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Unspliced HIV-1 RNAs function as messenger RNAs for Gag or Gag-Pol polyproteins and progeny genomes packaged into virus particles. Recently, it has been reported that fate of the RNAs might be primarily determined, depending on transcriptional initiation sites among three consecutive deoxyguanosine residues (GGG tract) downstream of TATA-box in the 5' long terminal repeat (LTR). Although HIV-1 RNA transcription starts mostly from the first deoxyguanosine of the GGG tract and often from the second or third deoxyguanosine, RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine, were predominant in HIV-1 particles. Despite selective packaging of G1-form RNAs into virus particles, its biological impact during viral replication remains to be determined. In this study, we revealed that G1-form RNAs are primarily selected as a template for provirus DNA rather than other RNAs. In competitions between HIV-1 and lentiviral vector transcripts in virus-producing cells, approximately 80% of infectious particles were found to generate provirus using HIV-1 transcripts, while lentiviral vector transcripts were conversely selected when we used HIV-1 mutants in which the third deoxyguanosine in the GGG tract was replaced with deoxythymidine or deoxycytidine (GGT or GGC mutants, respectively). In the other analyses of proviral sequences after infection with an HIV-1 mutant in which the GGG tract in 3' LTR was replaced with TTT, most proviral sequences of the GGG-tract region in 5' LTR were found to be TTG, which is reasonably generated using the G1-form transcripts. Our results indicate that the G1-form RNAs serve as a dominant genome to establish provirus DNA.IMPORTANCESince the promoter for transcribing HIV-1 RNA is unique, all viral elements including genomic RNA and viral proteins have to be generated by the unique transcripts through ingenious mechanisms including RNA splicing and frameshifting during protein translation. Previous studies suggested a new mechanism for diversification of HIV-1 RNA functions by heterogeneous transcriptional initiation site usage; HIV-1 RNAs whose transcription initiates from a certain nucleotide were predominant in virus particles. In this study, we established two methods to analyze heterogenous transcriptional initiation site usage by HIV-1 during viral infection and showed that RNAs beginning with one guanosine (G1-form RNAs), whose transcription initiates from the third deoxyguanosine of the GGG tract in 5' LTR, were primarily selected as viral genome in infectious particles and thus are used as a template to generate provirus for continuous replication. This study provides insights into the mechanism for diversification of unspliced RNA functions and requisites of lentivirus infectivity.
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VIH-1 , Provirus , Desoxiguanosina/genética , Guanosina/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/fisiología , Provirus/genética , ARN Viral/genética , Secuencias Repetidas TerminalesRESUMEN
Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.
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Virus de la Hepatitis A , Hepatitis A , Macrófagos , Esparcimiento de Virus , Animales , Ratones , Linfocitos T CD8-positivos , Heces , Virus de la Hepatitis A/fisiología , Inflamación , Macrófagos/virología , Receptor de Interferón alfa y beta/genética , ARN Viral/genética , Ratones NoqueadosRESUMEN
Parasites of the Babesiadivergens Asia lineage, which are closely related to B. divergens in Europe and Babesia sp. strain MO1 in the United States, were recently reported in sika deer (Cervus nippon) in eastern Japan. To identify the tick vector(s) for this parasite, we conducted a field survey in Hokkaido, Japan, where the infection rate in sika deer is the highest in the country. A specific PCR system which detects and discriminates between lineages within B. divergens and between those lineages and Babesia venatorum showed that Ixodes persulcatus (11/822), but not sympatric Ixodes ovatus (0/595) or Haemaphysalis sp. (0/163) ticks, carried B. divergens Asia lineage. Genomic DNA was archived from salivary glands of partially engorged I. persulcatus females and three isolates of B. divergens Asia lineage were newly described. The 18S rRNA gene sequence of the isolates formed the Asia lineage cluster with those previously described in sika deer isolates. One salivary gland also contained parasites of Babesia microti U.S. lineage, which were subsequently isolated in a hamster in vivoB. venatorum (strain Etb5) was also detected in one I. persulcatus tick. The 18S rRNA sequence of Etb5 was 99.7% identical to that of B. venatorum (AY046575) and was phylogenetically positioned in a taxon composed of B. venatorum isolates from Europe, China, and Russia. The geographical distribution of I. persulcatus is consistent with that of B. divergens in sika deer in Japan. These results suggest that I. persulcatus is a principal vector for B. divergens in Japan and Eurasia, where I. persulcatus is predominantly distributed.IMPORTANCE The Babesiadivergens Asia lineage of parasites closely related to B. divergens in Europe and Babesia sp. MO1 in the United States was recently reported in Cervus nippon in eastern Japan. In this study, specific PCR for the Asia lineage identified 11 positives in 822 host-seeking Ixodes persulcatus ticks, a principal vector for many tick-borne disease agents. Gene sequences of three isolates obtained from DNA in salivary glands of female ticks were identical to each other and to those in C. nippon We also demonstrate the coinfection of B. divergens Asia lineage with Babesia microti U.S. lineage in a tick salivary gland and, furthermore, isolated the latter in a hamster. These results suggest that I. persulcatus is the principal vector for B. divergens as well as for B. microti, and both parasites may be occasionally cotransmitted by I. persulcatus This report will be important for public health, since infection may occur through transfusion.
