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Highlights • The genome sequence and phylogenetic analysis of two novel orthoreovirus strains are described. • Strain 2533/4/1-10 is probably a triple reassortant. • Strain 2533/6/1-10 might have been acquired from an unknown natural host species. Abstract Complete genomic sequences of two orthoreovirus strains, D2533/4/1-10 and D2533/6/1-10, isolated from Pekin ducklings in Germany have been determined. Pairwise sequence comparisons and phylogenetic analyses indicated that strain D2533/4/1-10 might have acquired its genomic segments from three different origins, from classical and novel waterfowl reoviruses, and a yet unknown orthoreovirus strain. D2533/6/1-10 proved to be only distantly related to previously described orthoreoviruses. Reassortment, host species transmission events, and successful adaptation of novel variants may signify a challenge for animal health and maintenance of economic production. [ABSTRACT FROM AUTHOR]

Highlights • We developed a DNA-launched infectious clone for ZIKV isolate ZG01 (Asian lineage). • Two substitutions deactivating E. coli promoter enable the cloning of ZG01 genome. • Four mutations (4m) efficiently adapted recombinant ZG01 in vitro and in vivo. • ZG01_4m clone was stable after repetitive transformation-purification cycles in bacteria. • 5′-3′UTR base-paring may exist and the nucleotides involved were critical for ZIKV. Abstract Zika virus (ZIKV) is an Aedes mosquitoes-transmitted flavivirus, and its infection may cause severe neurological diseases. A genetically stable infectious clone is essential for ZIKV research, however the toxicity and instability of the viral cDNA in bacteria potentially due to its bacterial promoter activity are major challenges. Here, we constructed a full-length cDNA clone for isolate ZG01 by introducing non-coding changes T1865C/A1868G to reduce the bacterial promoter activity. Wild-type and recombinant ZG01 were highly attenuated in Vero cells, thus we serially passaged wild-type ZG01 through neonatal mice and Vero cells to generate high-titer virus, from which four mutations (4m, C2178T/G2913A/T4991C/T10561C) were identified. Addition of 4m greatly enhanced the infectivity, as ZG01_4m released ZIKV of 107.0–107.5 plaque-forming unit (PFU)/ml in infected Vero and A549 cells. ZG01_4m resembled the infectivity of high-titer ZG01 in vitro and in vivo. Notably, ZG01_4m plasmid was genetically stable after multiple rounds of transformation-purification in bacteria. Using ZG01_4m, we identified a potential RNA-RNA interaction between 5′UTR and 3′UTR and demonstrated that the nucleotides involved were essential for ZIKV production. The genetically stable ZG01 cDNA clone provides a reliable tool for the study of this important virus, and the strategy used here is feasible for the development of reverse genetics systems for other ZIKV isolates and related flaviviruses. [ABSTRACT FROM AUTHOR]

The current study investigated nucleotide variability and phylogeny in high-risk HPV45 collected from Chinese women. Fifty-one samples positive for single infections of HPV45 were collected for DNA extraction and HPV typing. The E6 and E7 genes of HPV45 were sequenced, and then the phylogenetic tree was reconstructed by the maximum likelihood method implemented in IQ-TREE under the HKY nucleotide substitution model. The selection pressures of the E6 and E7 genes were estimated using PAML software. Eleven nucleotide polymorphism sites were observed in the HPV45 E6 sequences, with 6 synonymous (C134T, T163C, A284C, T341C, T482C, A497G) and 5 non-synonymous (A124C, C157T, T162A, G259T, G487A) mutations. Six nucleotide polymorphism sites were observed in the E7 sequences, with 5 non-synonymous (G600A, A603C, A769C, G808T, G832T) and 1 synonymous (A718C) mutation. Our investigation suggests that B2 was the most frequent HPV45 sublineage in Southwest China, followed by A2; no A1 or A3 variants were detected. Selective pressure analysis showed that these mutations could reflect positive selection in HPV45 E6 and E7 genes. [ABSTRACT FROM AUTHOR]

Avian leukosis virus subgroup J (ALV-J) can cause great economic losses to the poultry industry worldwide. Baicalin, one of the flavonoids present in S.baicalensis Georgi, has been shown to have antiviral activities. To investigate whether baicalin has antiviral effects on the infection of ALV-J in DF-1 cells, the cells were treated with baicalin at different time points. We found that baicalin could inhibit viral mRNA, protein levels and overall virus infection in a dose- and time-dependent manner using a variety of assays. Baicalin specifically targeted virus internalization and reduced the infectivity of ALV-J particles, but had no effect on the levels of major ALV-J receptor and virus binding to DF-1 cells. Collectively, these results suggest that baicalin might have potential to be developed as a novel antiviral agent for ALV-J infection. [ABSTRACT FROM AUTHOR]

