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Taylor, A., Herrero, L. J., Rudd, P. A., Mahalingam, S.
Part of the Togaviridae family, alphaviruses are arthropod-borne viruses that are widely distributed throughout the globe. Alphaviruses are able to infect a variety of vertebrate hosts, but in humans, infection can result in extensive morbidity and mortality. Symptomatic infection can manifest as fever, an erythematous rash and/or significant inflammatory pathologies such as arthritis and encephalitis. Recent overwhelming outbreaks of alphaviral disease have highlighted the void in our understanding of alphavirus pathogenesis and the re-emergence of alphaviruses has given new impetus to anti-alphaviral drug design. In this review, the development of viable mouse models of Old Word and New World alphaviruses is examined. How mouse models that best replicate human disease have been used to elucidate the immunopathology of alphavirus pathogenesis and trial novel therapeutic discoveries is also discussed.
Strang, B. L.
In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virus–host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with subnuclear structures are also discussed. The data reviewed here highlight advances in our understanding of HCMV biology and emphasize the complexity of HCMV replication and virus–host interactions in the nucleus.
ANIMAL: RNA VIRUSES
Song, D., Kim, H., Na, W., Hong, M., Park, S.-J., Moon, H., Kang, B., Lyoo, K.-S., Yeom, M., Jeong, D. G., An, D.-J., Kim, J.-K.
We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naïve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.
ANIMAL: RNA VIRUSES
Kong, W., Liu, L., Wang, Y., He, Q., Wu, S., Qin, Z., Wang, J., Sun, H., Sun, Y., Zhang, R., Pu, J., Liu, J.
H9N2 avian influenza viruses are enzootic around the world and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess a non-structural protein 1 (NS1) of 230 aa, 98 % of the H9N2 viruses isolated in China lack the 13 aa at the C terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. To examine the effect of NS1 C-terminal elongation in the influenza virus, we used reverse genetics to generate a wt avian influenza H9N2 virus containing a 217 aa NS1 (H9N2NS1217) and two mutant viruses with elongated NS1s of 230 and 237 aa (H9N2NS1230 and H9N2NS1237). C-terminal elongation of NS1 did not have a significant impact on virus replication in Madin–Darby canine kidney cells or DF-1 cells. The three variants exhibited similar replicability in mice; however, the H9N2NS1230 and H9N2NS1237 variants exhibited an upregulation in the level of inflammatory cytokines. In addition, both the H9N2NS1230 and H9N2NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared with the wt virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.
ANIMAL: RNA VIRUSES
Lebarbenchon, C., Pedersen, J. C., Sreevatsan, S., Ramey, A. M., Dugan, V. G., Halpin, R. A., Ferro, P. J., Lupiani, B., Enomoto, S., Poulson, R. L., Smeltzer, M., Cardona, C. J., Tompkins, S. M., Wentworth, D. E., Stallknecht, D. E., Brown, J. D.
Introductions of H7 influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAVs in Minnesota (MN) turkey farms during 2009 and 2011. The full genome was sequenced from eight isolates as well as the haemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum-likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAVs, estimated the time and geographical origin of the ancestral viruses, and determined the relatedness between the 2009 and 2011 turkey-origin H7N9 IAVs. Analyses supported that the 2009 and 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further supported that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAVs isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAVs could not be determined, our findings suggested that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi Flyway.
ANIMAL: RNA VIRUSES
Shao, Q., Xu, W., Guo, Q., Yan, L., Rui, L., Liu, J., Zhao, Y., Li, Z.
The retinoic acid-induced gene I (RIG-I) plays a crucial role in sensing viral RNA and IFN-β production. RIG-I varies in length and sequence between different species. We assessed the functional differences between RIG-I proteins derived from mammals and birds. The transfection of duck caspase recruitment domains (CARDs) and duck RIG-I (dCARDs and dRIG-I) and goose CARDs and goose RIG-I (gCARDs and gRIG-I) into chicken DF-1 cells increased the production of IFN-β mRNA and IFN-stimulated genes and decreased influenza A virus (IAV) replication; whereas human CARDs and RIG-I (hCARDs and hRIG-I) and mouse CARDs and RIG-I (mCARDs and mRIG-I) had no effect. In human 293T and A549 cells, hCARDs had the strongest IFN-inducing activity, followed by mCARDs, dCARDs and gCARDs. The IFN-inducing activity of hRIG-I was stronger than that of mRIG-I, dRIG-I and gRIG-I, in that order. The results showed that, although the ability of dCARDs to activate IFN was stronger than that of gCARDs in DF-1, 293T and A549 cells, dRIG-I had a weaker ability to activate IFN than gRIG-I in DF-1 cells with or without IAV infection. These data suggest that RIG-I proteins from different species have different amino acid sequences and functions. This genetic and functional diversity renders RIG-I flexible, adaptable and capable of recognizing many viruses in different species.