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Babesia/fisiología , Babesiosis/transmisión , Ciervos , Ixodes/parasitología , Animales , Babesia/genética , Babesiosis/parasitología , Secuencia de Bases , ADN Protozoario/análisis , Interacciones Huésped-Parásitos , Japón , ARN Ribosómico 18S/análisisRESUMEN
The U.S. lineage, one of the major clades in the Babesia microti group, is known as a causal agent of human babesiosis mostly in the northeastern and upper midwestern United States. This lineage, however, also is distributed throughout the temperate zone of Eurasia with several reported human cases, although convincing evidence of the identity of the specific vector(s) in this area is lacking. Here, the goal was to demonstrate the presence of infectious parasites directly in salivary glands of Ixodes persulcatus, from which U.S. lineage genetic sequences have been detected in Asia, and to molecularly characterize the isolates. Five PCR-positive specimens were individually inoculated into hamsters, resulting in infections in four; consequently, four strains were newly established. Molecular characterization, including 18S rRNA, ß-tubulin, and CCT7 gene sequences, as well as Western blot analysis and indirect fluorescent antibody assay, revealed that all four strains were identical to each other and to the U.S. lineage strains isolated from rodents captured in Japan. The 18S rRNA gene sequence from the isolates was identical to those from I. persulcatus in Russia and China, but the genetic and antigenic profiles of the Japanese parasites differ from those in the United States and Europe. Together with previous epidemiological and transmission studies, we conclude that I. persulcatus is likely the principal vector for the B. microti U.S. lineage in Japan and presumably in northeastern Eurasia. IMPORTANCE: The major cause of human babesiosis, the tick-borne blood parasite Babesia microti, U.S. lineage, is widely distributed in the temperate Northern Hemisphere. However, the specific tick vector(s) remains unidentified in Eurasia, where there are people with antibodies to the B. microti U.S. lineage and cases of human babesiosis. In this study, the first isolation of B. microti U.S. lineage from Ixodes persulcatus ticks, a principal vector for many tick-borne diseases, is described in Japan. Limited antigenic cross-reaction was found between the Japan and United States isolates. Thus, current serological tests based on U.S. isolates may underestimate B. microti occurrence outside the United States. This study and previous studies indicate that I. persulcatus is part of the B. microti U.S. lineage life cycle in Japan and, presumably, northeastern Eurasia. This report will be important for public health, especially since infection may occur through transfusion, and also to researchers in the field of parasitology.
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Vectores Arácnidos/parasitología , Babesia microti/aislamiento & purificación , Babesiosis/parasitología , Babesiosis/transmisión , Ixodes/parasitología , Animales , Antígenos de Protozoos/genética , Babesia microti/clasificación , Babesia microti/genética , Babesiosis/epidemiología , China/epidemiología , Cricetinae , ADN Protozoario/genética , Femenino , Humanos , Ixodes/anatomía & histología , Japón/epidemiología , Medio Oeste de Estados Unidos/epidemiología , Reacción en Cadena de la Polimerasa , ARN Ribosómico 18S/genética , Roedores/parasitología , Federación de Rusia/epidemiología , Glándulas Salivales/parasitología , Tubulina (Proteína)/genéticaRESUMEN
Since 2019, many studies on coronavirus disease 2019, which has caused extensive damage as a pandemic, have been ongoing on a global scale. These include serological and biochemical studies using sera from patients and animal models. Testing with these sera must be performed after inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Heat treatment, UV irradiation, and/or gamma-ray irradiation have been used to inactivate viruses in the serum. Determining the inactivation conditions that ensure the inactivation of viruses and minimize the effect on test results after inactivation is important to ensure worker safety and the accuracy of test results. In this study, serum samples containing SARS-CoV-2 were subjected to heat, UV irradiation, and gamma irradiation to determine optimal inactivation conditions. The viral titers were below the detection limit after heating at 56°C for 1 h or 60°C for 15 min, UV-B irradiation with a transilluminator for 30 min, or gamma-ray irradiation with 60 Co at 10 kGy. These results provide useful information for safe serological and biochemical experiments.