In 2007, numerous hamadryas baboons ( Papio hamadryas ) died suddenly in an aviary of a primate institute in Sochi, Russia, in the absence of prior clinical signs. Necropsies were suggestive of encephalomyocarditis virus infection, but RT-PCR assays with commonly used primers were negative. Here we report the histopathological results obtained during necropsies and the isolation and genomic characterization of a divergent strain of encephalomyocarditis virus 1 (EMCV-1) from heart tissue of one of the succumbed hamadryas baboons. Phylogenetic analysis indicates that the isolated virus belongs to the newly proposed EMCV-1 lineage G, which clusters alongside lineage C (“Mengo virus”). This study is the first report describing a lineage G strain of EMCV-1 as the etiological agent of a lethal disease outbreak among captive nonhuman primates in Europe. [ABSTRACT FROM AUTHOR]

This manuscript reviews the reasons why Integrase inhibitors should now routinely constitute a part of first line antiretroviral therapy for the treatment of HIV disease. The use of these drugs that are generally well tolerated has resulted in far less drug resistance than was the case with most other categories of antiviral compounds. In addition, the integrase inhibitor family of drugs has been less prone to the problem of transmitted drug resistance which is due to a wide variety of substitutions in the HIV genome that can be sexually transmitted from one person to another. However, the use of integrase inhibitors in first line therapy may unfortunately not soon happen in developing country settings where non-nucleoside reverse transcriptase inhibitors continue to be a mainstay of initial therapy, primarily for reasons of cost. As long as this situation continues, problems of drug resistance and transmitted drug resistance will be common in such settings. Current evidence also suggests that the use of dolutegravir as a first line integrase inhibitor may limit development of drug resistance to an extent that exceeds the use of other members of this family of drugs. This may be due to particular patterns of resistance involving dolutegravir, whereby the mutations that are associated with resistance against this compound may actually diminish both HIV replication capacity as well as integrase enzymatic activity in a far-reaching and unique manner. This gives potential hope that the use of dolutegravir in first line therapy could actually form part of the long-sought goal of attainment of a functional cure for HIV disease. [ABSTRACT FROM AUTHOR]

The accurate classification of viral dark matter – metagenomic sequences that originate from viruses but do not align to any reference virus sequences – is one of the major obstacles in comprehensively defining the virome. Depending on the sample, viral dark matter can make up from anywhere between 40 and 90% of sequences. This review focuses on the specific nature of dark matter as it relates to viral sequences. We identify three factors that contribute to the existence of viral dark matter: the divergence and length of virus sequences, the limitations of alignment based classification, and limited representation of viruses in reference sequence databases. We then discuss current methods that have been developed to at least partially circumvent these limitations and thereby reduce the extent of viral dark matter. [ABSTRACT FROM AUTHOR]

Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for the study of viromes, sample preparation methodologies use different approaches to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focus on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density gradient centrifugation. However, recently a new approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was developed to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we discuss the different methods, their applications and evolution, for selective sequencing based virome analysis and also propose refinements needed to harness the full potential of HTS for virome analysis. [ABSTRACT FROM AUTHOR]

Rapidly evolving RNA viruses prevail within a host as a collection of closely related variants, referred to as viral quasispecies. Advances in high-throughput sequencing (HTS) technologies have facilitated the assessment of the genetic diversity of such virus populations at an unprecedented level of detail. However, analysis of HTS data from virus populations is challenging due to short, error-prone reads. In order to account for uncertainties originating from these limitations, several computational and statistical methods have been developed for studying the genetic heterogeneity of virus population. Here, we review methods for the analysis of HTS reads, including approaches to local diversity estimation and global haplotype reconstruction. Challenges posed by aligning reads, as well as the impact of reference biases on diversity estimates are also discussed. In addition, we address some of the experimental approaches designed to improve the biological signal-to-noise ratio. In the future, computational methods for the analysis of heterogeneous virus populations are likely to continue being complemented by technological developments. [ABSTRACT FROM AUTHOR]

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the m Sample Preparation Systems RNA kit with automated extraction by the m2000 sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5 kb, and subsequent recovery of near-complete viral genomes. Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3 kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml. [ABSTRACT FROM AUTHOR]

The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NS M ) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NS M proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NS M of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NS M , but not that of the CSNV, TCSV and TSWV NS M proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NS M mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NS M was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the ‘30K family’. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior. [ABSTRACT FROM AUTHOR]

