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1.
Viruses ; 16(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38932159

RESUMO

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an "infectious clone". This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.


Assuntos
Caliciviridae , Genética Reversa , Genética Reversa/métodos , Caliciviridae/genética , Genoma Viral , Animais , Humanos , Replicação Viral
2.
Emerg Infect Dis ; 18(12): 2009-12, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23171812

RESUMO

Outbreaks of rabbit hemorrhagic disease have occurred recently in young rabbits on farms on the Iberian Peninsula where rabbits were previously vaccinated. Investigation identified a rabbit hemorrhagic disease virus variant genetically related to apathogenic rabbit caliciviruses. Improved antivirus strategies are needed to slow the spread of this pathogen.


Assuntos
Infecções por Caliciviridae/veterinária , Surtos de Doenças/veterinária , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Animais , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Vírus da Doença Hemorrágica de Coelhos/classificação , Vírus da Doença Hemorrágica de Coelhos/genética , Dados de Sequência Molecular , Filogenia , Coelhos , Espanha/epidemiologia
3.
Phytother Res ; 26(10): 1513-20, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22318977

RESUMO

The antiherpes virus properties of Phyllanthus orbicularis Kunth, a Cuban-endemic medicinal plant, have been reported previously but data on its phytochemical profile and identification of antiviral metabolites as well as their mechanisms of action are still lacking. In this work, a bioactivity-guided phytochemical analysis was performed in order to isolate anti HSV-2 compounds. P. orbicularis contained mainly phenolic acids derivatives and flavonoids. The antiviral effects were attributed to (-)-epicatechin-3-O-gallate (EC(50) = 11.7 µg/mL), procyanidins B1 and B2 (EC(50) = 32.8 µg/mL and 24.2 µg/mL, respectively) as well as oligomeric and polymeric procyanidins and their gallate derivatives. The antiviral mechanisms of the active P. orbicularis extracts and fractions were also investigated and the inhibition of several HSV-2 early replication events and DNA synthesis were observed. This is the first study of extensive fractionation and phytochemical characterization of phenolic compounds from this species.


Assuntos
Antivirais/farmacologia , Herpesvirus Humano 2/efeitos dos fármacos , Phyllanthus/química , Extratos Vegetais/farmacologia , Animais , Catequina/análogos & derivados , Catequina/isolamento & purificação , Catequina/farmacologia , Fracionamento Químico , Chlorocebus aethiops , Extratos Vegetais/química , Plantas Medicinais/química , Proantocianidinas/isolamento & purificação , Proantocianidinas/farmacologia , Células Vero
4.
Microbiol Spectr ; 10(3): e0026922, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35506680

RESUMO

The antimicrobial properties of photocatalysts have long been studied. However, most of the available literature describes their antibacterial properties, while knowledge of their antiviral activity is rather scarce. Since the outset of the coronavirus disease 2019 (COVID-19) pandemic, an increasing body of research has suggested their antiviral potential and highlighted the need for further research in this area. In this study, we investigated the virucidal properties of a commercial TiO2-coated photocatalytic glass against a model human coronavirus. Our findings demonstrate that the TiO2-coated glass consistently inactivates coronaviruses upon contact under daylight illumination, in a time-dependent manner. A 99% drop in virus titer was achieved after 3.9 h. The electron micrographs of virus-covered TiO2-glass showed a reduced number of virions compared to control glass. Morphological alterations of TiO2-exposed viruses included deformation, disruption of the viral envelope, and virion ghosts, endorsing the application of this material in the construction of protective elements to mitigate the transmission of viruses. To the best of our knowledge, this is the first report showing direct visual evidence of human coronaviruses being damaged and morphologically altered following exposure to this photocatalyst. IMPORTANCE Surface contamination is an important contributor to SARS-CoV-2 spread. The use of personal protective elements and physical barriers (i.e., masks, gloves, and indoor glass separators) increases safety and has proven invaluable in preventing contagion. Redesigning these barriers so that the virus cannot remain infectious on them could make a difference in COVID-19 epidemiology. The introduction of additives with virucidal activity could potentiate the protective effects of these barriers to serve not only as physical containment but also as virus killers, reducing surface contamination after hand touch or aerosol deposition. We performed in-depth analysis of the kinetics of photocatalysis-triggered coronavirus inactivation on building glass coated with TiO2. This is the first report showing direct visual evidence (electron microscopy) of coronaviruses being morphologically damaged following exposure to this photocatalyst, demonstrating the high potential of this material to be incorporated into daily-life high-touch surfaces, giving them an added value in decelerating the virus spread.


Assuntos
COVID-19 , Vírus , Antivirais/farmacologia , COVID-19/prevenção & controle , Humanos , Pandemias , SARS-CoV-2
5.
Vaccines (Basel) ; 9(9)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34579243

RESUMO

The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.

6.
Virol J ; 7: 49, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20187925

RESUMO

BACKGROUND: Myxoma virus (MV) has been endemic in Europe since shortly after its deliberate release in France in 1952. While the emergence of more resistant hosts and more transmissible and attenuated virus is well documented, there have been relatively few studies focused on the sequence changes incurred by the virus as it has adapted to its new host. In order to identify regions of variability within the MV genome to be used for phylogenetic studies and to try to investigate causes of MV strain attenuation we have molecularly characterised nine strains of MV isolated in Spain between the years 1992 and 1995 from wide ranging geographic locations and which had been previously graded for virulence by experimental infection of rabbits. RESULTS: The findings reported here show the analysis of 16 genomic regions accounting for approximately 10% of the viral genomes. Of the 20 genes analysed 5 (M034L, M069L, M071L, M130R and M135R) were identical in all strains and 1 (M122R) contained only a single point mutation in an individual strain. Four genes (M002L/R, M009L, M036L and M017L) showed insertions or deletions that led to disruption of the ORFs. CONCLUSIONS: The findings presented here provide valuable tools for strain differentiation and phylogenetic studies of MV isolates and some clues as to the reasons for virus attenuation in the field.