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COVID-19 , Rayos gamma , Calor , SARS-CoV-2 , Rayos Ultravioleta , Inactivación de Virus , Humanos , Inactivación de Virus/efectos de la radiación , Suero/virología , Suero/efectos de la radiaciónRESUMEN
Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1-HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis. IMPORTANCE: Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.
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Governing dual-use research of concern (DURC) in the life sciences has become difficult owing to the diversification of scientific domains, digitalization of potential threats, and the proliferation of actors. This paper proposes three approaches to realize bottom-up governance of DURC from laboratory operation to institutional decision-making levels. First, a technological approach can predict and monitor the dual-use nature of the research target pathogens and their information. Second, an interactive approach is proposed in which diverse stakeholders proactively discuss and examine dual-use issues through research practice. Third, a personnel approach can identify the right persons involved in DURC. These approaches suggest that, going beyond self-governance by researchers, collaborative and networked governance involving diverse actors should become essential. This mode of governance can also be seen in light of the management of research use. Therefore, program design by funding agencies and publication screening by journal publishers continuously contribute to governance at the meso-level. Bottom-up governance may be realized by using an appropriately integrated design of these three approaches at the micro-level, such as dual-use prediction and monitoring, stakeholder dialogue, and background checks. Given that the term 'open science' has been promoted to the research community as part of top-down governance, paying due attention on site to research subjects, research practices, and persons involved in research will provide an opportunity to develop a more socially conscious open science.
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BACKGROUND: During the fourth COVID-19 wave in Japan, marked differences became apparent in the scale of the epidemic between metropolitan Tokyo in eastern Japan and Osaka prefecture in western Japan. METHODS: Public epidemic data were analyzed, with performance of mathematical simulations using simplified SEIR models. RESULTS: The increase in the number of infected persons per 100,000 population during the fourth wave of expansion was greater in Osaka than in Tokyo. The basic reproduction number in Osaka was greater than in Tokyo. Particularly, the number of infected people in their 20 s increased during the fourth wave: The generation-specific reproduction number for people in their 20 s was higher than for people of other generations. Both Tokyo and Osaka were found to have strong correlation between the increase in the number of infected people and the average number of people using the main downtown stations at night. Simulations showed vaccination of people in their 60 s and older reduced the number of infected people among the high-risk elderly population in the fourth wave. However, age-specific vaccination of people in their 20 s reduced the number of infected people more than vaccination of people in their 60 s and older. CONCLUSIONS: Differences in the epidemic between Tokyo and Osaka are explainable by different behaviors of the most socially active generation. When vaccine supplies are adequate, priority should be assigned to high-risk older adults, but if vaccine supplies are scarce, simulation results suggest consideration of vaccinating specific groups among whom the epidemic is spreading rapidly.
RESUMEN
Several studies have reported on the effectiveness of various disinfection methods for severe acute respiratory syndrome coronavirus 2 and their applicability to the disinfection of N95 respirators and surgical masks. To date, there have been no reports on the decontamination of deep in the intermediate layers of the multilayered structure. In this study, the conditions required for decontamination of such layers were simulated by considering the thickness and shape of N95 respirators or surgical masks (samples). After applying heat (steam, dry heat, or hot water) at 75°C for 60 min or chemical (benzalkonium chloride or laundry detergent) treatment, the collection efficiency of the samples was evaluated. Following the dry heat treatment, the time between the treatment and heat reaching the intermediate layer of the filter fiber was extended by 10 min. A dry heat disinfection method that combines hot water and a closed container was also evaluated, and satisfactory conditions were extended by 60 min. For each heat treatment, there was almost no effect on the collection efficiency, although there were cases where deformation was caused by mechanical stress. In contrast, chemical treatment resulted in a reduction in the collection efficiency of smaller particles.
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COVID-19 , Máscaras , COVID-19/prevención & control , Desinfección/métodos , Humanos , Respiradores N95 , AguaRESUMEN
Enterovirus 71 (EV71) is one of the causative agents of hand-foot-and-mouth disease, which in some circumstances could lead to severe neurological diseases. Despite of its importance for human health, little is known about the early stages of EV71 infection. EV71 starts uncoating with its receptor, human scavenger receptor B2 (hSCARB2), at low pH. We show that EV71 was not targeted to lysosomes in human rhabdomyosarcoma cells overexpressing hSCARB2 and that the autophagic pathway is not essential for EV71 productive uncoating. Instead, EV71 was efficiently uncoated 30â min after infection in late endosomes (LEs) containing hSCARB2, mannose-6-phosphate receptor (M6PR), RAB9, bis(monoacylglycero)phosphate and lysosomal associated membrane protein 2 (LAMP2). Furthering the notion that mature LEs are crucial for EV71 uncoating, cation-dependent (CD)-M6PR knockdown impairs EV71 infection. Since hSCARB2 interacts with cation-independent (CI)-M6PR through M6P-binding sites and CD-M6PR also harbor a M6P-binding site, CD-M6PR is likely to play important roles in EV71 uncoating in LEs.