Emergence of the porcine epidemic diarrhea virus (PEDV) as a global threat to the swine industry underlies the urgent need for deeper understanding of this virus. To date, we have yet to identify functions for all the major gene products, much less grasp their implications for the viral life cycle and pathogenic mechanisms. A major reason is the lack of genetic tools for studying PEDV. In this review, we discuss the reverse genetics approaches that have been successfully used to engineer infectious clones of PEDV as well as other potential and complementary methods that have yet to be applied to PEDV. The importance of proper cell culture for successful PEDV propagation and maintenance of disease phenotype are addressed in our survey of permissive cell lines. We also highlight areas of particular relevance to PEDV pathogenesis and disease that have benefited from reverse genetics studies and pressing questions that await resolution by such studies. In particular, we examine the spike protein as a determinant of viral tropism, entry and virulence, ORF3 and its association with cell culture adaptation, and the nucleocapsid protein and its potential role in modulating PEDV pathogenicity. Finally, we conclude with an exploration of how reverse genetics can help mitigate the global impact of PEDV by addressing the challenges of vaccine development. [ABSTRACT FROM AUTHOR]

• Due to a lack of an efficient reverse genetic (RG) system, reovirus biology studies have lagged behind those of other RNA pathogens. • Recently, several entirely plasmid-based RG systems are developed for several members of the Reovirales. • It is less likely to develop a universal RG system for the Reovirales viruses. Reovirales are segmented double-strand RNA viruses with a broad host range that pose a serious threat to human and animal health. However, there are numerous viral species within the Reovirales , some of which have lagged behind other RNA viruses in the study of their biology due to the lack of an effective reverse genetics (RG) system. The RG systems are the most powerful tools for studying viral protein function, viral gene expression regulation, viral pathogenesis, and the generation of engineered vaccines. Recently, several entirely plasmid-based RG systems have been developed for several members of the Reovirales. This review outlines the development and future direction of the RG system for the best studied Reovirales viruses. [ABSTRACT FROM AUTHOR]

Modelling of gene networks is widely used in systems biology to study the functioning of complex biological systems. Most of the existing mathematical modelling techniques are useful for analysis of well-studied biological processes, for which information on rates of reactions is available. However, complex biological processes such as those determining the phenotypic traits of organisms or pathological disease processes, including pathogen-host interactions, involve complicated cross-talk between interacting networks. Furthermore, the intrinsic details of the interactions between these networks are often missing. In this study, we developed an approach, which we call mosaic network modelling , that allows the combination of independent mathematical models of gene regulatory networks and, thereby, description of complex biological systems. The advantage of this approach is that it allows us to generate the integrated model despite the fact that information on molecular interactions between parts of the model (so-called mosaic fragments) might be missing. To generate a mosaic mathematical model, we used control theory and mathematical models, written in the form of a system of ordinary differential equations (ODEs). In the present study, we investigated the efficiency of this method in modelling the dynamics of more than 10,000 simulated mosaic regulatory networks consisting of two pieces. Analysis revealed that this approach was highly efficient, as the mean deviation of the dynamics of mosaic network elements from the behaviour of the initial parts of the model was less than 10%. It turned out that for construction of the control functional, data on perturbation of one or two vertices of the mosaic piece are sufficient. Further, we used the developed method to construct a mosaic gene regulatory network including hepatitis C virus (HCV) as the first piece and the tumour necrosis factor (TNF)-induced apoptosis and NF-κB induction pathways as the second piece. Thus, the mosaic model integrates the model of HCV subgenomic replicon replication with the model of TNF-induced apoptosis and NF-κB induction. Analysis of the mosaic model revealed that the regulation of TNF-induced signaling by the HCV network is crucially dependent on the RIP1, TRADD, TRAF2, FADD, IKK, IκBα, c-FLIP, and BAR genes. Overall, the developed mosaic gene network modelling approach demonstrated good predictive power and allowed the prediction of new regulatory nodes in HCV action on apoptosis and the NF-κB pathway. Those theoretical predictions could be a basis for further experimental verification. [ABSTRACT FROM AUTHOR]

To investigate the genetic variability and evolution of rabbit hemorrhagic disease virus (RHDV) strains in China, VP60 gene sequences of eight new isolates collected from farms with RHD occurrences in China between 2009 and 2014 were analyzed, and compared with the reference sequence of the vaccine strain WF/China/2007. We conducted a comprehensive analysis of the Chinese RHDV strains, including hemagglutination tests, western blot and immunosassays of capsid proteins, and phylogenetic analysis, and identified a new distinct antigenic variant. Specifically, strain HB/2014 collected in North China was identified as a non-hemagglutinating strain, and belongs to the original RHDV (G1–G5) group. The other seven isolates were classified in genogroup G6 (RHDVa), which was widely distributed across China before 2014, and was thought to replace the earlier groups. Antigenic characterization of the VP60 genes revealed a large degree of nucleotide sequence divergence between HB/2014 and the other Chinese strains. However, the current vaccine showed complete cross-protection against HB/2014 challenge in inoculated rabbits. Collectively, these data provide new tools and insight for further understanding the molecular evolution of RHDV in China. [ABSTRACT FROM AUTHOR]