ANIMAL: RNA VIRUSES
Pitcher, T. J., Sarathy, V. V., Matsui, K., Gromowski, G. D., Huang, C. Y.- H., Barrett, A. D. T.
The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants.
ANIMAL: RNA VIRUSES
Das, T., Hoarau, J. J., Bandjee, M. C. J., Maquart, M., Gasque, P.
Chikungunya virus (CHIKV) has recently affected millions of people in the Indian Ocean, with rare cases of encephalopathy and encephalitis occurring in neonates. In the study described herein, the capacity of mouse brain cells to control infection through innate immune antiviral responses was assessed. In vitro, CHIKV principally infected a subpopulation of mouse GFAP+ primary astrocytes. Oligodendrocytes and neurons could also be infected. An innate immune response was engaged by CHIKV-infected astrocytes with elevated expression of mRNAs for IFN-α–β, inflammatory cytokines (e.g. IL-1β, IL-12, IL-10, IL-24) and proapoptotic factors (e.g. TNF-α, FasL, Lymphotoxin B). Programmed cell death through the intrinsic caspase-9 pathway was observed by immunofluorescence in infected astrocytes and neurons but not in oligodendrocytes. Interestingly, microglia did not replicate CHIKV but responded by elevated mitogen-activated protein kinase (MAPK) activity. Intracerebroventricular injection of CHIKV in neonate mice led to the infection of astrocytes. The astrogliosis response was accompanied by a dendritic CD206+ cell mobilization restricted to the site of infection. The results of this study support the paradigm that a multifaceted innate immune response can be mobilized by both professional immune and glial cells to control CHIKV neuroinfection events in neonates.
ANIMAL: RNA VIRUSES
Afzal, M. S., Alsaleh, K., Farhat, R., Belouzard, S., Danneels, A., Descamps, V., Duverlie, G., Wychowski, C., Zaidi, N. u. S. S., Dubuisson, J., Rouille, Y.
Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle.
ANIMAL: RNA VIRUSES
Bengs, S., Marttila, J., Susi, P., Ilonen, J.
Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within the family Picornaviridae. To investigate target proteins recognized by T-cells in human enterovirus B infections, virus-encoded structural [VP0 (VP4 and VP2), VP1, VP3] and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in Escherichia coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognized enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of the 19 (79 %) donor lines. Non-structural 2C protein was recognized in 11 of the 19 (58 %) lines, and 11 of the 19 (58 %) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.
ANIMAL: RNA VIRUSES
Pratelli, A., Colao, V.
Coronaviruses are enveloped RNA viruses that have evolved complex relationships with their host cells, and modulate their lipid composition, lipid synthesis and signalling. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes in viral infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to lipid rafts. Because cholesterol-rich microdomains appear to be a general feature of the entry mechanism of non-eneveloped viruses and of several coronaviruses, the purpose of this study was to analyse the contribution of lipids to the infectivity of canine coronavirus (CCoV). The CCoV life cycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-β-cyclodextrin (MβCD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from the cell membrane resulted in a dose-dependent reduction, but not abolishment, of virus infectivity, and at a concentration of 15 mM, the reduction in the infection rate was about 68 %. MβCD treatment was used to verify if cholesterol in the envelope was required for CCoV infection. This resulted in a dose-dependent inhibitory effect, and at a concentration of 9 mM MβCD, infectivity was reduced by about 73 %. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cell membranes, or interfering with lipid metabolism, may have strong antiviral potential. It will be interesting in the future to analyse the membrane microdomains in the CCoV envelope.
ANIMAL: RNA VIRUSES
Gauchan, P., Sasaki, E., Nakagomi, T., Do, L. P., Doan, Y. H., Mochizuki, M., Nakagomi, O.
Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA–RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.
ANIMAL: DNA VIRUSES
Hough, K. P., Rogers, A. M., Zelic, M., Paris, M., Heilman, D. W.
