Inactivación del virus Zika con medio de transporte de ácidos nucleicos, potencialmente aplicable a muestras clínicas para el diagnóstico molecular de SARS-CoV-2 / Inactivation of Zika virus with nucleic acids transport media, potentially applicable to clinical samples for the molecular diagnosis of SARS-CoV-2

RESUMEN Introducción: Los medios de colecta de muestras clínicas con capacidad de desnaturalizar virus reducen los riesgos de contagio durante el transporte y procesamiento. Objetivo: Emplear el medio de transporte de ácidos nucleicos (TAN) en muestras de exudado nasofaríngeo colectadas para el diagnóstico de SARS-CoV-2. Métodos: Se realizó un estudio experimental para demostrar la capacidad del medio de inactivar la infectividad viral. Se tomó como modelo de virus envuelto el virus Zika (VZk), cuyo nivel de bioseguridad es 2. Se evaluó el desempeño clínico del medio TAN para el diagnóstico de SARS-CoV-2. Se empleó una cepa del VZk propagada en la línea celular Vero y, previo a la infección de las células, el VZk se puso en contacto a intervalos de tiempo diferentes (2; 15 y 30 min) con el medio TAN puro; y luego se realizaron diluciones seriadas (10-1-10-4). La inactivación viral se evaluó por RT-PCR, en el sobrenadante y células colectadas, al culminar el periodo de propagación. El desempeño clínico del medio TAN se estimó tomando como referencia el CITOSWAB® VTM, en 30 exudados nasofaríngeos colectados para diagnóstico de la infección por SARS-CoV-2. Resultados: El VZk preservó su infectividad a diluciones del inóculo ≥ 10-2, independientemente del tiempo de contacto. La sensibilidad y especificidad clínica del medio TAN para el diagnóstico de SARS-CoV-2 fueron del 100 %, respectivamente. Conclusiones: Los resultados sugieren que muestras clínicas positivas a VZk en diluciones ≤ 10-1 del medio TAN pueden ser manipuladas de forma segura, lo que pudiera aplicarse potencialmente al diagnóstico molecular del SARS-CoV-2.

ABSTRACT Introduction: Collection media of clinical samples with the capacity to denature viruses reduce the risk of contagion during transportation and processing. Objective: To use the nucleic acids transport media (NATM) in nasopharyngeal swab samples collected for the diagnosis of SARS-CoV-2. Methods: An experimental study was conducted to demonstrate the medium capacity to inactivate viral infectivity. Zika virus (ZIKV), of biosafety level 2, was used as an enveloped virus model. The clinical performance of the NATM for the diagnosis of SARS-CoV-2 was evaluated. A ZIKV strain propagated in the Vero cell line was used and, prior to cells infection, ZIKV was in contact at different intervals (2; 15, and 30 min) with pure NATM; subsequently, serial dilutions (10-1-10-4) were performed. Viral inactivation was evaluated by RT-PCR in the supernatant and the collected cells when the propagation period was completed. CITOSWAB® VTM was used as reference to estimate the clinical performance of the NATM in 30 nasopharyngeal swabs collected for the diagnosis of SARS-CoV-2 infection. Results: ZIKV remained infectious at inoculum dilutions of ≥ 10-2, regardless of contact time. Clinical specificity and sensitivity of the NATM for the diagnosis of SARS-CoV-2 were 100%, respectively. Conclusions: Results suggest that ZIKV positive clinical samples at dilutions ≤ 10-1 of the NATM can be safely handled, which could potentially be applied to the molecular diagnosis of SARS-CoV-2.

Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model.

Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dose-dependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes.

Inactivation of avian influenza viruses by hydrostatic pressure as a potential vaccine development approach.

Vaccines are a recommended strategy for controlling influenza A infections in humans and animals. Here, we describe the effects of hydrostatic pressure on the structure, morphology and functional characteristics of avian influenza A H3N8 virus. The effect of hydrostatic pressure for 3 h on H3N8 virus revealed that the particles were resistant to this condition, and the virus displayed only a discrete conformational change. We found that pressure of 3 kbar applied for 6 h was able to inhibit haemagglutination and infectivity while virus replication was no longer observed, suggesting that full virus inactivation occurred at this point. However, the neuraminidase activity was not affected at this approach suggesting the maintenance of neutralizing antibody epitopes in this key antigen. Our data bring important information for the area of structural virology of enveloped particles and support the idea of applying pressure-induced inactivation as a tool for vaccine production.

In vitro and in vivo evaluation of sodium dodecyl sulfate (SDS) as an inactivator of caprine lentivirus (CLV) in colostrum and milk / Avaliação in vitro e in vivo do dodecil sulfato de sódio (SDS) como inativador do lentivírus caprino (LVC) em colostro e leite

The aim of this study was to evaluate in vitro and in vivo the effect of sodium dodecyl sulfate (SDS) on the caprine lentivirus (CLV) in colostrum and milk. This was performed to develop a practical and efficient method of blocking the lactogenic transmission of the virus. In the in vitro experiment, colostrum and milk were treated with 0.25%; 0.50% and 1% SDS. Then, somatic cells of colostrum and milk were submitted to co-culture with caprine synovial membrane cells (CSM). In the in vivo test, goats were fed with colostrum and milk provided from CLV-positive goats treated with SDS in the same concentrations used in the in vitro experiment. Animals were tested by nested polymerase chain reaction (nPCR) and Western blot (WB) assays. In the in vitro experiment, inhibitory activity against CLV without inactivation occurred in colostrum with all SDS concentrations. However, concentrations of 0.25 and 0.5% SDS presented only inhibitory activity against CLV in milk cells, and 1% concentration provided inactivation of the virus. In the in vivo tests, none of the three concentrations of SDS was effective in inactivating LVC in colostrum or goat milk, which was confirmed by seroconversion and presence of proviral DNA in animals afterwards.(AU)