The prevalence of Enterovirus G (EV-G) and Sapelovirus A (PSV-1) was investigated in Spanish swine herds by means of cross-sectional studies. Faecal samples from clinically healthy pigs were collected from six farms, and analysed by RT-PCR. The results indicated a high prevalence of EV-G detected in nearly all the animals older than 3 weeks of age. Otherwise, PSV-1 was only detected in 3-week-old piglets from one of the farms. Genetic analyses performed in the VP1 region of the EV-G indicated circulation of diverse strains in the same farm, related to genotypes G1, G2, G3, G4, G6, G9, G12, G13 and G14. Moreover, co-infection of several PSV-1 variants in the same animal was evident, typical of viral quasispecies. Evolutionary pressure analysis indicated that microevolution of PSV-1 seems to be driven by negative selection. This study gives further insights in the epidemiology of EV-G and PSV-1. [ABSTRACT FROM AUTHOR]

In order to detect phleboviruses’ natural infection in sandflies, an entomological survey was carried out, from May to October in 2007 and 2008, in Arrábida region in the south of Portugal. The isolation of a new phlebovirus was achieved after inoculation of a sandfly pool homogenate in Vero E6 cells. Based on the phylogenetic analysis of the complete genome sequences from the Small, Medium and Large, segments obtained with Next Generation sequencing, we can assume that the new phlebovirus , provisionally named Arrabida virus, is closely related to Massilia, Granada and Punique viruses. This is the first isolation of a sandfly-borne phlebovirus from the Sandfly Naples Fever Virus group in Portugal. Further investigation is needed in order to assess the importance of this phlebovirus for Public Health. [ABSTRACT FROM AUTHOR]

Our objective here is to review the novel delivery platform based on Bacteriophage MS2 virus-like particles (VLPs), including introduction to their structure, their potential as a delivery platform, and their expected use in medicine and other fields. Bacteriophage MS2 VLPs are nanoparticles devoid of viral genetic material and can self-assemble from the coat protein into an icosahedral capsid. As a novel delivery platform, they possess numerous features that make them suitable and attractive for targeted delivery of RNAs or DNAs, epitope peptides, and drugs within the protein capsid. In short, as a novel delivery platform, MS2 VLPs are suitable for delivery of targeted agents and hold promise for use in diagnostics, vaccines, and therapeutic modalities. [ABSTRACT FROM AUTHOR]

Giant viruses infect protozoa, especially amoebae of the genus Acanthamoeba . These viruses possess genetic elements named Mobilome. So far, this mobilome comprises provirophages which are integrated into the genome of their hosts, transpovirons, and Maverick/Polintons. Virophages replicate inside virus factories within Acanthamoeba and can decrease the infectivity of giant viruses. The virophage infecting CroV was found to be integrated in the host of CroV, Cafeteria roenbergensis , thus protecting C. roenbergensis by reduction of CroV multiplication. Because of this unique property, assessment of the mechanisms of replication of virophages and their relationship with giant viruses is a key element of this investigation. This work aimed at evaluating the presence and the dynamic of these mobile elements in sixteen Acanthamoeba genomes. No significant traces of the integration of genomes or sequences from known virophages were identified in all the available Acanthamoeba genomes. These results brought us to hypothesize that the interactions between mimiviruses and their virophages might occur through different mechanisms, or at low frequency. An additional explanation could be that our knowledge of the diversity of virophages is still very limited. [ABSTRACT FROM AUTHOR]

The Caliciviridae family of small positive sense RNA viruses contains a diverse range of pathogens of both man and animals. The molecular mechanisms of calicivirus genome replication and translation have not been as widely studied as many other RNA viruses. With the relatively recent development of robust cell culture and reverse genetics systems for several members of the Caliciviridae family, a more in-depth analysis of the finer detail of the viral life cycle has now been obtained. As a result, the identification and characterization of the role of RNA structures in the calicivirus life cycle has also been possible. This review aims to summarize the current state of knowledge with respect to the role of RNA structures at the termini of calicivirus genomes. [ABSTRACT FROM AUTHOR]

A novel recombinant pseudorabies virus (PRV) expressing porcine circovirus type 2 (PCV2) capsid protein and IL-18 was constructed. The PCV2 open reading frame 2 (ORF2) and porcine IL-18 genes were amplified by PCR and then inserted into the PRV transfer vector (pG) to produce a recombinant plasmid (pGO18). Plasmid pGO18 was transfected into porcine kidney cell (PK15) pre-infected with PRV HB98 vaccine strain to generate a recombinant virus. The recombinant virus PRV-ORF2-IL18 was purified by green fluorescent plaque purification and the inserts were confirmed by PCR, enzyme digestion, sequencing, and Western blot. The humoral and cellular responses induced by the recombinant virus were assessed in mice. Mice ( n = 10) were immunized with PRV-ORF2-IL18, PRV-ORF2, PRV HB98, or inactivated PCV2. PRV-ORF2-IL18 elicited high titers of ELISA and serum neutralizing antibodies and strong cell-mediated immune responses in mice as indicated by anti-PCV2 ELISA, PRV-neutralizing assay, PCV2 specific lymphocyte proliferation assay, CD3 + , CD4 + and CD8 + T lymphocytes analysis, respectively. And PRV-ORF2-IL18 immunization protected mice against a lethal challenge of a virulent PRV Fa strain and significantly reduced the amount of PCV2 viremia. These results suggest an adjuvant effect of IL-18 on cellular immune responses. The recombinant virus might be an attractive candidate vaccine for preventing PCV2 and PRV infections in pigs. [ABSTRACT FROM AUTHOR]

