recombinant viruses containing CRF01_AE, CRF02_AG, and/or subtype B gene fragments were also detected. Several major DRMs against non-nucleoside reverse transcriptase inhibitors were found among antiretroviral therapy (ART)-experienced subjects, whereas ART-naive subjects did not possess any transmitted drug resistance. The prevalence of DRMs was very high among ART-experienced subjects; therefore, further surveillance is required in this region.

Effective SARS-CoV-2 replication of monolayers of intestinal epithelial cells differentiated from human induced pluripotent stem cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe acute respiratory symptoms in humans. Controlling the coronavirus disease pandemic is a worldwide priority. The number of SARS-CoV-2 studies has dramatically increased, and the requirement for analytical tools is higher than ever. Here, we propose monolayered-intestinal epithelial cells (IECs) derived from human induced pluripotent stem cells (iPSCs) instead of three-dimensional cultured intestinal organoids as a suitable tool to study SARS-CoV-2 infection. Differentiated IEC monolayers express high levels of angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), host factors essential for SARS-CoV-2 infection. SARS-CoV-2 efficiently grows in IEC monolayers. Using this propagation system, we confirm that TMPRSS2 inhibition blocked SARS-CoV-2 infection in IECs. Hence, our iPSC-derived IEC monolayers are suitable for SARS-CoV-2 research under physiologically relevant conditions.

Genotype Diversity of Enteric Viruses in Wastewater Amid the COVID-19 Pandemic.

Viruses remain the leading cause of acute gastroenteritis (AGE) worldwide. Recently, we reported the abundance of AGE viruses in raw sewage water (SW) during the COVID-19 pandemic, when viral AGE patients decreased dramatically in clinics. Since clinical samples were not reflecting the actual state, it remained important to determine the circulating strains in the SW for preparedness against impending outbreaks. Raw SW was collected from a sewage treatment plant in Japan from August 2018 to March 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major gastroenteritis viruses by RT-PCR. Genotypes and evolutionary relationships were evaluated through sequence-based analyses. Major AGE viruses like rotavirus A (RVA), norovirus (NoV) GI and GII, and astrovirus (AstV) increased sharply (10-20%) in SW during the COVID-19 pandemic, though some AGE viruses like sapovirus (SV), adenovirus (AdV), and enterovirus (EV) decreased slightly (3-10%). The prevalence remained top in the winter. Importantly, several strains, including G1 and G3 of RVA, GI.1 and GII.2 of NoV, GI.1 of SV, MLB1 of AstV, and F41 of AdV, either emerged or increased amid the pandemic, suggesting that the normal phenomenon of genotype changing remained active over this time. This study crucially presents the molecular characteristics of circulating AGE viruses, explaining the importance of SW investigation during the pandemic when a clinical investigation may not produce the complete scenario.

The Emergence and Widespread Circulation of Enteric Viruses Throughout the COVID-19 Pandemic: A Wastewater-Based Evidence.

Growing evidence shed light on the importance of wastewater-based epidemiology (WBE) during the pandemic, when the patients rarely visited the clinics despite the fact that the infections were still prevalent in the community as before. The abundance of infections in the community poses a constant threat of the emergence of new epidemic strains. Herein, we investigated enteric viruses in raw sewage water (SW) from Japan's Tohoku region and compared them to those from the Kansai region to better understand the circulating strains and their distribution across communities during the COVID-19 pandemic. Raw SW was collected between 2019 and 2022, concentrated by polyethylene-glycol-precipitation method, and investigated for major AGE viruses by RT-PCR. Sequence-based analyses were used to assess genotypes and evolutionary relationships. The most commonly detected enteric virus was rotavirus A (RVA) at 63.8%, followed by astrovirus (AstV) at 61.1%, norovirus (NoV) GII and adenovirus (AdV) at 33.3%, sapovirus (SV) at 25.0%, enterovirus (EV) at 19.4%, and NoV GI at 13.9%. The highest prevalence (46.0%) was found in the spring. Importantly, enteric viruses did not decline during the pandemic. Rather, several strains like NoV GII.2, DS-1-like human G3 (equine) RVA, MLB1 AstV, and different F41 HAdV emerged throughout the pandemic and spread widely over the Tohoku and Kansai regions. Tohoku's detection rate remained lower than that of the Kansai area (36 vs 58%). This study provides evidence for the emergence and spread of enteric viruses during the pandemic.