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Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Animales , Cationes/metabolismo , Endosomas/metabolismo , Enterovirus/metabolismo , Enterovirus Humano A/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas/química , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismoRESUMEN
The risk of SARS-CoV-2 infection when people handle linens is uncertain. We examined the presence of SARS-CoV-2 on linens, in the air, and on personal protective equipment (PPE) to assess potential infection risk among individuals who handle linens used by SARS-CoV-2-infected people. Patients in a hospital and an accommodation facility who tested positive for SARS-CoV-2 participated in this study in 2020. Linen samples before washing or disinfection, rinse water after washing or disinfection, air in the workplace at the hospital and an accommodation facility, and the PPE worn by linen-handling people were tested for SARS-CoV-2 RNA and viable viruses. Among 700 samples from 13 SARS-CoV-2-infected participants and their surrounding environment, SARS-CoV-2 RNA was detected from 14% (52/362) of the linens used by COVID-19 patients (cycle threshold [Ct] value: 33-40). SARS-CoV-2 RNA was detected from 8% (2/26) of rinse water after washing or disinfection, from 15% (16/104) of air samples in the workspace, and from 10% (5/52) of gowns worn by linen-handling people, all with high Ct values (> 36). No SARS-CoV-2 was isolated from any samples. The potential risk of SARS-CoV-2 infection from handling linens used by SARS-CoV-2-infected people exists but appears to below.
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COVID-19 , Ropa de Cama y Ropa Blanca , COVID-19/prevención & control , Humanos , ARN Viral , SARS-CoV-2 , AguaRESUMEN
Babesia divergens is the major causal agent of zoonotic human babesiosis across Europe. Previously, we reported the detection of a B. divergens Asia lineage in wild sika deer (Cervus nippon) in Japan which was genetically closely related to the European B. divergens. To further elucidate its etiology, we conducted a large epidemiological survey by combining lineage-specific PCR system and blood direct PCR. The infection rate of the Asia lineage was 6.6% (116/1,747) throughout Japan, where Hokkaido (45%), Nagano (17%), Iwate (12%), Gunma (11%), and Yamanashi (11%) were highly enzootic (> 10%) among the 30 prefectures examined. European B. divergens was not detected. A geographical information system (GIS) map revealed dense populations of PCR-positive deer in the mountains including the Japanese Alps in eastern Honshu, and Hokkaido. These areas markedly overlapped with the major habitats of Ixodes persulcatus, a principal tick vector responsible for the transmission of the Asia lineage. Other areas in southern Japan including Miyazaki, Kagoshima, and Shimane Prefectures, where positive sika deer were sporadically detected, may be habitats for other tick species involved in the enzootic cycle as I. persulcatus were scarce. The rise in human babesiosis cases is occasionally attributed to healthy blood donors who were unaware of tick bites and Babesia infection. Therefore, there is an urgent need to investigate whether infections in humans have occurred in Japan.
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Babesia/clasificación , Babesiosis/epidemiología , Babesiosis/parasitología , Ciervos/parasitología , Animales , Asia , ADN Protozoario/genética , Ixodes/parasitología , Japón/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADNRESUMEN
Mammalian androgenetic embryos can be produced by pronuclear exchange of fertilized oocytes or by dispermic in vitro fertilization of enucleated oocytes. Here, we report a new technique for producing mouse androgenetic embryos by injection of two round spermatid nuclei into oocytes, followed by female chromosome removal. We found that injection of round spermatids resulted in high rates of oocyte survival (88%). Androgenetic embryos thus produced developed into mid-gestation fetuses at various rates, depending on the mouse strain used. All the fetuses examined maintained paternally specific genomic imprinting memories. This technique also enabled us to produce complete heterozygous F1 embryos by injecting two spermatids from different strains. The best rate of fetal survival (12% per embryos transferred) was obtained with C57BL/6 x DBA/2 androgenetic embryos. We also generated embryonic stem cell lines efficiently with the genotype of Mus musculus domesticus x M. m. molossinus. Thus, injection of two round spermatid nuclei followed by maternal enucleation is an effective alternative method of producing androgenetic embryos that consistently develop into blastocysts and mid-gestation fetuses.
