RESUMEN
Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses.
Asunto(s)
Atadenovirus/crecimiento & desarrollo , Técnicas de Cultivo de Célula , Herpesvirus Suido 1/crecimiento & desarrollo , Virus de la Parainfluenza 3 Bovina/crecimiento & desarrollo , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Cultivo de Virus/métodos , Animales , Atadenovirus/fisiología , Atadenovirus/ultraestructura , Bovinos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Chlorocebus aethiops , Perros , Herpesvirus Suido 1/fisiología , Herpesvirus Suido 1/ultraestructura , Células de Riñón Canino Madin Darby , Virus de la Parainfluenza 3 Bovina/fisiología , Virus de la Parainfluenza 3 Bovina/ultraestructura , Porcinos , Células Vero , Virus de la Estomatitis Vesicular Indiana/fisiología , Virus de la Estomatitis Vesicular Indiana/ultraestructura , Replicación ViralRESUMEN
Previous studies of egg drop syndrome virus (EDSV) is restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10 min post-infection and that integrated endocytic vesicles formed at 20 min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-ß-cyclodextrin (MßCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MßCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5.
Asunto(s)
Atadenovirus/fisiología , Clatrina/metabolismo , Endocitosis , Fibroblastos/virología , Interacciones Huésped-Patógeno , Internalización del Virus , Animales , Atadenovirus/ultraestructura , Membrana Celular/ultraestructura , Membrana Celular/virología , Células Cultivadas , Pollos , Patos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Hepatocitos/virología , Microscopía Electrónica de Transmisión , Factores de Tiempo , Replicación ViralRESUMEN
UNLABELLED: Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE: Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins-one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.
Asunto(s)
Atadenovirus/genética , Proteínas de la Cápside/genética , ADN Viral/genética , Genoma Viral , Lagartos/virología , Virión/genética , Secuencia de Aminoácidos , Animales , Atadenovirus/clasificación , Atadenovirus/ultraestructura , Composición de Base , Secuencia de Bases , Proteínas de la Cápside/ultraestructura , ADN/genética , Expresión Génica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Virión/ultraestructuraRESUMEN
A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-A resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent "knobs" on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.
Asunto(s)
Adenovirus Humanos/ultraestructura , Atadenovirus/ultraestructura , Virión/ultraestructura , Secuencia de Aminoácidos , Atadenovirus/química , Microscopía por Crioelectrón , Imagenología Tridimensional , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Proteínas Virales/químicaRESUMEN
Ovine adenovirus serotype 7 (OAdV), the prototype atadenovirus, has gene homologues for most mastadenovirus structural proteins but lacks proteins V and IX. Instead, OAdV has structural proteins of 32 and 42 kDa although the gene encoding the latter had not previously been identified. The presently reported studies of OAdV virions have now identified a minor structural polypeptide of approximately 40 kDa as the product of the L1 52/55-kDa gene and, more surprisingly, shown that the 42-kDa protein is encoded by LH3. This gene product was previously thought to be a homologue of mastadenovirus E1B 55 kDa, which is a multi-functional, non-structural protein that cooperates with E1A in cell transformation. The lack of transforming activity previously demonstrated for OAdV combined with a structural role for the LH3 product indicates that the protein has a different function in atadenoviruses. We discuss the abundance and likely core location of LH3 in the virion and the possible derivation of the E1B 55-kDa gene from the LH3 gene.