Several members of the family Circoviridae have been shown to encode proteins with apoptotic activity. For example, both porcine circovirus type 2 (PCV2) and chicken anemia virus (CAV) encode a third viral protein (VP3) that has been shown to be cytotoxic. Interestingly, in the case of the CAV protein (designated apoptin), apoptosis is specific to transformed cell types. Similarities in genome structure and organization suggest that PCV type 1 (PCV1) may also contain a third ORF, which codes for a protein with homologous activity. To investigate this, ORF prediction followed by gene expression analyses were conducted on a gene found to be homologous to CAV and PCV2 VP3. Our data presented herein elucidate a putative ORF3 that codes for a viral protein with functional similarity to that of apoptin and PCV2 VP3. Unlike its homologues, sequence analysis revealed a highly hydrophobic, extended C-terminal domain in PCV1 VP3, which harbours a strong nuclear export signal. Subcellular localization analysis demonstrated divergent PCV1 VP3 localization patterns compared with that of CAV VP3. Interestingly, cytotoxicity studies revealed evidence that apoptosis may be selective to transformed cell types, similar to apoptin; however, PCV1 VP3 induced a dramatic G1 cell cycle arrest as opposed to the G2/M arrest observed with apoptin. These results indicate that nuclear localization of PCV1 VP3 is necessary neither for induction of apoptosis nor for transformed cell selectivity, and suggest a mechanism of action distinct from that of apoptin.
ANIMAL: DNA VIRUSES
Gerna, G., Lilleri, D., Fornara, C., Bruno, F., Gabanti, E., Cane, I., Furione, M., Revello, M. G.
The comparative long-term kinetics of human cytomegalovirus (HCMV) load and HCMV-specific antibody responses in the immunocompetent and immunocompromised solid-organ transplanted host during primary HCMV infection was investigated. In total, 40 immunocompetent subjects and 17 transplanted patients were examined for viral load as well as for IgG antibody responses to HCMV glycoproteins gH/gL/pUL128L, gH/gL and gB, and neutralizing antibodies in ARPE-19 epithelial cells and human fibroblasts. In parallel, the CD4+ and CD8+ HCMV-specific T-cell responses were determined by cytokine flow cytometry. Transplanted patients reached significantly higher viral DNA peaks, which persisted longer than in immunocompetent subjects. The ELISA-IgG responses to the pentamer, gH/gL and gB were significantly higher in primary infections of the immunocompetent until six months after onset, with the two antibody levels then overlapping from six to 12 months. Antibody levels neutralizing infection of epithelial cells were significantly higher in transplanted patients after six months, persisting for up to a year after transplantation. This trend was not observed for antibodies neutralizing infection of human fibroblasts, which showed higher titres in the immunocompetent over the entire one-year follow-up. In conclusion, in immunocompromised patients the viral load peak was much higher, while the neutralizing antibody response exceeded that detected in the immunocompetent host starting six months after onset of follow-up, often concomitantly with a lack of specific CD4+ T cells. In this setting, the elevated antibody response occurred in the presence of differentiated follicular helper T cells in the blood, which decreased in number as did antibody titres upon reappearance of HCMV-specific CD4+ T cells.
ANIMAL: DNA VIRUSES
Tweedy, J., Spyrou, M. A., Hubacek, P., Kuhl, U., Lassner, D., Gompels, U. A.
Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1–1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.
ANIMAL: DNA VIRUSES
Orba, Y., Sasaki, M., Yamaguchi, H., Ishii, A., Thomas, Y., Ogawa, H., Hang'ombe, B. M., Mweene, A. S., Morikawa, S., Saijo, M., Sawa, H.
Human monkeypox is a viral zoonosis caused by monkeypox virus, an orthopoxvirus (OPXV). The majority of human monkeypox cases have been reported in moist forested regions in West and Central Africa, particularly in the Democratic Republic of the Congo (DRC). In this study we investigated zoonotic OPXV infection among wild animals in Zambia, which shares a border with DRC, to assess the geographical distribution of OPXV. We screened for OPXV antibodies in sera from non-human primates (NHPs), rodents and shrews by ELISA, and performed real-time PCR to detect OPXV DNA in spleen samples. Serological analysis indicated that 38 of 259 (14.7 %) rodents, 14 of 42 (33.3 %) shrews and 4 of 188 (2.1 %) NHPs had antibodies against OPXV. The OPXV DNA could not be detected in spleens from any animals tested. Our results indicated that wild animals living in rural human habitation areas of Zambia have been infected with OPXV.
ANIMAL: DNA VIRUSES
Mazzon, M., Castro, C., Roberts, L. D., Griffin, J. L., Smith, G. L.
Vaccinia virus (VACV) is a large DNA virus that replicates in the cytoplasm and encodes about 200 proteins of which approximately 50 % may be non-essential for viral replication. These proteins enable VACV to suppress transcription and translation of cellular genes, to inhibit the innate immune response, to exploit microtubule- and actin-based transport for virus entry and spread, and to subvert cellular metabolism for the benefit of the virus. VACV strain WR protein C16 induces stabilization of the hypoxia-inducible transcription factor (HIF)-1α by binding to the cellular oxygen sensor prolylhydroxylase domain-containing protein (PHD)2. Stabilization of HIF-1α is induced by several virus groups, but the purpose and consequences are unclear. Here, 1H-NMR spectroscopy and liquid chromatography-mass spectrometry are used to investigate the metabolic alterations during VACV infection in HeLa and 2FTGH cells. The role of C16 in such alterations was examined by comparing infection to WT VACV (strain WR) and a derivative virus lacking gene C16L (vC16). Compared with uninfected cells, VACV infection caused increased nucleotide and glutamine metabolism. In addition, there were increased concentrations of glutamine derivatives in cells infected with WT VACV compared with vC16. This indicates that C16 contributes to enhanced glutamine metabolism and this may help preserve tricarboxylic acid cycle activity. These data show that VACV infection reprogrammes cellular energy metabolism towards increased synthesis of the metabolic precursors utilized during viral replication, and that C16 contributes to this anabolic reprogramming of the cell, probably via the stabilization of HIF-1α.
ANIMAL: DNA VIRUSES
Portugal, R., Coelho, J., Hoper, D., Little, N. S., Smithson, C., Upton, C., Martins, C., Leitao, A., Keil, G. M.
Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.
ANIMAL: RNA VIRUSES
Grisenti, M., Vazquez, A., Herrero, L., Cuevas, L., Perez-Pastrana, E., Arnoldi, D., Rosa, R., Capelli, G., Tenorio, A., Sanchez-Seco, M. P., Rizzoli, A.
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 transcription-nested-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.
ANIMAL: RNA VIRUSES
Atsumi, G., Tomita, R., Yamashita, T., Sekine, K.-T.
In this study, we identified a novel virus from gentian (Gentiana triflora) that causes ring-spots on ovaries. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. Pollen grains carrying the virus were used to infect host plants by hand-pollination. RNA extracted from purified virions indicated that the virus had two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1–3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence similarity and phylogenetic analysis suggested that the virus is closely related to members of the genera Pecluvirus and Hordeivirus but distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus gentian ovary ring-spot virus.
Sasaki, M., Orba, Y., Ueno, K., Ishii, A., Moonga, L., Hang'ombe, B. M., Mweene, A. S., Ito, K., Sawa, H.
Shrews are small insectivorous mammals that are distributed worldwide. Similar to rodents, shrews live on the ground and are commonly found near human residences. In this study, we investigated the enteric virome of wild shrews in the genus Crocidura using a sequence-independent viral metagenomics approach. A large portion of the shrew enteric virome was composed of insect viruses, whilst novel viruses including cyclovirus, picornavirus and picorna-like virus were also identified. Several cycloviruses, including variants of human cycloviruses detected in cerebrospinal fluid and stools, were detected in wild shrews at a high prevalence rate. The identified picornavirus was distantly related to human parechovirus, inferring the presence of a new genus in this family. The identified picorna-like viruses were characterized as different species of calhevirus 1, which was discovered previously in human stools. Complete or nearly complete genome sequences of these novel viruses were determined in this study and then were subjected to further genetic characterization. Our study provides an initial view of the diversity and distinctiveness of the shrew enteric virome and highlights unique novel viruses related to human stool-associated viruses.
Mattila, S., Oksanen, H. M., Bamford, J. K. H.
PRD1 is a Gram-negative bacteria infecting complex tailless icosahedral virus with an inner membrane. This type virus of the family Tectiviridae contains at least 18 structural protein species, of which several are membrane associated. Vertices of the PRD1 virion consist of complexes recognizing the host cell, except for one special vertex through which the genome is packaged. Despite extensive knowledge of the overall structure of the PRD1 virion and several individual proteins at the atomic level, the locations and interactions of various integral membrane proteins and membrane-associated proteins still remain a mystery. Here, we demonstrated that blue native PAGE can be used to probe protein–protein interactions in complex membrane-containing viruses. Using this technique and PRD1 as a model, we identified the known PRD1 multiprotein vertex structure composed of penton protein P31, spike protein P5, receptor-binding protein P2 and stabilizing protein P16 linking the vertex to the internal membrane. Our results also indicated that two transmembrane proteins, P7 and P14, involved in viral nucleic acid delivery, make a complex. In addition, we performed a zymogram analysis using mutant particles devoid of the special vertex that indicated that the lytic enzyme P15 of PRD1 was not part of the packaging vertex, thus contradicting previously published results.
Endersen, L., Guinane, C. M., Johnston, C., Neve, H., Coffey, A., Ross, R. P., McAuliffe, O., O'Mahony, J.
Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multidrug-resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full-genome sequence of Cronobacter phage vB_CsaP_Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage vB_CsaP_Ss1 is composed of 42 205 bp of dsDNA with a G+C content of 46.1 mol%. A total of 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterized proteins. The genome of Cronobacter phage vB_CsaP_Ss1 showed little similarity to any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis of the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, and this endolysin thus represents a novel candidate with potential for use against Gram-negative pathogens.
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