To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.The final step in the lytic cycle of enveloped viruses involves the budding of the newly formed particles from cellular membranes. Previous to this step, all viral structural components should have been transported, either individually or as preassembled complexes, to the cellular membrane, where viral proteins will drive the budding process.A number of studies have focused on the assembly and budding processes of viruses (arena-, alpha-, rhabdo-, paramyxo-, orthomyxo-, and retroviruses) that obtain their envelope from the plasma membrane (reviewed in references 2, 13, and 19). For the alphavirus Semliki Forest virus, it has been established that virus budding is strictly dependent on interactions between the transmembrane spike protein and the internal nucleocapsid (46). In retroviruses, however, interactions between the cytoplasmic tail of external virus proteins (Env) and the internal virus components (Gag polyprotein) are not a prerequisite for virus budding since expression of the Gag protein alone is sufficient to drive budding of virus-like particles (VLPs) (7,14). A different mechanism, which directs the assembly and release of coronavirus particles, which assemble at intracellular membranes, has been described (47). In this case, expression of viral membrane proteins alone is sufficient to drive the assembly and budding of VLPs (47).It is widely accepted that the matrix protein plays a pivotal role as an assembly organizer for RNA viruses containing a single negative-strand genomic RNA molecule (such as rhabdo-and paramyxoviruses) (reviewed in reference 25). In fact, rabies and measles viruses modified by reverse genetics technology to lack the matrix gene grow poorly, and the released matrix-less particles show drastically altered morphologies (3, 31). Moreover, it has been shown that the M1 proteins of vesicular stomatits virus (VSV) and human parainfluenza virus type 1 have intrinsic budding activity when expressed alone (5, 22, 26), an observation ...
BackgroundDuring recent years, numerous novel ‘insect flaviviruses’ have been discovered in natural mosquito populations. In a previous study we described the presence of flavivirus DNA sequences integrated in Aedes albopictus (Asian tiger mosquito) populations from Northern Italy in 2007.MethodsDuring 2008 we collected and tested Aedes females for flavivirus presence and developed phylogenetic analysis, virus isolation, electron microscopy studies and RNAse treatments.ResultsWe detected a high prevalence of flavivirus in Ae. albopictus (77.5%). The phylogenetic analysis identified the insect flavivirus sequences as Aedes flavivirus (AEFV) recently described in Japan, and that may have been introduced in Italy travelling with the tiger mosquito. Some of these pools grew in C6/36 cells, producing cytopathic effects, and the RNase treatment results showed the presence of the detected sequences in RNA forms. Furthermore, we detected a new insect flavivirus in one pool of Aedes cinereus/geminus mosquitoes. Phylogenetic analysis of this virus shows that it forms a distinct cluster within the clade of insect flavivirus.ConclusionsThis is the first study to report a high prevalence, to describe the seasonal activity and an isolation of the insect flavivirus Aedes flavivirus in Europe. Moreover we describe the detection of a new insect flavivirus detected from Ae. cinereus mosquitoes from Italy. These flavivirus may be common, ubiquitous and diverse in nature and we discuss the implications of the insect flavivirus group in virus evolution and transmission.
The pattern of flavivirus infection in mosquitoes belonging to the genera Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for flaviviruses using a generic reverse transcriptionnested-PCR targeted on a region of the non-structural NS5 gene. The phylogenetic analysis was performed on a fragment of~1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes flavivirus (AeFV) in Aedes albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus. Interestingly, we detected AeFV in C. pipiens, for the first time to our knowledge, in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. Co-infections with different flaviviruses were not detected. INTRODUCTIONThe genus Flavivirus (family Flaviviridae) comprises .70 viruses that, according to their mechanism of transmission, are included in one of the following three groups: (1) those infecting a range of vertebrate hosts through mosquito or tick bites, called 'arthropod-borne viruses', (2) those spread without a known vector, presumed to be limited to infecting vertebrates only, and (3) those apparently limited to insects alone, called 'insect-specific flaviviruses' (ISFs) (Ishikawa & Konishi, 2011;Huhtamo et al., 2012 and references therein). The flavivirus genome contains genes coding for three structural proteins (capsid, premembrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Lindenbach et al., 2006). Regions encoding envelope, NS3 and NS5 are the most frequently used for phylogenetic analysis of flaviviruses. The inclusion of ISFs in the genus Flavivirus is supported by similarities with other flaviviruses in terms of genomic organization, polyprotein hydropathy profiles and cleavage sites, but they are not able to replicate in mammalian cells and they have been isolated only in mosquito-derived cells (Kuno, 2007;Hoshino et al., 2009;Bolling et al., 2011;Cook et al., 2012;Haddow et al., 2013).The 'arthropod-borne viruses' group includes some important emerging human and animal pathogens, such 3These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession numbers for representative sequences obtained in this work are KM871198-KM871202. Lelli et al., 2008;Manarolla et al., 2010;Tamba et al., 2011; Vázquez et al., 2011;Cerutti et al., 2012;Ravanini et al., 2012;Buchebner et al., 2013;Calzolari et al., 2013a; Höfle et al., 2013). In Europe, USUV has been recognized as a candidate human pathogen in Austria (Weissenböck et al., 2007), Italy (Cavrini et al., 2009;Pecorari et al., ...
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