Propagation of Brazilian Zika virus strains in static, microcarrier-based and suspension cultures using BHK and Vero cells

The spread of Zika virus (ZIKV) in the Americas results in an urgent need for the development of a ZIKV vaccine. Current strategies for ZIKV propagation in animal cells rely mainly on adherent Vero and C6/36 cells. This work focused on understanding ZIKV replication in animal cell culture to develop an inactivated or live-attenuated ZIKV vaccine in microcarrier culture or, preferably, in suspension cells, so that low cell-specific yields can be overcome by the establishment of high-cell density processes. First, adherent cells (Vero and BHK-21) were infected with different Brazilian ZIKV isolates. Comparing both cell lines, maximum infectious titers and cell-specific yields (1–48 PFU/cell) of respective virus strains were similar, whereas process yields across different strains strongly varied by two log-scales. Scale-up of Vero cells in bioreactors using 6 g/L Cytodex 1 resulted in maximum cell concentrations of 5.3 × 106 cells/mL. However, low cell-specific yields of 0.0002 PFU/cell indicated poor virus replication. Using suspension-adapted BHK-21 cells grown in a chemically-defined medium, higher virus titers were achieved when infections were initiated at the mid/late exponential growth phase at MOI 0.001. Nevertheless, cell-specific yields did not exceed 0.0002 PFU/cell. Subsequent RT-qPCR data indicated a poor virus release as intracellular viral RNA levels were 20-fold higher than extracellular levels. At small-scale, centrifugal spinoculation was evaluated to enhance ZIKV infection in suspension BHK-21 cells, with no significant improvements. In a further investigation with these cells in a perfusion bioreactor using an ATF-2 filtration system, a maximum cell concentration of 14 × 106 cells/mL was achieved with a final titer of 4.6 × 106 PFU/mL and an increased cell-specific yield of 0.09 PFU/cell. Overall, the present results demonstrate that ZIKV propagation in microcarrier- and suspension-based systems is challenging regarding virus yields. Future investigations will focus on improving cell-specific yields by adapting Zika virus isolates to suspension cell lines, and on increasing maximum titers by process intensification

Epidemiología y relaciones evolutivas entre cepas de HIV subtipo F rumano y brasileño

La clasificación inicial del virus del HIV-1 como las cepas Africanas u Occidentales han sido reemplazadas por la subtipificación filogenética que usa datos de secuencia de nucleótidos. En HIV-1 han sido definidos ocho linajes o subtipos distintos de A a H. Considerando que el valor de la evolución del genoma del HIV-1 se estima en 0,5% a 1% al año y que la distancia genética promedio entre los subtipos de HIV-1 es aproximadamente 20%, es probable que estos subtipos se originaran antes de la pandemia del HIV-1. El mosaico global de los subtipos del HIV-1 es consistente con la hipótesis que la mayoría de las epidemias regionales comenzaron con la introducción de uno o de unas pocas variantes que se diversificaron localmente más que mediante olas radiantes de subtipos de HIV-1 ya diversificados que se diseminaron desde el lugar de origen. En este informe, nos abocamos a las relaciones evolutivas epidemiológicas entre el subtipo F del virus del HIV-1 recientemente identificados en dos regiones geográficamente distintas, Rumania y Brasil.Facultad de Ciencias Veterinaria

Interactions between Nef and AIP1 proliferate multivesicular bodies and facilitate egress of HIV-1

BACKGROUND: Nef is an accessory protein of primate lentiviruses, HIV-1, HIV-2 and SIV. Besides removing CD4 and MHC class I from the surface and activating cellular signaling cascades, Nef also binds GagPol during late stages of the viral replicative cycle. In this report, we investigated further the ability of Nef to facilitate the replication of HIV-1. RESULTS: To this end, first the release of new viral particles was much lower in the absence of Nef in a T cell line. Since the same results were obtained in the absence of the viral envelope using pseudo-typed viruses, this phenomenon was independent of CD4 and enhanced infectivity. Next, we found that Nef not only possesses a consensus motif for but also binds AIP1 in vitro and in vivo. AIP1 is the critical intermediate in the formation of multivesicular bodies (MVBs), which play an important role in the budding and release of viruses from infected cells. Indeed, Nef proliferated MVBs in cells, but only when its AIP1-binding site was intact. Finally, these functions of Nef were reproduced in primary macrophages, where the wild type but not mutant Nef proteins led to increased release of new viral particles from infected cells. CONCLUSION: We conclude that by binding GagPol and AIP1, Nef not only proliferates MVBs but also contributes to the egress of viral particles from infected cells

