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    Virology Journal - Latest Articles

  • Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus

  • Background: In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus. Methods: We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414–535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution. Results: Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed. Conclusions: Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.

  • Fecal virome analysis of three carnivores reveals a novel nodavirus and multiple gemycircularviruses

  • Background: More knowledge about viral populations in wild animals is needed in order to better understand and assess the risk of zoonotic diseases. In this study we performed viral metagenomic analysis of fecal samples from three healthy carnivores: a badger (Meles meles), a mongoose (Herpestes ichneumon) and an otter (Lutra lutra) from Portugal. Results: We detected the presence of novel highly divergent viruses in the fecal material of the carnivores analyzed, such as five gemycircularviruses. Four of these gemycircularviruses were found in the mongoose and one in the badger. In addition we also identified an RNA-dependent RNA polymerase gene from a putative novel member of the Nodaviridae family in the fecal material of the otter. Conclusions: Together these results underline that many novel viruses are yet to be discovered and that fecal associated viruses are not always related to disease. Our study expands the knowledge of viral species present in the gut, although the interpretation of the true host species of such novel viruses needs to be reviewed with great caution.

  • Evolutionary relationships of West Nile virus detected in mosquitoes from a migratory bird zone of Colombian Caribbean

  • Background: West Nile virus (WNV) is a member of the genus Flavivirus, and it is transmitted between Culex sp. mosquitoes and avian hosts. Equids and humans are commonly infected with WNV as dead-end hosts, and the signs and symptoms of infection range from mild illness to neurologic symptoms as encephalitis, meningitis and sometimes death. Previous phylogenetic studies have classified WNV into six genetically distinct lineages and provided valuable insight on WNV dispersal patterns within the Americas and its emergence in different geographic areas. In this study, we isolated, sequenced and genetically characterized the NS5 and envelope genes for two WNV strains detected from Northern of Colombia. Herein we describe the evolutionary relationships with representative WNV-strains isolated in a variety of epidemic outbreaks and countries, to define the phylogeographic origin and possible implications in the epidemiology of this emergent virus in Colombia.FindingsFragments of the NS5 and Envelope genes were amplified with RT-PCR and sequenced to obtain 1186-nt and 1504-nt portions, respectively. Our sequences were aligned with 46 sequences from WNV-strains collected in the U.S., Mexico and Argentina for phylogenetic reconstruction using Bayesian methods. Sequence analyses identified unique non-synonymous substitutions in the envelope gene of the WNV strains we detected, and our sequences clustered together with those from the attenuated Texas– 2002 genotype. Conclusions: A new strain closely related to attenuated strains collected in Texas during 2002 was identified from Colombia by phylogenetic analysis. This finding may explain the absence of human/equine cases of WNV-encephalitis or severe disease in Colombia and possibly other regions of South America. Follow-up studies are needed in ecosystems used by migratory birds areas and virological/entomological surveillance.

  • Prevalence of classic, MLB-clade and VA-clade Astroviruses in Kenya and The Gambia

  • Background: Infectious diarrhea leads to significant mortality in children, with 40 % of these deaths occurring in Africa. Classic human astroviruses are a well-established etiology of diarrhea. In recent years, seven novel astroviruses have been discovered (MLB1, MLB2, MLB3, VA1/HMO-C, VA2/HMO-B, VA3/HMO-A, VA4); however, there have been few studies on their prevalence or potential association with diarrhea. Methods: To investigate the prevalence and diversity of these classic and recently described astroviruses in a pediatric population, a case–control study was performed. Nine hundred and forty nine stools were previously collected from cases of moderate-to-severe diarrhea and matched controls of patients less than 5 years of age in Kenya and The Gambia. RT-PCR screening was performed using pan-astrovirus primers. Results: Astroviruses were present in 9.9 % of all stool samples. MLB3 was the most common astrovirus with a prevalence of 2.6 %. Two subtypes of MLB3 were detected that varied based on location in Africa. In this case–control study, Astrovirus MLB1 was associated with diarrhea in Kenya, whereas Astrovirus MLB3 was associated with the control state in The Gambia. Classic human astrovirus was not associated with diarrhea in this study. Unexpectedly, astroviruses with high similarity to Canine Astrovirus and Avian Nephritis Virus 1 and 2 were also found in one case of diarrhea and two control stools respectively. Conclusions: Astroviruses including novel MLB- and VA-clade members are commonly found in pediatric stools in Kenya and The Gambia. The most recently discovered astrovirus, MLB3, was the most prevalent and was found more commonly in control stools in The Gambia, while astrovirus MLB1 was associated with diarrhea in Kenya. Furthermore, a distinct subtype of MLB3 was noted, as well as 3 unanticipated avian or canine astroviruses in the human stool samples. As a result of a broadly reactive PCR screen for astroviruses, new insight was gained regarding the epidemiology of astroviruses in Africa, where a large proportion of diarrheal morbidity and mortality occur.