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae , was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV. [ABSTRACT FROM AUTHOR]

Multiple genetically and antigenically distinct hemagglutinin genes of the H1 and H3 influenza A virus (IAV) subtypes co-circulate in North American swine. This diversity has evolved by repeated transmission of IAVs from humans to swine and subsequent antigenic drift in swine. To understand the evolutionary dynamics of these diverse HA lineages in North American swine, we undertook a phylogenetic analysis of 1576 H1 and 607 H3 HA gene segments, as well as 834 N1 and 1293 N2 NA gene segments, and 2126 M gene segments. These data revealed yearly co-circulation of H1N1, H1N2, and H3N2 viruses, with three HA clades representing the majority of the HA sequences: of the H1 viruses, 42% were classified as H1δ1 and 40.6% were classified as H1γ; and of the H3 viruses 53% were classified as cluster IV-A H3N2. We detected a genetically distinct minor clade consisting of 37 H1 viruses isolated between 2003 and 2013, which we classified as H1γ-2. We estimated that this clade circulated in swine since approximately 1995, but it was not detected in swine until 2003. Though this clade only represents 1.07% of swine H1 sequences reported over the past 10 years, hemagglutination inhibition (HI) assays demonstrated that representatives of this clade of viruses are antigenically distinct, and, when measured using antigenic cartography, were as many as 7 antigenic units from other H1γ viruses. Therefore vaccines against the contemporary H1γ viruses are not likely to cross-protect against γ-2 viruses. The long-term circulation of these γ-2 viruses suggests that minor populations of viruses may be underreported in the national dataset given the long branch lengths and gaps in detections. The identification of these γ-2 viruses demonstrates the need for robust surveillance to capture the full diversity IAVs in swine in the USA and the importance of antigenic drift in the diversification and emergence of new antigenic variants in swine, which complicates vaccine design. [ABSTRACT FROM AUTHOR]

Toscana virus (TOSV) was originally isolated in 1971 from a pool of Phlebotomus perniciosus sandflies collected in Grosseto province (Central Italy). Since its first isolation, several studies have been conducted in Italy and other Mediterranean countries in order to identify its possible animal reservoirs, spread of infection and genetic variability. Phylogenetic analysis conducted on TOSV genome demonstrated the co-circulation of two major lineages in the Mediterranean areas, TOSV A and TOSV B. This study reports the results of the genetic analysis of 32 viral strains isolated in Italy in the last 30 years from patients hospitalized with neurological disease, from sandflies and from the brain of a bat. The genetic diversity of TOSV was investigated by determining the sequences of the whole S segment. Phylogenetic analysis showed that TOSV A lineage represents the lineage circulating in Italy. Moreover, the current variability of lineage A is similar to that of lineage B. [ABSTRACT FROM AUTHOR]

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last 13 years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV). [ABSTRACT FROM AUTHOR]

Highlights: [•] First, a detailed overview on the early attempts to serologically detect hantavirus infections in Africa is presented. [•] Since 2006, genetic material of various novel hantaviruses from Africa has been molecularly identified not only in rodents but also shrews and bats. [•] Only Sangassou virus from Guinea has been isolated and functionally characterized in cell culture yet. [•] Serological studies in West Africa, based on a battery of screening and confirmatory assays, led to the detection of hantavirus antibodies in the human population and in patients with putative hantavirus disease. [•] In a current investigation, a human seroprevalence rate of 1.0% (n =1442) was detected in the South African Cape Region. [Copyright &y& Elsevier]

Highlights: [•] Isolation of a granulovirus from Phthorimaea operculella (Zeller). [•] PCR amplification and sequencing of the grn, lef-8, egt genes. [•] Phylogenetic comparison of the egt gene sequence using GenBank data. [•] Analysis of the egt gene structure. [ABSTRACT FROM AUTHOR]

Highlights: [•] Description of a sequence of structural events for VACV assembly that provides key clues about VACV morphogenesis. [•] Electron microscopy (EM) studies with the WR mutants M65 and M101. [•] Intermediate stages in poxvirus morphogenesis. [ABSTRACT FROM AUTHOR]

Highlights: [•] We review information about BTV in Caribbean, Central and South America. [•] There is the lack of information about the virus in the region. [•] Surveillance programs and control networks are needed in that region. [•] It is proposed a genetic continuity for the whole continent. [ABSTRACT FROM AUTHOR]