O objetivo da pesquisa foi avaliar in vitro e in vivo o efeito do dodecil sulfato de sódio (SDS) sobre o lentivírus caprino (LVC) no colostro e no leite, a fim de desenvolver um método prático e eficiente no bloqueio da via de transmissão lactogênica do vírus. No experimento in vitro, o colostro e o leite de cabras positivas foram tratados com SDS a 0,25%, 0,50% e 1,0%. Em seguida, as células somáticas do colostro e do leite foram obtidas e direcionadas ao cocultivo com células de membrana sinovial caprina (MSC). No teste in vivo, os cabritos foram alimentados com colostro e leite providos de cabras positivas para LVC, tratados com SDS nas mesmas concentrações usadas no teste in vitro. Os animais foram acompanhados pelos testes de reação em cadeia da polimerase nested (nPCR) e western blot (WB). Nos resultados in vitro, no colostro, observou-se que, em todas as concentrações de SDS, ocorreu uma atividade inibitória contra o LVC, sem a inativação. Em relação às células do leite, o SDS apresentou, nas concentrações de 0,25 e 0,5%, atividade inibitória contra o LVC, e na concentração de 1%, houve inativação viral. Nos testes in vivo, as três concentrações de SDS testadas não foram efetivas na inativação do LVC no colostro e no leite caprino, o que se comprovou pela soroconversão e pela presença de DNA proviral nos animais.(AU)

In vitro and in vivo evaluation of sodium dodecyl sulfate (SDS) as an inactivator of caprine lentivirus (CLV) in colostrum and milk / Avaliação in vitro e in vivo do dodecil sulfato de sódio (SDS) como inativador do lentivírus caprino (LVC) em colostro e leite

The aim of this study was to evaluate in vitro and in vivo the effect of sodium dodecyl sulfate (SDS) on the caprine lentivirus (CLV) in colostrum and milk. This was performed to develop a practical and efficient method of blocking the lactogenic transmission of the virus. In the in vitro experiment, colostrum and milk were treated with 0.25%; 0.50% and 1% SDS. Then, somatic cells of colostrum and milk were submitted to co-culture with caprine synovial membrane cells (CSM). In the in vivo test, goats were fed with colostrum and milk provided from CLV-positive goats treated with SDS in the same concentrations used in the in vitro experiment. Animals were tested by nested polymerase chain reaction (nPCR) and Western blot (WB) assays. In the in vitro experiment, inhibitory activity against CLV without inactivation occurred in colostrum with all SDS concentrations. However, concentrations of 0.25 and 0.5% SDS presented only inhibitory activity against CLV in milk cells, and 1% concentration provided inactivation of the virus. In the in vivo tests, none of the three concentrations of SDS was effective in inactivating LVC in colostrum or goat milk, which was confirmed by seroconversion and presence of proviral DNA in animals afterwards.(AU)

O objetivo da pesquisa foi avaliar in vitro e in vivo o efeito do dodecil sulfato de sódio (SDS) sobre o lentivírus caprino (LVC) no colostro e no leite, a fim de desenvolver um método prático e eficiente no bloqueio da via de transmissão lactogênica do vírus. No experimento in vitro, o colostro e o leite de cabras positivas foram tratados com SDS a 0,25%, 0,50% e 1,0%. Em seguida, as células somáticas do colostro e do leite foram obtidas e direcionadas ao cocultivo com células de membrana sinovial caprina (MSC). No teste in vivo, os cabritos foram alimentados com colostro e leite providos de cabras positivas para LVC, tratados com SDS nas mesmas concentrações usadas no teste in vitro. Os animais foram acompanhados pelos testes de reação em cadeia da polimerase nested (nPCR) e western blot (WB). Nos resultados in vitro, no colostro, observou-se que, em todas as concentrações de SDS, ocorreu uma atividade inibitória contra o LVC, sem a inativação. Em relação às células do leite, o SDS apresentou, nas concentrações de 0,25 e 0,5%, atividade inibitória contra o LVC, e na concentração de 1%, houve inativação viral. Nos testes in vivo, as três concentrações de SDS testadas não foram efetivas na inativação do LVC no colostro e no leite caprino, o que se comprovou pela soroconversão e pela presença de DNA proviral nos animais.(AU)

Water treatment with exceptional virus inactivation using activated carbon modified with silver (Ag) and copper oxide (CuO) nanoparticles.

Continuous flow experiments (450 mL min-1) were performed in household filter in order to investigate the removal and/or inactivation of T4 bacteriophage, using granular activated carbon (GAC) modified with silver and/or copper oxide nanoparticles at different concentrations. GAC and modified GAC were characterized by X-ray diffractometry, specific surface area, pore size and volume, pore average diameter, scanning electron microscopy, transmission electron microscopy, zeta potential and atomic absorption spectroscopy. The antiviral activity of the produced porous media was evaluated by passing suspensions of T4 bacteriophage (∼105 UFP/mL) through filters. The filtered water was analyzed for the presence of the bacteriophage and the release of silver and copper oxide. The porous media containing silver and copper oxide nanoparticles showed high inactivation capacity, even reaching reductions higher than 3 log. GAC6 (GAC/Ag0.5%Cu1.0%) was effective in the bacteriophage inactivation, reaching 5.53 log reduction. The levels of silver and copper released in filtered water were below the recommended limits (100 ppb for silver and 1000 ppb for copper) in drinking water. From this study, it is possible to conclude that activated carbon modified with silver and copper oxide nanoparticles can be used as a filter for virus removal in the treatment of drinking water.