RESUMEN
Human noroviruses (hNoV) are the primary cause of foodborne disease in the USA. Most studies on inactivation kinetics of hNoV and its surrogates are performed in monoculture, while the microbial ecosystem effect on virus inactivation remains limited. This study investigated the persistence of hNoV surrogates, murine norovirus (MNV) and Tulane virus (TuV), along with Aichi virus (AiV) under thermal and chemical inactivation in association with Gram-negative (Enterobacter cloacae) bacteria. Thermal inactivation of viruses in co-culture with E. cloacae revealed no protective effects of bacteria. At 56 °C, AiV with and without bacteria was completely inactivated by 10 min with decimal reduction values (D-values) of 41 and 43 s, respectively. Similar results were also observed for TuV. Conversely, MNV with bacteria was completely inactivated by 10 min while MNV alone remained stable up to 30 min at 56 °C. Both MNV and TuV were slightly more stable than AiV at 63 °C with TuV detection up to 2 min without bacteria. For chemical inactivation on stainless steel surfaces, viruses alone and in association with bacteria were treated with 1000 ppm sodium hypochlorite. Virus association with bacteria had no significant effect (p > 0.05) on virus resistance to bleach inactivation compared to virus alone. Specifically, exposure to 1000 ppm bleach for 5 min resulted in an average of 3.86, 2.14, and 0.94 log10 PFU/ml reductions for TuV, MNV, and AiV without bacteria, respectively. Reductions in TuV, MNV, and AiV were 3.50, 1.88, and 0.61 log10 PFU/ml when associated with E. cloacae, respectively.
Asunto(s)
Enterobacter cloacae/efectos de los fármacos , Kobuvirus/efectos de los fármacos , Norovirus/efectos de los fármacos , Hipoclorito de Sodio/farmacología , Técnicas de Cocultivo , Enterobacter cloacae/química , Enterobacter cloacae/crecimiento & desarrollo , Calor , Kobuvirus/química , Kobuvirus/crecimiento & desarrollo , Norovirus/química , Norovirus/crecimiento & desarrollo , Inactivación de Virus/efectos de los fármacosRESUMEN
Ultrafiltration (UF) membranes are increasingly being used for wastewater reclamation treatment for their high removal of pathogens and suspended solids. However, breakage of UF membrane fibers could allow leakage of pathogens into the permeate and create health risks in the use of reclaimed water. Here, we assessed the log10 reduction value (LRV) of human enteric viruses and microbial indicators of new and aged UF modules in a pilot-scale UF process to evaluate the influence of fiber breakage. Norovirus genotypes I and II, Aichi virus, and Escherichia coli were not detected in any permeate samples of intact UF modules, but were detected in samples of damaged UF modules. LRVs of all microorganisms assayed decreased as fiber breakage of new UF modules increased, with maximum decreases of > 3.3 log10. Fiber breakage in the aged UF modules did not decrease LRVs of somatic coliphages and MS2, but breakage in the new UF modules did decrease them. Intact new UF modules gave higher LRVs than intact aged UF modules. When the LRV of intact UF module was assumed to be 1 or 2 log10, increasing fiber breakage did not significantly decrease the predicted LRV, but when it was ≥ 3 log10, it did decrease LRV, in good agreement with measured LRVs in the degraded UF modules. These results suggest that the LRV of intact UF modules affects the decrease in LRV and confirm the leakage of human enteric viruses following fiber breakage in UF modules of different ages in the UF process of wastewater reclamation.
Asunto(s)
Ultrafiltración/métodos , Aguas Residuales/química , Purificación del Agua/métodos , Adsorción , Escherichia coli/química , Escherichia coli/aislamiento & purificación , Kobuvirus/química , Kobuvirus/aislamiento & purificación , Membranas Artificiales , Norovirus/química , Norovirus/aislamiento & purificación , Ultrafiltración/instrumentación , Aguas Residuales/microbiología , Aguas Residuales/virología , Purificación del Agua/instrumentaciónRESUMEN
Phosphatidylinositol 4-kinase III beta (PI4KIIIß) is an essential enzyme in mediating membrane transport, and plays key roles in facilitating viral infection. Many pathogenic positive-sense single-stranded RNA viruses activate PI4KIIIß to generate phosphatidylinositol 4-phosphate (PI4P)-enriched organelles for viral replication. The molecular basis for PI4KIIIß activation during viral infection has remained largely unclear. We describe the biochemical reconstitution and characterization of the complex of PI4KIIIß with the Golgi protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3) and Aichi virus 3A protein on membranes. We find that 3A directly activates PI4KIIIß, and this activation is sensitized by ACBD3. The interfaces between PI4KIIIß-ACBD3 and ACBD3-3A were mapped with hydrogen-deuterium exchange mass spectrometry (HDX-MS). Determination of the crystal structure of the ACBD3 GOLD domain revealed a unique N terminus that mediates the interaction with 3A. Rationally designed complex-disrupting mutations in both ACBD3 and PI4KIIIß completely abrogated the sensitization of 3A activation by ACBD3.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Kobuvirus/metabolismo , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Activación Enzimática , Humanos , Kobuvirus/química , Espectrometría de Masas , Proteínas de la Membrana/química , Modelos Moleculares , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Unión Proteica , Conformación Proteica , Replicación ViralRESUMEN
Aichi virus (AiV), an unusual and poorly characterized picornavirus, classified in the genus Kobuvirus, can cause severe gastroenteritis and deaths in children below the age of five years, especially in developing countries1,2. The seroprevalence of AiV is approximately 60% in children under the age of ten years and reaches 90% later in life3,4. There is no available vaccine or effective antiviral treatment. Here, we describe the structure of AiV at 3.7â Å. This first high-resolution structure for a kobuvirus is intermediate between those of the enteroviruses and cardioviruses, with a shallow, narrow depression bounded by the prominent VP0 CD loops (linking the C and D strands of the ß-barrel), replacing the depression known as the canyon, frequently the site of receptor attachment in enteroviruses. VP0 is not cleaved to form VP2 and VP4, so the 'VP2' ß-barrel structure is complemented with a unique extended structure on the inside of the capsid. On the outer surface, a polyproline helix structure, not seen previously in picornaviruses is present at the C terminus of VP1, a position where integrin binding motifs are found in some other picornaviruses. A peptide corresponding to this polyproline motif somewhat attenuates virus infectivity, presumably blocking host-cell attachment. This may guide cellular receptor identification.