Assuntos
Myxoma virus/química , Myxoma virus/patogenicidade , Polimorfismo Genético , Infecções por Poxviridae/veterinária , Infecções Tumorais por Vírus/veterinária , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes Virais , Mutação INDEL , Dados de Sequência Molecular , Myxoma virus/isolamento & purificação , Mutação Puntual , Infecções por Poxviridae/virologia , Coelhos , Alinhamento de Sequência , Análise de Sequência de DNA , Espanha , Infecções Tumorais por Vírus/virologia , Virulência
7.
Front Microbiol ; 11: 596245, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33304341

RESUMO

Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5'-cap structure was added to the 5' end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

8.
J Gen Virol ; 90(Pt 11): 2724-2730, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19605586

RESUMO

The mechanisms of calicivirus attachment and internalization are not well understood, mainly due to the lack of a reliable cell-culture system for most of its members. In this study, rabbit vesivirus (RaV) virions were shown to bind annexin A2 (ANXA2) in a membrane protein fraction from HEK293T cells, using a virus overlay protein-binding assay and matrix-assisted laser desorption/ionization time-of-flight analysis. A monoclonal anti-ANXA2 antibody and small interfering RNA-mediated knockdown of ANXA2 expression in HEK293T cells reduced virus infection significantly, further supporting the role of ANXA2 in RaV attachment and/or internalization.


Assuntos
Anexina A2/metabolismo , Receptores Virais/metabolismo , Vesivirus/fisiologia , Internalização do Vírus , Animais , Anexina A2/antagonistas & inibidores , Anticorpos Monoclonais/imunologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Ligação Proteica , Coelhos , Receptores Virais/antagonistas & inibidores
9.
Transbound Emerg Dis ; 66(6): 2218-2226, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31322320

RESUMO

The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host-pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis-like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5-month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next-generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population.


Assuntos
Lebres/virologia , Myxoma virus/genética , Infecções por Poxviridae/virologia , Animais , DNA Viral/genética , Genoma Viral , Mutagênese Insercional , Filogenia , Reação em Cadeia da Polimerase , Infecções por Poxviridae/veterinária , Coelhos , Espanha , Sequenciamento Completo do Genoma
10.
Vet Microbiol ; 169(1-2): 67-73, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24461551

RESUMO

Rabbit hemorrhagic disease outbreaks in young rabbits have been recently observed in Spain. In this study we have tracked the spread of variant RHDV in samples collected from rabbit farms over a period of more than one year using RT-PCR and antigen-capture ELISA. The isolates were sequenced and compared to classic and variant RHDV strains and phylogenetic analyses were conducted. Mortalities have been observed in kits as young as 11 days. More than 50% of the dead rabbits had been previously vaccinated against RHDV using commercially available inactivated vaccines indicating a putative lack of protection against the variant RHDV. The large majority of the studied outbreaks (94.5%) in Spanish farms during 2012 were due to variant RHDV and only 3 out of the 55 farms were affected by classic RHDV. The data demonstrates that the variant RHDV has spread through a large number of Spanish provinces in a relatively short period of time, largely replacing the previously predominant G1 RHDV genotypes. Considering the lack of efficient vaccines against the variant RHDV strains strict disease control and greater vigilance measures should be put in place.


Assuntos
Infecções por Caliciviridae/veterinária , Surtos de Doenças/veterinária , Vírus da Doença Hemorrágica de Coelhos , Coelhos/virologia , Animais , Sequência de Bases , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Genoma Viral , Genótipo , Vírus da Doença Hemorrágica de Coelhos/classificação , Vírus da Doença Hemorrágica de Coelhos/genética , Vírus da Doença Hemorrágica de Coelhos/imunologia , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espanha/epidemiologia , Vacinação/veterinária , Vacinas Virais/administração & dosagem
11.
J Med Food ; 15(6): 581-7, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22424460

RESUMO

The anti-herpes simplex virus type 1 and anti-herpes simplex virus type 2 effects of apple pomace, a by-product from the cider-processing industry, were investigated. The mechanisms of antiviral action were assessed using a battery of experiments targeting sequential steps in the viral replication cycle. The anti-herpetic mechanisms of apple pomaces included the inhibition of virus attachment to the cell surface and the arrest of virus entry and uncoating. Quercitrin and procyanidin B2 were found to play a crucial role in the antiviral activity.


Assuntos
Antivirais/farmacologia , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Malus , Preparações de Plantas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Indústria Alimentícia , Frutas , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/patogenicidade , Fitoterapia , Preparações de Plantas/uso terapêutico , Células Vero
12.
J Clin Microbiol ; 42(6): 2668-74, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15184450

RESUMO

From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%), Campylobacter spp. (28.8%), and Salmonella spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and Yersinia spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.


Assuntos
Gastroenterite/virologia , Norovirus/classificação , Doença Aguda , Adolescente , Sequência de Bases , Criança , Pré-Escolar , Genótipo , Humanos , Lactente , Dados de Sequência Molecular , Norovirus/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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