RESUMEN
The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCE The emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.
Asunto(s)
COVID-19 , Reinfección , Animales , Cricetinae , Mesocricetus , ARN Viral , SARS-CoV-2/genéticaRESUMEN
A community cluster of influenza A(H3N2) caused by viruses with an E199G substitution in PA was detected in Nara, Japan, between February and March 2023. The three patients with these mutant viruses had not received antiviral treatment before specimen collection but patients in the same hospital had. The sequences of the mutant viruses were closely related, suggesting clonal spread in Nara. They showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear.
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Gripe Humana , Tiepinas , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Subtipo H3N2 del Virus de la Influenza A/genética , Oxazinas/farmacología , Piridinas/farmacología , Japón , Tiepinas/farmacología , Tiepinas/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia Viral/genéticaRESUMEN
In 2019, influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic gene, which confers reduced susceptibility to baloxavir, were detected in Japan in an infant without baloxavir exposure and a baloxavir-treated sibling. These viruses' whole-genome sequences were identical, indicating human-to-human transmission. Influenza virus isolates should be monitored for baloxavir susceptibility.
Asunto(s)
Antivirales/farmacología , Susceptibilidad a Enfermedades , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/transmisión , Gripe Humana/virología , Oxazinas/farmacología , Piridinas/farmacología , Tiepinas/farmacología , Triazinas/farmacología , Adolescente , Adulto , Antivirales/uso terapéutico , Niño , Dibenzotiepinas , Humanos , Lactante , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Japón/epidemiología , Persona de Mediana Edad , Morfolinas , Mutación , Oxazinas/uso terapéutico , Piridinas/uso terapéutico , Piridonas , Tiepinas/uso terapéutico , Triazinas/uso terapéutico , Adulto JovenRESUMEN
The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.
Asunto(s)
Antivirales/uso terapéutico , Endonucleasas/antagonistas & inhibidores , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/tratamiento farmacológico , Oxazinas/uso terapéutico , Piridinas/uso terapéutico , Tiepinas/uso terapéutico , Triazinas/uso terapéutico , Sustitución de Aminoácidos/genética , Antivirales/administración & dosificación , Dibenzotiepinas , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Endonucleasas/genética , Humanos , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , Japón , Pruebas de Sensibilidad Microbiana , Morfolinas , Piridonas , Resultado del TratamientoRESUMEN
In January 2019, two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA), which confers reduced susceptibility to baloxavir, were detected from epidemiologically unrelated hospitalised children in Japan. The viruses exhibited reduced susceptibility to baloxavir but were susceptible to neuraminidase inhibitors. Only one of the two children had been treated with baloxavir. An epidemiological analysis suggests possible transmission of the PA I38T mutant A(H3N2) virus among humans.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Gripe Humana/tratamiento farmacológico , Oxazinas/farmacología , Piridinas/farmacología , Tiepinas/farmacología , Triazinas/farmacología , Adolescente , Adulto , Antivirales/uso terapéutico , Niño , Dibenzotiepinas , Inhibidores Enzimáticos/farmacología , Humanos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , Pacientes Internos , Japón , Persona de Mediana Edad , Morfolinas , Oxazinas/uso terapéutico , Reacción en Cadena de la Polimerasa , Piridinas/uso terapéutico , Piridonas , Tiepinas/uso terapéutico , Resultado del Tratamiento , Triazinas/uso terapéutico , Adulto JovenRESUMEN
BACKGROUND: Influenza A(H3N2) virus rapidly evolves to evade human immune responses, resulting in changes in the antigenicity of haemagglutinin (HA). Therefore, continuous genetic and antigenic analyses of A(H3N2) virus are necessary to detect antigenic mutants as quickly as possible. AIM: We attempted to phylogenetically and antigenically capture the epidemic trend of A(H3N2) virus infection in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons. METHODS: We determined the HA sequences of A(H3N2) viruses detected in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons to identify amino acid substitutions and the loss or gain of potential N-glycosylation sites in HA, both of which potentially affect the antigenicity of HA. We also examined the antigenicity of isolates using ferret antisera obtained from experimentally infected ferrets. RESULTS: Influenza A(H3N2) viruses belonging to six clades (clades 3C.2A1, 3C.2A1a, 3C.2A1b, 3C.2A2, 3C.2A3 and 3C.2A4) were detected during the 2016/17 influenza season, whereas viruses belonging to two clades (clades 3C.2A1b and 3C.2A2) dominated during the 2017/18 influenza season. The isolates in clades 3C.2A1a and 3C.2A3 lost one N-linked glycosylation site in HA relative to other clades. Antigenic analysis revealed antigenic differences among clades, especially clade 3C.2A2 and 3C.2A4 viruses, which showed distinct antigenic differences from each other and from other clades in the antigenic map. CONCLUSION: Multiple clades, some of which differed antigenically from others, co-circulated in Yokohama, Japan during the 2016/17 and 2017/18 influenza seasons.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/diagnóstico , ARN Viral/genética , Epidemias , Variación Genética , Hemaglutininas/genética , Humanos , Gripe Humana/epidemiología , Japón/epidemiología , Datos de Secuencia Molecular , Filogenia , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estaciones del Año , Análisis de Secuencia de ADNRESUMEN
In Japan, influenza activity was low throughout the COVID-19 pandemic until the 2022-23 season, when the first influenza outbreak occurred since the 2020-21 season. In our influenza surveillance during the COVID-19 pandemic, co-infection with SARS-CoV-2 and influenza virus had not been detected; however, in January 2024, we identified three pediatric outpatients co-infected with these viruses: one with SARS-CoV-2 Omicron EG.5 sublineage HK.3 and influenza A(H3N2) and two with SARS-CoV-2 Omicron BA.2.86 sublineage JN.1.5 and influenza A(H1N1)pdm09. We evaluated the susceptibility of SARS-CoV-2 against RNA-dependent RNA polymerase inhibitors (remdesivir and molnupiravir) and 3C-like protease inhibitors (nirmatrelvir and ensitrelvir), and that of influenza viruses against neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir) and the cap-dependent endonuclease inhibitor baloxavir. All viruses tested were susceptible to these antiviral drugs and did not possess amino acid substitutions associated with reduced antiviral susceptibility. The patients were treated with anti-influenza drugs and did not develop severe symptoms despite the co-infection. Since SARS-CoV-2 and influenza viruses continue to evolve, continuous monitoring of their circulation remains essential to assess public health measures and support clinical management.
Asunto(s)
Antivirales , COVID-19 , Coinfección , Gripe Humana , SARS-CoV-2 , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Antivirales/uso terapéutico , Antivirales/farmacología , Coinfección/virología , Coinfección/tratamiento farmacológico , SARS-CoV-2/efectos de los fármacos , Niño , COVID-19/epidemiología , COVID-19/virología , Masculino , Preescolar , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Femenino , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Japón/epidemiología , Tratamiento Farmacológico de COVID-19RESUMEN
BACKGROUND: Influenza viruses can cause zoonotic infections that pose public health risks. Surveillance of influenza A and B viruses is conducted globally; however, information on influenza C and D viruses is limited. Longitudinal monitoring of influenza C virus in humans has been conducted in several countries, but there has been no long-term monitoring of influenza D virus in humans. The public health risks associated with the influenza D virus therefore remain unknown. METHODS: We established a duplex real-time RT-PCR to detect influenza C and D viruses and analyzed respiratory specimens collected from 2144 patients in Japan with respiratory diseases between January 2018 and March 2023. We isolated viruses and conducted hemagglutination inhibition tests to examine antigenicity and focus reduction assays to determine susceptibility to the cap-dependent endonuclease inhibitor baloxavir marboxil. RESULTS: We detected three influenza C viruses belonging to the C/Kanagawa- or C/Sao Paulo-lineages, which recently circulated globally. None of the specimens was positive for the influenza D virus. The C/Yokohama/1/2022 strain, isolated from the specimen with the highest viral RNA load and belonging to the C/Kanagawa-lineage, showed similar antigenicity to the reference C/Kanagawa-lineage strain and was susceptible to baloxavir. CONCLUSIONS: Our duplex real-time RT-PCR is useful for the simultaneous detection of influenza C and D viruses from the same specimen. Adding the influenza D virus to the monitoring of the influenza C virus would help in assessing the public health risks posed by this virus.