  • HIV infection and antiretroviral therapy lead to unfolded protein response activation

  • Background: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1). These three proteins’ synergic action elicits translation and transcriptional downstream pathways, leading to less protein production and activating genes that encode important proteins in folding processes, including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However, HIV infection’s effect on the UPR has scarcely been investigated. Methods: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs’ influence on this stress response was also considered. Results: In in vitro and in vivo analyses, our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR’s activation profile. Conclusions: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus, two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs’ pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented.

  • Development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus

  • Background: Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by severe enteritis, vomiting and watery diarrhea in swine. Recently, the outbreak of the epidemic disease has been a serious problem in swine industry. The objective of this study is to develop a rapid, sensitive, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of porcine epidemic diarrhea virus (PEDV) in less equipped laboratories. Results: The optimal reaction condition of the current real-time RT-LAMP for PEDV was 62 °C for 45 min. It was capable of detecting PEDV from clinical samples and differentiating PEDV from several related porcine viruses, while it did not require additional expensive equipment. The minimum detection limit of the real-time RT-LAMP assay was 0.07PFU per reaction for PEDV RNA, making this assay approximately 100-fold more sensitive than that of one-step RT-PCR. By screening a panel of clinical specimens, the results showed that this method presented a similar sensitivity with real-time RT-PCR and was somewhat sensitive than one-step RT-PCR in detection of clinical samples. Conclusions: In this study, we have developed a new real-time RT-LAMP method, which is rapid, sensitive and efficient to detect PEDV.This method holds great promises not only in laboratory detection and discrimination of PEDV but also in large scale field and clinical studies.

  • Dilution testing using rapid diagnostic tests in a HIV diagnostic algorithm: a novel alternative for confirmation testing in resource limited settings

  • Background: Current WHO testing guidelines for resource limited settings diagnose HIV on the basis of screening tests without a confirmation test due to cost constraints. This leads to a potential risk of false positive HIV diagnosis. In this paper, we evaluate the dilution test, a novel method for confirmation testing, which is simple, rapid, and low cost. The principle of the dilution test is to alter the sensitivity of a rapid diagnostic test (RDT) by dilution of the sample, in order to screen out the cross reacting antibodies responsible for falsely positive RDT results. Methods: Participants were recruited from two testing centres in Ethiopia where a tiebreaker algorithm using 3 different RDTs in series is used to diagnose HIV. All samples positive on the initial screening RDT and every 10th negative sample underwent testing with the gold standard and dilution test. Dilution testing was performed using Determine™ rapid diagnostic test at 6 different dilutions. Results were compared to the gold standard of Western Blot; where Western Blot was indeterminate, PCR testing determined the final result. Results: 2895 samples were recruited to the study. 247 were positive for a prevalence of 8.5 % (247/2895). A total of 495 samples underwent dilution testing. The RDT diagnostic algorithm misclassified 18 samples as positive. Dilution at the level of 1/160 was able to correctly identify all these 18 false positives, but at a cost of a single false negative result (sensitivity 99.6 %, 95 % CI 97.8-100; specificity 100 %, 95 % CI: 98.5-100). Concordance between the gold standard and the 1/160 dilution strength was 99.8 %. Conclusion: This study provides proof of concept for a new, low cost method of confirming HIV diagnosis in resource-limited settings. It has potential for use as a supplementary test in a confirmatory algorithm, whereby double positive RDT results undergo dilution testing, with positive results confirming HIV infection. Negative results require nucleic acid testing to rule out false negative results due to seroconversion or misclassification by the lower sensitivity dilution test. Further research is needed to determine if these results can be replicated in other settings.Trial registrationClinicalTrials.gov, NCT01716299.