Epidemiología y relaciones evolutivas entre cepas de HIV subtipo F rumano y brasileño

La clasificación inicial del virus del HIV-1 como las cepas Africanas u Occidentales han sido reemplazadas por la subtipificación filogenética que usa datos de secuencia de nucleótidos. En HIV-1 han sido definidos ocho linajes o subtipos distintos de A a H. Considerando que el valor de la evolución del genoma del HIV-1 se estima en 0,5% a 1% al año y que la distancia genética promedio entre los subtipos de HIV-1 es aproximadamente 20%, es probable que estos subtipos se originaran antes de la pandemia del HIV-1. El mosaico global de los subtipos del HIV-1 es consistente con la hipótesis que la mayoría de las epidemias regionales comenzaron con la introducción de uno o de unas pocas variantes que se diversificaron localmente más que mediante olas radiantes de subtipos de HIV-1 ya diversificados que se diseminaron desde el lugar de origen. En este informe, nos abocamos a las relaciones evolutivas epidemiológicas entre el subtipo F del virus del HIV-1 recientemente identificados en dos regiones geográficamente distintas, Rumania y Brasil.Facultad de Ciencias Veterinaria

Epidemiología y relaciones evolutivas entre cepas de HIV subtipo F rumano y brasileño

La clasificación inicial del virus del HIV-1 como las cepas Africanas u Occidentales han sido reemplazadas por la subtipificación filogenética que usa datos de secuencia de nucleótidos. En HIV-1 han sido definidos ocho linajes o subtipos distintos de A a H. Considerando que el valor de la evolución del genoma del HIV-1 se estima en 0,5% a 1% al año y que la distancia genética promedio entre los subtipos de HIV-1 es aproximadamente 20%, es probable que estos subtipos se originaran antes de la pandemia del HIV-1. El mosaico global de los subtipos del HIV-1 es consistente con la hipótesis que la mayoría de las epidemias regionales comenzaron con la introducción de uno o de unas pocas variantes que se diversificaron localmente más que mediante olas radiantes de subtipos de HIV-1 ya diversificados que se diseminaron desde el lugar de origen. En este informe, nos abocamos a las relaciones evolutivas epidemiológicas entre el subtipo F del virus del HIV-1 recientemente identificados en dos regiones geográficamente distintas, Rumania y Brasil.Facultad de Ciencias Veterinaria

Selective regimes and evolutionary rates of HIV-1 subtype B V3 variants in the Brazilian epidemic

Half of subtype B Brazilian HIV-1 harbors the V3 tip GWGR instead of the GPGR. To investigate the evolution of GW variants, we analyzed 81 env sequences and 5 full-length GW genomes from antiretroviral-naive individuals sampled between 1983 and 1999. Phylogenetic analysis indicated that GW strains intermingle in the tree with other subtype B sequences. the mean d(N)/d(S) values of GW strains were Proximal to those of the other sequences, regardless of sampling years of clinical status. in sequences from patients with CD4+ T cell counts >= 200 cells/mu L, the mean d(N)/d(S) ratio was greater than one, suggesting a positive selection. the prevalence of GW variants was lower among individuals in whom disease progressed. This is probably attributable to the fact that tryptophan is replaced by other amino acids over time, whereas the GP motif does not evolve as rapidly. (C) 2008 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, São Paulo, BrazilHemoctr São Paulo, Fdn Prosangue, São Paulo, BrazilUniv Fed Rio de Janeiro, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilFAPESP: 98/14381-4Web of Scienc