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Dibenzotiepinas , Gammainfluenzavirus , Gripe Humana , Piridonas , Triazinas , Humanos , Japón/epidemiología , Gripe Humana/virología , Gripe Humana/epidemiología , Triazinas/farmacología , Masculino , Femenino , Gammainfluenzavirus/aislamiento & purificación , Gammainfluenzavirus/genética , Persona de Mediana Edad , Adulto , Anciano , Antivirales/uso terapéutico , Antivirales/farmacología , Morfolinas , Pruebas de Inhibición de Hemaglutinación , Preescolar , Niño , Adolescente , Adulto Joven , Thogotovirus/genética , Thogotovirus/aislamiento & purificación , Thogotovirus/clasificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Lactante , Anciano de 80 o más AñosRESUMEN
We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.
RESUMEN
The emergence and spread of antiviral-resistant SARS-CoV-2 is of great concern. In this study, we evaluated the propensity of Omicron variants to escape from RNA-dependent RNA polymerase (RdRP) inhibitors and 3C-like protease (3CLpro) inhibitors. SARS-CoV-2 Delta and Omicron variants were serially passaged in vitro in the presence of RdRP inhibitors (remdesivir and molnupiravir) and 3CLpro inhibitors (nirmatrelvir and lufotrelvir) to detect SARS-CoV-2 escape mutants. After five passages with 3CLpro inhibitors, mutant viruses that escaped from 3CLpro inhibitors emerged; however, in the presence of RdRP inhibitors all variants disappeared within 2-4 passages. Our findings suggest that the frequency of SARS-CoV-2 mutant escape from RdRP inhibitors is lower than that from 3CLpro inhibitors. We also found that Delta variants were more likely to acquire amino acid substitutions associated with resistance to 3CLpro inhibitors under the selective pressure of this drug compared with Omicron variants.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/genética , Antivirales/farmacología , Leucina , ARN Polimerasa Dependiente del ARN/genética , Inhibidores de Proteasas/farmacologíaRESUMEN
The circulation of avian influenza A viruses in poultry is a public health concern due to the potential transmissibility and severity of these viral infections. Monitoring the susceptibility of these viruses to antivirals is important for developing measures to strengthen the level of preparedness against influenza pandemics. However, drug susceptibility information on these viruses is limited. Here, we determined the susceptibilities of avian influenza A(H5N1), A(H5N2), A(H5N8), A(H7N7), A(H7N9), A(H9N1), and A(H9N2) viruses isolated in Japan to the antivirals approved for use there: an M2 inhibitor (amantadine), neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir) and RNA polymerase inhibitors (baloxavir and favipiravir). Genotypic methods that detect amino acid substitutions associated with antiviral resistance and phenotypic methods that assess phenotypic viral susceptibility to drugs have revealed that these avian influenza A viruses are susceptible to neuraminidase and RNA polymerase inhibitors. These results suggest that neuraminidase and RNA polymerase inhibitors currently approved in Japan could be a treatment option against influenza A virus infections in humans.
Asunto(s)
Farmacorresistencia Viral , Gripe Aviar , Gripe Humana , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , ARN Polimerasas Dirigidas por ADN , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N7 del Virus de la Influenza A/efectos de los fármacos , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Subtipo H9N2 del Virus de la Influenza A/efectos de los fármacos , Gripe Aviar/epidemiología , Gripe Aviar/virología , Gripe Humana/epidemiología , Gripe Humana/virología , Japón/epidemiología , Neuraminidasa/genética , Neuraminidasa/metabolismo , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Aves de CorralRESUMEN
Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10- to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD(50)) of 10(2.5) PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10(5) PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10(5) PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.
Asunto(s)
Antígeno Carcinoembrionario/metabolismo , Glicoproteínas/metabolismo , Inmunidad Innata , Virus de la Hepatitis Murina/patogenicidad , Internalización del Virus , Alelos , Animales , Antígeno Carcinoembrionario/genética , Moléculas de Adhesión Celular , Glicoproteínas/genética , Homocigoto , Dosificación Letal Mediana , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de SupervivenciaRESUMEN
BACKGROUND: The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir. OBJECTIVES: Development of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA. METHODS: Three assays were developed based on RNase H2-dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro-transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season. RESULTS: The assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity. CONCLUSIONS: Our assays should be considered a powerful tool for monitoring the emergence of baloxavir-resistant influenza viruses.
Asunto(s)
Sustitución de Aminoácidos/efectos de los fármacos , Antivirales/farmacología , Dibenzotiepinas/farmacología , Farmacorresistencia Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Morfolinas/farmacología , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/genética , Piridonas/farmacología , Triazinas/farmacología , Sustitución de Aminoácidos/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/enzimología , Subtipo H3N2 del Virus de la Influenza A/genética , Virus de la Influenza B/efectos de los fármacos , Virus de la Influenza B/enzimología , Virus de la Influenza B/genética , Gripe Humana/diagnóstico , Gripe Humana/virología , Japón , Límite de Detección , Técnicas de Diagnóstico Molecular/normas , Orthomyxoviridae/enzimología , Ribonucleasa H/genética , Sensibilidad y EspecificidadRESUMEN
Human-to-human transmission of PA I38 mutant influenza A(H3N2) viruses with reduced baloxavir susceptibility has been reported in Japan. In December 2019, we detected a PA E23K mutant A(H1N1)pdm09 virus from a child without baloxavir treatment. The PA E23K mutant virus exhibited reduced baloxavir susceptibility but remained susceptible to neuraminidase inhibitors. Epidemiological data suggest possible transmission of this PA E23K mutant virus among humans, although its growth capability relative to that of the wild-type virus was reduced. Therefore, baloxavir susceptibility monitoring of influenza viruses is essential.
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Antivirales/farmacología , Dibenzotiepinas/farmacología , Farmacorresistencia Viral/genética , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Morfolinas/farmacología , Piridonas/farmacología , Triazinas/farmacología , Proteínas Virales/genética , Sustitución de Aminoácidos , Animales , Niño , Perros , Humanos , Gripe Humana/transmisión , Gripe Humana/virología , Japón , Células de Riñón Canino Madin DarbyRESUMEN
Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.
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Farmacorresistencia Viral/efectos de los fármacos , VIH-1/efectos de los fármacos , Esparsomicina/farmacología , Replicación Viral/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , VIH-1/genética , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Mutación , Streptococcaceae/metabolismo , TransfecciónRESUMEN
The integration of reverse transcribed proviral DNA into a host genome is an essential event in the human immunodeficiency virus type 1 (HIV-1) replication life cycle. Therefore, the viral enzyme integrase (IN), which plays a crucial role in the integration event, has been an attractive target of anti-retroviral drugs. Several IN inhibitory compounds have been reported previously, yet none has been successful in clinical use. To find a new, more successful IN inhibitor, we screened a diverse library of 12 000 small molecular weight compounds randomly by in vitro strand-transfer assay. We identified a series of substituted carbazoles that exhibit strand-transfer inhibitory activity at low micromolar concentrations. Of these, the most potent compound exhibited an IC50 of 5.00+/-3.31 microM (CA-0). To analyse the structural determinants of strand-transfer inhibitory activity of the carbazole derivatives, we selected 23 such derivatives from our compound library and performed further analyses. Of these 23 compounds, six showed strong strand-transfer inhibition. The inhibition kinetics analyses and ethidium bromide displacement assays indicated that the carbazole derivatives are competitive inhibitors and not intercalators. An HeLa4.5/LTR-nEGFP cell line was employed to evaluate in vitro virus replication inhibition of the carbazole derivatives, and IC50 levels ranged from 0.48-1.52 microM. Thus, it is possible that carbazole derivatives, which possess structures different from previously-reported IN inhibitors, may become novel lead compounds in the development of IN inhibitors.
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Fármacos Anti-VIH/farmacología , Carbazoles/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/uso terapéutico , Secuencia de Bases , Carbazoles/química , Carbazoles/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Técnicas de Transferencia de Gen , Humanos , Pruebas de Sensibilidad Microbiana , Oligodesoxirribonucleótidos , Relación Estructura-ActividadAsunto(s)
Ácidos Carbocíclicos/uso terapéutico , Antivirales/uso terapéutico , Farmacorresistencia Viral , Guanidinas/uso terapéutico , Virus de la Influenza B/genética , Gripe Humana/diagnóstico , Adulto , Dibenzotiepinas/uso terapéutico , Humanos , Indonesia , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Japón , Masculino , Pruebas de Sensibilidad Microbiana , Morfolinas/uso terapéutico , Mutación , Neuraminidasa/metabolismo , Piranos/uso terapéutico , Piridonas/uso terapéutico , Ácidos Siálicos/uso terapéutico , Triazinas/uso terapéutico , Zanamivir/uso terapéuticoRESUMEN
Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.