Highlights: [•] Four viroid species infecting Coleus blumei (CbVd-1, 3, 5, and 6) were studied. [•] Bioassays using infectious viroid cDNA clones were conducted for all four species. [•] Infectivity assay for CbVd-5 and CbVd-6 was reported for the first time. [•] Genetic diversity of CbVd populations may depend on the infected coleus plants and the viroid genotype. [•] CbVd-5 and CBVd-6 were confirmed to be transmitted via seeds. [ABSTRACT FROM AUTHOR]

Highlights: [•] First study describing the full-length genome of a begomovirus infecting carrot. [•] Ageratum enation virus (AEV), detected from carrot is referred as AEV-Car. [•] Symptomatic ageratum also showed the presence of AEV. [•] Ageratum yellow leaf curl betasatellite (AYLCB) was detected from symptomatic carrot and ageratum samples. [•] Agroinoculation of the AEV-Car plus AEV-Age plus AYLCB resulted in enhancement of symptoms and a higher level of viral DNA accumulation. [Copyright &y& Elsevier]

Highlights: [•] The most recent common ancestor of PPV strains existed about 250 years ago. [•] The 127-nt repeat in the 3′NTR was present in viruses of some subclades that evolved about 80 years ago. [•] PPV NS1 and VP2 proteins were under purifying selection. [•] Recombination did not contribute to the genetic diversity of PPV. [Copyright &y& Elsevier]

Highlights: [•] First publication of complete genomes of Type 1 PRRSV isolated in Denmark. [•] Genetic and antigenetic analysis of complete genomes of PRRSV isolated over a period of 10 years. [•] Two genetic different viruses are circulating in Danish pig herds. [•] A thorough examination of ORF5 diversity. [Copyright &y& Elsevier]

Highlights: [•] Rubus yellow net virus (RYNV) complete genomic sequence. [•] Sequences indicate RYNV is a distantly related member of the genus Badnavirus. [•] A full length clone confirmed validity of the genomic sequence. [•] Detected virus-derived small RNA (vsRNA) in RYNV infected raspberry tissue. [•] Mapping vsRNA showed uneven genome-wide clustering in both RYNV strands. [Copyright &y& Elsevier]

Highlights: [•] Virus associated with blackberry yellow vein disease. [•] First ampelovirus infecting small fruit crops. [•] Among largest closteroviruses known to date. [•] High degree of diversity identified among isolates. [•] Virus genes under stringent purification selection. [Copyright &y& Elsevier]

Highlights: [•] The 16 reported DHAV isolates (15 DHAV-1 strains and one DHAV-3) were isolated from infected ducks with clinical symptoms in China between 2009 and 2012. [•] The various DHAV-1 strains have different lethal doses, and duck eggs were more susceptible to DHAV than chicken eggs. [•] We report the genetic characterization of the DHAVs isolated in clinical samples in China, which will be helpful for understanding the epidemiology and evolution of DHAVs. [Copyright &y& Elsevier]

Highlights: [•] We identify 13 recombinants from 32 PCV2 positive clinical samples from the diseased and dead pigs. [•] 13 recombinants are classified into two new clusters in the phylogenetic tree of PCV2. [•] Two cluster recombinants are derived from different patterns of natural inter-genotype recombination between two PCV2 parental strains with the breakpoint located within ORF2. [Copyright &y& Elsevier]

Highlights: [•] Feline infectious peritonitis (FIP) is caused by a mutant enteric coronavirus. [•] Susceptibility to FIP appears to be heritable in pedigreed cats. [•] Birman cats suffer a high incidence of FIP and are highly inbred. [•] Heritable factors were interrogated in Birman cats by genome wide association study. [•] Five relevant candidate genes were strongly associated with FIP susceptibility. [Copyright &y& Elsevier]

Highlights: [•] Genomic sequencing was performed on resistant isolates obtained after antiviral treatment. [•] A common mutation was found in the NS5B region of the genome in all resistant isolates. [•] Two isolates had an additional mutation in the same region. [•] Observed mutations did not largely alter growth curves for mutant isolates. [•] Compound appears to act by binding to the region of NS5B exhibiting the mutations. [Copyright &y& Elsevier]

Abstract: Macrobrachium rosenbergii nodavirus (MrNV) is responsible for the newly emerging catastrophic disease known as white tail disease (WTD) in M. rosenbergii. The complete sequence of RNA2 (1175bp) and 3126bp region of RNA1 of an Indian strain of MrNV was generated. Sequence analysis of RNA2 revealed the presence of a single ORF encoding a capsid protein of 371 amino acids with a predicted molecular mass and pI of 41.5kDa and 8.97 respectively. RNA1 contained two ORFs, one encoding a partial RNA dependent RNA polymerase (RdRp) of length 1034 amino acids and another a B2-like protein with a length 133 amino acids. A phylogenetic analysis based on the amino acid sequence of the capsid protein, to related nodavirus sequences suggests the establishment of new genotypes within the Nodaviridae family and we suggest the name should be genus Gammanodavirus. A new reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been developed and optimized for the detection of shrimp nodavirus with a sensitivity to detect up to 24 copy numbers of plasmid construct. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type-2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases and has 4 main ORFs, ORF1 (Rep gene), ORF2 (Cap gene), ORF3 within ORF1, and ORF4, which is overlapped with ORF3, and 1 origin (Ori) of replication located between ORF1 and ORF2. The chimeric PCV1-2, containing the PCV2 capsid, PCV1 rep, and Ori genes, is attenuated in pigs. In order to verify the role of the Rep gene or Ori in the virulence of PCV2, 3 chimeric viruses [PCV2b-Ori1 (PCV1 Ori gene cloned into the backbone of PCV2b), PCV2b-rep1 (PCV1 Rep gene cloned into the backbone of PCV2b), and PCV2b-rep1-Ori1 (PCV1 Rep and Ori genes cloned into the backbone of PCV2b)] and 2 wild-type recombinant PCV2b and PCV1 were constructed and identified. The experimental results in piglets showed that clinical symptoms, viremia, viral load, lesions in lymphoid and lung tissues, and IL-10 and TNF-α expression levels in PBMCs in the PCV2b-rep1-Ori1 and PCV2b-rep1 groups were significantly decreased, compared to PCV2-infected piglets. Meanwhile, histological lesions of lymphoid and lung tissues, viral loads in lymphoid tissues, viremia, and TNF-α expression in PBMCs were not significantly different between groups PCV2b-Ori1 and PCV2b, suggesting that the Rep gene (ORF1) likely contributes to viral pathogenicity in vivo. [Copyright &y& Elsevier]

Abstract: Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 106 EID50 of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds. [Copyright &y& Elsevier]

Abstract: The early steps of HIV-1 infection starting after virus entry into cells up to integration of its genome into host chromosomes are poorly understood. From seminal work showing that HIV-1 and oncoretroviruses follow different steps in the early stages post-entry, significant advances have been made in recent years and an important role for the HIV-1 capsid (CA) protein, the constituent of the viral core, has emerged. CA appears to orchestrate several events, such as virus uncoating, recognition by restriction factors and the innate immune system. It also plays a role in nuclear import and integration of HIV-1 and has become a novel target for antiretroviral drugs. Here we describe the different functions of CA and how they may be integrated into one or more coherent models that illuminate the early events in HIV-1 infection and their relations with the host cell. [Copyright &y& Elsevier]

Abstract: One of the hallmarks of HIV infection is the rapid development of a genetically complex population (quasispecies) from an initially limited number of infectious particles. Genetic diversity remains one of the major obstacles to eradication of HIV. The viral quasispecies can respond rapidly to selective pressures, such as that imposed by the immune system and antiretroviral therapy, and frustrates vaccine design efforts. Two unique features of retroviral replication are responsible for the unprecedented variation generated during infection. First, mutations are frequently introduced into the viral genome by the error prone viral reverse transcriptase and through the actions of host cellular factors, such as the APOBEC family of nucleic acid editing enzymes. Second, the HIV reverse transcriptase can utilize both copies of the co-packaged viral genome in a process termed retroviral recombination. When the co-packaged viral genomes are genetically different, retroviral recombination can lead to the shuffling of mutations between viral genomes in the quasispecies. This review outlines the stages of the retroviral life cycle where genetic variation is introduced, focusing on the principal mechanisms of mutation and recombination. Understanding the mechanistic origin of genetic diversity is essential to combating HIV. [Copyright &y& Elsevier]

Abstract: We describe the full genetic characterization of an insect-specific flavivirus (ISF) from Culex theileri (Theobald) mosquitoes collected in Portugal. This represents the first isolation and full characterization of an ISF from Portuguese mosquitoes. The virus, designated CTFV, for Culex theileri flavivirus, was isolated in the C6/36 Stegomyia albopicta (=Aedes albopictus) cell line, and failed to replicate in vertebrate (Vero) cells in common with other ISFs. The CTFV genome encodes a single polyprotein with 3357 residues showing all the features expected for those of flaviviruses. Phylogenetic analyses based on all ISF sequences available to date, place CTFV among Culex-associated flaviviruses, grouping with recently published NS5 partial sequences documented from mosquitoes collected in the Iberian Peninsula, and with Quang Binh virus (isolated in Vietnam) as a close relative. No CTFV sequences were found integrated in their host''s genome using a range of specific PCR primers designed to the prM/E, NS3, and NS5 region. [Copyright &y& Elsevier]

Abstract: This review summarizes the molecular studies pertaining to porcine circovirus (PCV) transcription and DNA replication. The genome of PCV is circular, single-stranded DNA and contains 1759–1768 nucleotides. Both the genome-strand (packaged in the virus particle) and the complementary-strand (synthesized in the new host) encode viral proteins. Among a multitude of RNAs synthesized by alternate splicing, only rep and rep′ are essential for virus DNA replication via the rolling-circle replication (RCR) mechanism. In contrast to other RCR biological systems which utilize only one multi-functional protein, Rep, to replicate their respective genomes, PCV requires two proteins, Rep and Rep′. During DNA synthesis, the PCV origin of DNA replication (Ori), which contains a pair of inverted repeats (palindrome), exists in a destabilized four-stranded configuration (the melting-pot model) and permits both the palindromic-strand and the complementary-strand to serve as templates simultaneously for initiation and termination. Inherent in the “melting-pot” model is the template-strand-switching mechanism. This mechanism is the basis for the “correction or conversion” of any mutated nucleotide sequences engineered into either arm of the palindrome and the incorporation of “illegitimate recombination” (addition or deletion of nucleotides) events that are commonly observed at the Ori of other RCR biological systems during DNA replication. [Copyright &y& Elsevier]

Abstract: Porcine circovirus-associated disease (PCVAD) encompasses a group of complex, multi-factorial syndromes, which are dependent on infection with porcine circovirus type 2 (PCV2). Current strains of PCV2 circulating in the field are classified into two groups, termed PCV2a and PCV2b. Outbreaks of severe PCVAD in North America and other countries are often linked to a shift from PCV2a to PCV2b as the predominant genotype. Therefore, genotype-specific differences in pathogenesis and antigenicity have been suggested. Overall, evidence suggests that virulence is a function of the specific PCV2 isolate, regardless of genotype. In addition, only minor antigenic differences have been reported. In terms of immunopathogenesis, a conserved decoy epitope, located in the C-terminal region of the capsid protein, provides an explanation for the inability to identify pathogenic differences between genotypes. Finally, genetic variation in PCV2 and the resulting consequences with respect to vaccination and diagnostics are discussed. [Copyright &y& Elsevier]

Abstract: This review discusses the molecular interaction of proteins encoded by porcine circoviruses type 1 (PCV1) and type 2 (PCV2) with respect to the putative role for disease induction. Besides protein:protein interactions also proteins binding to PCV DNA are covered. Moreover, new approaches are considered which have been generated by genomic and proteomic techniques. PCV are still an enigma, when communication with the host and induction of disease are concerned. This is remarkably, since comparison of two viruses with a size of less than 2000 nts should easily identify the molecular trigger responsible for pathogenicity of PCV2. Since scientific life isn’t all beer and skittles we have to accept that this conundrum may be solved only in a long time range. The good news is that some common themes become visible and that certain aspects of the cell life cycle have been identified to be involved in interaction with the pathogen. [Copyright &y& Elsevier]

Abstract: Porcine circovirus type 2 (PCV2) has been recognized as an important vial pathogen for global swine industry. The virus is associated with postweaning multisystemic wasting syndrome (PMWS) and other syndrome diseases collectively known as porcine circovirus-associated disease (PCVAD). PCV2 infection and PMWS have caused great impact on the pig production in China during the past 10 years. This review will involve in the history of PCVAD, serological epidemiology of PCV2 infection, and clinical aspect of PCVAD, and the genomic characteristics, variation and genotyping of PCV2, as well as control situation of PCVAD in China. [Copyright &y& Elsevier]

Abstract: PCV2 has been highly prevalent in the pig population for decades, prior to the emergence of associated clinical disease manifestations that severely affected the pig production worldwide in the late 90s. PCV2 can be further subdivided into several genotypes. From descriptive epidemiologic data, there is evidence of a global shift of the main PCV2 genotypes in different countries from PCV2a to PCV2b, which is generally associated with more severe disease. In addition, from analytic epidemiologic studies, the modified within-herd PCV2 dynamics of infection is strongly related to the increased incidence of clinical disorders associated with PCV2 infection. Because PCV2 is shed for a long time by an extremely large variety of routes, it easily spreads within the population both through horizontal and vertical transmission. Even if airborne transmission cannot be formally excluded, direct contact is certainly the most efficient infectious route due to the simultaneous exposure of susceptible pigs to contaminated respiratory, digestive, and urinary secretions since the probability of transmission is strongly limited by the distance between infectious and susceptible animals. Consequently, farm to farm transmission is restricted to the introduction of infected animals or infected animal products such as semen. More information would be required to assess the risk of other vehicles such as vaccines or feed ingredients since the probability of these products to be contaminated by PCV2 is unknown. However, owing to its transmission characteristics, PCV2 is able to be maintained within pig farms for years without any further need for re-introduction due to the population dynamics of modern pig operations, which continually renew the pool of the susceptible population through replacements and pig movements between compartments. [Copyright &y& Elsevier]