  • Frequency of coreceptor tropism in PBMC samples from HIV-1 recently infected blood donors by massively parallel sequencing: the REDS II study

  • Background: The interaction of HIV-1 and target cells involves sequential binding of the viral gp120 Env protein to the CD4 receptor and a chemokine co-receptor (either CCR5 or CXCR4). CCR5 antagonists have proved to be an effective salvage therapy in patients with CCR5 using variants (R5) but not with variants capable of using CXCR4 (×4) phenotype. Thus, it is critically important to determine cellular tropism of a country’s circulating HIV strains to guide a management decision to improve treatment outcome. In this study, we report the prevalence of R5 and×4 HIV strains in 45 proviral DNA massively parallel sequencing“MPS” data from recently infected Brazilian blood donors. Methods: The MPS data encompassing the tropism-related V3 loop region of the HIV‐1 env gene was extracted from our recently published HIV-1 genomes sequenced by a paired-end protocol (Illumina). HIV‐1 tropism was inferred using Geno2pheno[coreceptor] algorithm (3.5 % false-positive rate). V3 net charge and 11/25 rules were also used for coreceptor prediction. Results: Among the 45 samples for which tropism were determined, 39 were exclusively R5 variants, 5×4 variants, and one dual-tropic or mixed (D/M) populations of R5 and×4 viruses, corresponding to 86.7, 11.1 and 2.2 %, respectively. Thus, the proportion of all blood donors that harbor CXCR4-using virus was 13.3 % including individuals with D/M-tropic viruses. Conclusions: The presence of CCR5-tropic variants in more than 85 % of our cohort of antiretroviral-naïve blood donors with recent HIV-1 infection indicates a potential benefit of CCR5 antagonists as a therapeutic option in Brazil. Therefore, determination of viral co-receptor tropism is an important diagnostic prerequisite.

  • A transgenic ginseng vaccine for bovine viral diarrhea

  • Background: Bovine viral diarrhea virus (BVDV) infections are endemic in cattle populations worldwide and cause major economic losses. Thus, an effective vaccine is needed against the transmission of BVDV. The glycoprotein Erns is one of the envelope proteins of this virus and shows BVDV-related immunogenicity. Here, we report the use of Panax ginseng as an alternative production platform for the expression of glycoprotein Erns via Agrobacterium-mediated transformation.ResultPolymerase chain reaction (PCR) and reverse transcription (RT)-PCR analyses showed that pBI121-Erns was stably integrated into the chromosome of transformants. ELISA assay and Western blot analysis confirmed the antigenicity of plant-derived Erns glycoprotein. Immunogenicity was evaluated subcutaneously in deer using a soluble protein extract of dried transgenic ginseng hairy roots. Specific humoral and cell-mediated immune responses against BVDV were detected following immunization. Conclusion: These results demonstrated that the Erns glycoprotein could be expressed in ginseng hairy roots and that plant-derived glycoprotein Erns retained its antigenicity and immunogenicity.

  • The I22V and L72S substitutions in West Nile virus prM protein promote enhanced prM/E heterodimerisation and nucleocapsid incorporation

  • Background: Amino acid substitutions I22V and L72S in the prM protein of West Nile virus Kunjin strain (WNVKUN) were previously shown to enhance virus secretion and virulence, but a mechanism by which this occurred was not determined.FindingsUsing pulse-chase experiments followed by co-immunoprecipitation with anti-E antibody, we demonstrated that the I22V and L72S substitutions enhanced prM/E heterodimerization for both the E-glycosylated and E-unglycosylated virus. Furthermore, analysis of secreted particles revealed that I22V and L72S substitutions also enhanced nucleocapsid incorporation. Conclusions: We have demonstrated mechanistically that improved secretion of virus particles in the presence of I22V and L72S substitutions was contributed by more efficient prM/E heterodimerization.
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