Asunto(s)
Fármacos Anti-VIH/farmacología , Genes Reporteros , VIH-1/efectos de los fármacos , Luciferasas/metabolismo , Linfocitos T/virología , Línea Celular , Farmacorresistencia Viral , VIH-1/patogenicidad , Humanos , Luciferasas/genética , Pruebas de Sensibilidad Microbiana , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Transfección , Replicación ViralRESUMEN
The mouse hepatitis virus (MHV) receptor (MHVR), CEACAM1, has two different functions for MHV entry into cells: binding to MHV spike protein (S protein) and activation of the S protein to execute virus-cell membrane fusion, the latter of which is accompanied by conformational changes of the S protein. The MHVR comprising the N-terminal and fourth domains [R1(1,4)] displays these two activities, and the N domain is thought to be critical for binding to MHV. In this study, we have addressed whether or not the N domain alone is sufficient for these activities. We examined three types of soluble form MHVR (soMHVR), one consisting of the N domain alone [soR1(1)], one with the N and second domains [soR1(1,2)], and one [soR1(1,4)] expressed by recombinant baculoviruses. We assessed the abilities of these three types of soMHVR to bind to MHV, activate fusogenicity, and induce conformational changes of the S protein. All three types of soMHVR similarly bound to MHV, as examined by a solid-phase binding assay and neutralized MHV infectivity. They also activated S protein fusogenicity and induced its conformational changes with similar levels of efficiency. However, R1(1) expressed on the BHK cell surface failed to serve as a receptor in spite of a sufficient level of expression. The inability of expressed R1(1) to work as a receptor was due to the inaccessibility of virions to R1(1); however, these were accessible using the MHVR-specific monoclonal antibody CC1. These results collectively indicated that the N domain retains all biological activities necessary for receptor function.
Asunto(s)
Antígenos CD/química , Antígenos de Diferenciación/química , Fusión de Membrana , Glicoproteínas de Membrana/metabolismo , Virus de la Hepatitis Murina/patogenicidad , Receptores Virales/química , Proteínas del Envoltorio Viral/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígeno Carcinoembrionario , Moléculas de Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Citometría de Flujo , Glicoproteínas de Membrana/química , Virus de la Hepatitis Murina/metabolismo , Virus de la Hepatitis Murina/fisiología , Pruebas de Neutralización , Conformación Proteica , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/químicaRESUMEN
An increasing number of HIV-1-infected patients living in developing countries now have access to antiretroviral drugs. Information regarding the drug-resistant mutations of non-B subtype HIV-1 remains limited, however. The authors cross-sectionally compared patterns of the drug-resistant point mutations in patients infected with either subtype B or CRF01_AE (subtype E) among patients who acquired HIV by sexual transmission in Japan. Protease sequence data were available from 216 patients with a detectable level of RNA copies in plasma. Based on phylogenetic analysis of the protease and the C2V3 regions, 162 subtype B and 45 CRF01_AE cases were identified; 82 subtype B and 24 CRF01_AE patients had a treatment failure with nucleoside reverse transcriptase inhibitors; and 69 subtype B and 19 CRF01_AE patients had a treatment failure with a protease inhibitor. Antiretroviral drug history was similar in subtype B-infected and CRF01_AE-infected patients. The mutations T69N and V75M in reverse transcriptase and L10F, K20I, L33I, and N88S in protease were seen more frequently in patients infected with CRF01_AE than in patients with subtype B. The mutations, D30N, A71V, and N88D were found exclusively in patients with subtype B. Most of the characteristic mutation patterns were associated with a history of receiving nelfinavir. The pattern of drug resistance mutations differs between the subtypes. Data derived from subtype B drug-resistant genotypes may not always be applicable to non-B subtypes.