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Núcleo Celular , Embrión de Mamíferos , Técnicas de Transferencia Nuclear , Oocitos , Espermátides , Animales , Femenino , Masculino , RatonesRESUMEN
Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct--namely, magnesium pyrophosphate--without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 microM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.
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Colorimetría/métodos , Colorantes/química , ADN/análisis , ADN/química , Naftalenosulfonatos/análisis , Naftalenosulfonatos/química , Naftoles/química , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/análisis , ARN/químicaRESUMEN
Eleven monoclonal antibodies (MAbs) that were reactive against the hemagglutinating encephalomyelitis virus (HEV), as seen in the enzyme-linked immunosorbent assay, were obtained. All of these MAbs showed neutralizing activity (1:20,000 to 1:800,000) against the 67N strain of HEV. They also showed hemagglutination inhibition activity (1:400 to 1:409,600). Western blotting tests revealed that all of these 11 MAbs were specific for the S protein of HEV. All MAbs with neutralizing activity showed the same fluorescent staining pattern. Ten-day-old mice pups were immunized with MAb, inoculated with the 10(5) tissue culture-infective dose of HEV at 3 days after immunization, and then examined to determine the viral inhibition. The 1:800-diluted MAb120 inhibited the viral infection in mice pups, though the 1:1,000-diluted MAb120 failed to inhibit the viral infectivity. These MAbs would be a useful tool for rapid and specific diagnosis of HEV and also for antibody-based treatment of the disease.
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Anticuerpos Monoclonales/farmacología , Infecciones por Coronavirus/veterinaria , Coronavirus/inmunología , Enfermedades de los Porcinos/virología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Western Blotting/veterinaria , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Pruebas de Inhibición de Hemaglutinación/veterinaria , Pruebas de Hemaglutinación/veterinaria , Inmunización/veterinaria , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Pruebas de Neutralización/veterinaria , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & controlRESUMEN
An ultrasensitive protein detection system in situ named the ImmunoAT-tailing method was developed. It consists of three elementary processes: (i) detection of a protein by a primary antibody and a biotinylated secondary antibody; (ii) linking of biotinylated 15-base oligo(dA-dT) to the biotinylated immunocomplex via streptavidin; and (iii) self-priming elongation of oligo(dA-dT) by the Klenow fragment, 3' to 5' exo-. After the elongation reaction in the presence of dATP, dTTP, and dye-labeled dUTP, the protein was labeled with a large number of the dye molecules. The poly(dA-dT) elongated without the labeled nucleotides was detected by 4',6-diamidino-2-phenylindole (DAPI) staining. By combining the different labelings, double staining was possible. This ImmunoAT-tailing method has a specificity and sensitivity higher than that of tyramide signal amplification.
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Técnica del Anticuerpo Fluorescente Directa/métodos , Oligodesoxirribonucleótidos/química , Poli dA-dT/química , Proteínas/análisis , Proteínas/química , Animales , Chlorocebus aethiops , Historia Antigua , Microscopía Fluorescente/métodos , Proteínas/metabolismo , Coloración y Etiquetado , Células VeroRESUMEN
The 3' end region nucleotide sequence, including ORF7, of nine Japanese and two U.S.A. isolates of transmissible gastroenteritis virus (TGEV) were determined and compared. Nine Japanese TGEV strains have been isolated over the past 40 years (1956-1997). From the comparison of determined nucleotide sequences, we could divide the TGEV Japanese isolates into two groups and distinguish them from TGEV U.S.A. isolates.
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Sistemas de Lectura Abierta/genética , Virus de la Gastroenteritis Transmisible/genética , Animales , Secuencia de Bases , Electroforesis en Gel de Agar/veterinaria , Japón , Datos de Secuencia Molecular , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Homología de Secuencia de Ácido Nucleico , Porcinos , Virus de la Gastroenteritis Transmisible/aislamiento & purificación , Estados UnidosRESUMEN
A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1-ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.
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Norovirus/aislamiento & purificación , Compuestos Orgánicos/química , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Animales , Secuencia de Bases , Benzotiazoles , Línea Celular , Cartilla de ADN , Diaminas , Heces/virología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Norovirus/genética , Sistemas de Lectura Abierta , Plásmidos , Quinolinas , ARN Viral/genética , Reproducibilidad de los Resultados , Temperatura , Factores de TiempoRESUMEN
Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under isothermal conditions at 62°C for 90min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV.