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  • Investigations into the presence of nidoviruses in pythons
    Virol. J. (IF 2.464) Pub Date : 2020-01-17
    Silvia Blahak; Maria Jenckel; Dirk Höper; Martin Beer; Bernd Hoffmann; Kore Schlottau

    Pneumonia and stomatitis represent severe and often fatal diseases in different captive snakes. Apart from bacterial infections, paramyxo-, adeno-, reo- and arenaviruses cause these diseases. In 2014, new viruses emerged as the cause of pneumonia in pythons. In a few publications, nidoviruses have been reported in association with pneumonia in ball pythons and a tiger python. The viruses were found using new sequencing methods from the organ tissue of dead animals. Severe pneumonia and stomatitis resulted in a high mortality rate in a captive breeding collection of green tree pythons. Unbiased deep sequencing lead to the detection of nidoviral sequences. A developed RT-qPCR was used to confirm the metagenome results and to determine the importance of this virus. A total of 1554 different boid snakes, including animals suffering from respiratory diseases as well as healthy controls, were screened for nidoviruses. Furthermore, in addition to two full-length sequences, partial sequences were generated from different snake species. The assembled full-length snake nidovirus genomes share only an overall genome sequence identity of less than 66.9% to other published snake nidoviruses and new partial sequences vary between 99.89 and 79.4%. Highest viral loads were detected in lung samples. The snake nidovirus was not only present in diseased animals, but also in snakes showing no typical clinical signs. Our findings further highlight the possible importance of snake nidoviruses in respiratory diseases and proof multiple circulating strains with varying disease potential. Nidovirus detection in clinical healthy individuals might represent testing during the incubation period or reconvalescence. Our investigations show new aspects of nidovirus infections in pythons. Nidoviruses should be included in routine diagnostic workup of diseased reptiles.

  • Early fulminant BK polyomavirus-associated nephropathy in two kidney transplant patients with low neutralizing antibody titers receiving allografts from the same donor
    Virol. J. (IF 2.464) Pub Date : 2020-01-10
    Elias Myrvoll Lorentzen; Stian Henriksen; Amandeep Kaur; Grete Birkeland Kro; Clara Hammarström; Hans H. Hirsch; Karsten Midtvedt; Christine Hanssen Rinaldo

    BK Polyomavirus (BKPyV) causes premature graft failure in 1 to 15% of kidney transplant (KT) recipients. High-level BKPyV-viruria and BKPyV-DNAemia precede polyomavirus-associated nephropathy (PyVAN), and guide clinical management decisions. In most cases, BKPyV appears to come from the donor kidney, but data from biopsy-proven PyVAN cases are lacking. Here, we report the early fulminant course of biopsy-proven PyVAN in two male KT recipients in their sixties, receiving kidneys from the same deceased male donor. Both recipients received intravenous basiliximab induction, and maintenance therapy consisting of tacrolimus (trough levels 3–7 ng/mL from time of engraftment), mycophenolate mofetil 750 mg bid, and prednisolone. At 4 weeks post-transplant, renal function was satisfactory with serum creatinine concentrations of 106 and 72 μmol/L in recipient #1 and recipient #2, respectively. Plasma BKPyV-DNAemia was first investigated at 5 and 8 weeks post-transplant being 8.58 × 104 and 1.12 × 106 copies/mL in recipient #1 and recipient #2, respectively. Renal function declined and biopsy-proven PyVAN was diagnosed in both recipients at 12 weeks post-transplant. Mycophenolate mofetil levels were reduced from 750 mg to 250 mg bid while tacrolimus levels were kept below 5 ng/mL. Recipient #2 cleared BKPyV-DNAemia at 5.5 months post-transplant, while recipient #1 had persistent BKPyV-DNAemia of 1.07 × 105 copies/mL at the last follow-up 52 weeks post-transplant. DNA sequencing of viral DNA from early plasma samples revealed apparently identical viruses in both recipients, belonging to genotype Ib-2 with archetype non-coding control region. Retrospective serological work-up, demonstrated that the donor had high BKPyV-IgG-virus-like particle ELISA activity and a high BKPyV-genotype I neutralizing antibody titer, whereas both KT recipients only had low neutralizing antibody titers pre-transplantation. By 20 weeks post-transplant, the neutralizing antibody titer had increased by > 1000-fold in both recipients, but only recipient #2 cleared BKPyV-DNAemia. Low titers of genotype-specific neutralizing antibodies in recipients pre-transplant, may identify patients at high risk for early fulminant donor-derived BKPyV-DNAemia and PyVAN, but development of high neutralizing antibody titers may not be sufficient for clearance.

  • First report and genetic diversity of porcine bufavirus in China
    Virol. J. (IF 2.464) Pub Date : 2020-01-06
    Yan-Kuo Sun; Yong-Jie Chen; Yu Cai; Di-Hua Zhu; Hao-Ming Pan; Ying-Fang Wei; Xiao-Liang Han; Chi-Hai Ji; Gang Lu; Heng Wang; Chun-Quan Ma; Gui-Hong Zhang

    Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3–99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

  • Effect of a bacteriophage T5virus on growth of Shiga toxigenic Escherichia coli and Salmonella strains in individual and mixed cultures
    Virol. J. (IF 2.464) Pub Date : 2020-01-07
    Yan D. Niu; Hui Liu; Roger P. Johnson; Tim A. McAllister; Kim Stanford

    A previously isolated a bacteriophage, vB_EcoS_AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P < 0.05) at a lower multiplicity of infection and sampling time (6–10 h), when STEC O157:H7 was also included in the culture. This phage was able to simultaneously reduce numbers of STEC and Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

  • The forces driving clonal expansion of the HIV-1 latent reservoir
    Virol. J. (IF 2.464) Pub Date : 2020-01-07
    Runxia Liu; Francesco R. Simonetti; Ya-Chi Ho

    Despite antiretroviral therapy (ART) which halts HIV-1 replication and reduces plasma viral load to clinically undetectable levels, viral rebound inevitably occurs once ART is interrupted. HIV-1-infected cells can undergo clonal expansion, and these clonally expanded cells increase over time. Over 50% of latent reservoirs are maintained through clonal expansion. The clonally expanding HIV-1-infected cells, both in the blood and in the lymphoid tissues, contribute to viral rebound. The major drivers of clonal expansion of HIV-1-infected cells include antigen-driven proliferation, homeostatic proliferation and HIV-1 integration site-dependent proliferation. Here, we reviewed how viral, immunologic and genomic factors contribute to clonal expansion of HIV-1-infected cells, and how clonal expansion shapes the HIV-1 latent reservoir. Antigen-specific CD4+ T cells specific for different pathogens have different clonal expansion dynamics, depending on antigen exposure, cytokine profiles and exhaustion phenotypes. Homeostatic proliferation replenishes the HIV-1 latent reservoir without inducing viral expression and immune clearance. Integration site-dependent proliferation, a mechanism also deployed by other retroviruses, leads to slow but steady increase of HIV-1-infected cells harboring HIV-1 proviruses integrated in the same orientation at specific sites of certain cancer-related genes. Targeting clonally expanding HIV-1 latent reservoir without disrupting CD4+ T cell function is a top priority for HIV-1 eradication.

  • Decay of infectious adenovirus and coliphages in freshwater habitats is differentially affected by ambient sunlight and the presence of indigenous protozoa communities
    Virol. J. (IF 2.464) Pub Date : 2020-01-06
    Brian R. McMinn; Eric R. Rhodes; Emma M. Huff; Asja Korajkic

    Sanitary quality of recreational waters worldwide is assessed using fecal indicator bacteria (FIB), such as Escherichia coli and enterococci. However, fate and transport characteristics of FIB in aquatic habitats can differ from those of viral pathogens which have been identified as main etiologic agents of recreational waterborne illness. Coliphages (bacteriophages infecting E. coli) are an attractive alternative to FIB because of their many morphological and structural similarities to viral pathogens. In this in situ field study, we used a submersible aquatic mesocosm to compare decay characteristics of somatic and F+ coliphages to those of infectious human adenovirus 2 in a freshwater lake. In addition, we also evaluated the effect of ambient sunlight (and associated UV irradiation) and indigenous protozoan communities on decay of somatic and F+ coliphage, as well as infectious adenovirus. Our results show that decay of coliphages and adenovirus was similar (p = 0.0794), indicating that both of these bacteriophage groups are adequate surrogates for decay of human adenoviruses. Overall, after 8 days the greatest log10 reductions were observed when viruses were exposed to a combination of biotic and abiotic factors (2.92 ± 0.39, 4.48 ± 0.38, 3.40 ± 0.19 for somatic coliphages, F+ coliphages and adenovirus, respectively). Both, indigenous protozoa and ambient sunlight, were important contributors to decay of all three viruses, although the magnitude of that effect differed over time and across viral targets. While all viruses studied decayed significantly faster (p < 0.0001) when exposed to ambient sunlight, somatic coliphages were particularly susceptible to sunlight irradiation suggesting a potentially different mechanism of UV damage compared to F+ coliphages and adenoviruses. Presence of indigenous protozoan communities was also a significant contributor (p value range: 0.0016 to < 0.0001) to decay of coliphages and adenovirus suggesting that this rarely studied biotic factor is an important driver of viral reductions in freshwater aquatic habitats.

  • Marburg virus pathogenesis – differences and similarities in humans and animal models
    Virol. J. (IF 2.464) Pub Date : 2019-12-30
    Kyle Shifflett; Andrea Marzi

    Marburg virus (MARV) is a highly pathogenic virus associated with severe disease and mortality rates as high as 90%. Outbreaks of MARV are sporadic, deadly, and often characterized by a lack of resources and facilities to diagnose and treat patients. There are currently no approved vaccines or treatments, and the chaotic and infrequent nature of outbreaks, among other factors, makes testing new countermeasures during outbreaks ethically and logistically challenging. Without field efficacy studies, researchers must rely on animal models of MARV infection to assess the efficacy of vaccines and treatments, with the limitations being the accuracy of the animal model in recapitulating human pathogenesis. This review will compare various animal models to the available descriptions of human pathogenesis and aims to evaluate their effectiveness in modeling important aspects of Marburg virus disease.

  • Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16
    Virol. J. (IF 2.464) Pub Date : 2019-12-30
    Xin-na Li; Xin-xin Shen; Ming-hui Li; Ju-ju Qi; Rui-huan Wang; Qing-xia Duan; Rui-qing Zhang; Tao Fan; Xue-ding Bai; Guo-hao Fan; Yao Xie; Xue-jun Ma

    Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

  • Virus-based pharmaceutical production in plants: an opportunity to reduce health problems in Africa
    Virol. J. (IF 2.464) Pub Date : 2019-12-30
    Pingdwende Kader Aziz Bamogo; Christophe Brugidou; Drissa Sérémé; Fidèle Tiendrébéogo; Florencia Wendkuuni Djigma; Jacques Simpore; Séverine Lacombe

    Developing African countries face health problems that they struggle to solve. The major causes of this situation are high therapeutic and logistical costs. Plant-made therapeutics are easy to produce due to the lack of the safety considerations associated with traditional fermenter-based expression platforms, such as mammalian cells. Plant biosystems are easy to scale up and inexpensive, and they do not require refrigeration or a sophisticated medical infrastructure. These advantages provide an opportunity for plant-made pharmaceuticals to counteract diseases for which medicines were previously inaccessible to people in countries with few resources. The techniques needed for plant-based therapeutic production are currently available. Viral expression vectors based on plant viruses have greatly enhanced plant-made therapeutic production and have been exploited to produce a variety of proteins of industrial, pharmaceutical and agribusiness interest. Some neglected tropical diseases occurring exclusively in the developing world have found solutions through plant bioreactor technology. Plant viral expression vectors have been reported in the production of therapeutics against these diseases occurring exclusively in the third world, and some virus-derived antigens produced in plants exhibit appropriate antigenicity and immunogenicity. However, all advances in the use of plants as bioreactors have been made by companies in Europe and America. The developing world is still far from acquiring this technology, although plant viral expression vectors may provide crucial help to overcome neglected diseases. Today, interest in these tools is rising, and viral amplicons made in and for Africa are in progress. This review describes the biotechnological advances in the field of plant bioreactors, highlights factors restricting access to this technology by those who need it most and proposes a solution to overcome these limitations.

  • Roles of the highly conserved amino acids in the second receptor binding site of the Newcastle disease virus HN protein
    Virol. J. (IF 2.464) Pub Date : 2019-12-27
    Yaqing Liu; Miaomiao Chi; Ying Liu; Hongling Wen; Li Zhao; Yanyan Song; Na Liu; Zhiyu Wang

    The paramyxovirus haemagglutinin-neuraminidase (HN) is a multifunctional protein that is responsible for attachment to receptors, removal of receptors from infected cells to prevent viral self-aggregation (neuraminidase, NA) and fusion promotion. It is commonly accepted that there are two receptor binding sites in the globular head of HN, and the second receptor binding site is only involved in the function of receptor binding and fusion promotion. 10 conserved residues in the second receptor binding site of Newcastle disease virus (NDV) HN were chosen and substituted to alanine (A). The desired mutants were examined to detect the functional change in hemadsorption (HAD) ability, NA activity and fusion promotion ability. The HAD and fusion promotion ability of mutants C172A, R174A, C196A, D198A, Y526A and E547A were abolished. Compared with wild-type (wt) HN, the HAD of mutants T167A, S202A and R516A decreased to 55.81, 44.53, 69.02%, respectively, and the fusion promotion ability of these three mutants decreased to 54.74, 49.46, 65.26%, respectively; however, mutant G171A still maintained fusion promotion ability comparable with wt HN but had impaired HAD ability. All the site-directed mutations altered the NA activity of NDV HN without affecting protein cell surface expression. The data suggest that mutants C172A, R174A, C196A, D198A, Y526A and E547A do not allow the conformational change that is required for fusion promotion ability and HAD activity, while the other mutants only affect the conformational change to a limited extent, except mutant G171A with intact fusion promotion ability. Overall, the conserved amino acids in the second receptor binding site, especially residues C172, R174, C196, D198, Y526 and E547, are crucial to normal NDV HN protein function.

  • Inhibition effects of patchouli alcohol against influenza a virus through targeting cellular PI3K/Akt and ERK/MAPK signaling pathways
    Virol. J. (IF 2.464) Pub Date : 2019-12-23
    Yunjia Yu; Yang Zhang; Shuyao Wang; Wei Liu; Cui Hao; Wei Wang

    Patchouli alcohol (PA) is a tricyclic sesquiterpene extracted from Pogostemonis Herba, which is a traditional Chinese medicine used for therapy of inflammatory diseases. Recent studies have shown that PA has various pharmacological activities, including anti-bacterial and anti-viral effects. In this study, the anti-influenza virus (IAV) activities and mechanisms were investigated both in vitro and in vivo. The inhibitory effects of PA against IAV in vitro were evaluated by plaque assay and immunofluorescence assay. The neuraminidase inhibition assay, hemagglutination inhibition (HI) assay, and western blot assay were used to explore the anti-viral mechanisms. The anti-IAV activities in vivo were determined by mice pneumonia model and HE staining. The results showed that PA significantly inhibited different IAV strains multiplication in vitro, and may block IAV infection through inactivating virus particles directly and interfering with some early stages after virus adsorption. Cellular PI3K/Akt and ERK/MAPK signaling pathways may be involved in the anti-IAV actions of PA. Intranasal administration of PA markedly improved mice survival and attenuated pneumonia symptoms in IAV infected mice, comparable to the effects of Oseltamivir. Therefore, Patchouli alcohol has the potential to be developed into a novel anti-IAV agent in the future.

  • NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes
    Virol. J. (IF 2.464) Pub Date : 2019-12-21
    Megan L. Dickherber; Charlie Garnett-Benson

    Adenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear. The dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding. PMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells. Our data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

  • Changes in RNA secondary structure affect NS1 protein expression during early stage influenza virus infection
    Virol. J. (IF 2.464) Pub Date : 2019-12-21
    Irina Baranovskaya; Mariia Sergeeva; Artem Fadeev; Renata Kadirova; Anna Ivanova; Edward Ramsay; Andrey Vasin

    RNA secondary structures play a key role in splicing, gene expression, microRNA biogenesis, RNA editing, and other biological processes. The importance of RNA structures has been demonstrated in the life cycle of RNA-containing viruses, including the influenza virus. At least two regions of conserved secondary structure in NS segment (+) RNA are predicted to vary among influenza virus strains with respect to thermodynamic stability; both fall in the NS1 open reading frame. The NS1 protein is involved in multiple virus-host interaction processes, and its main function is to inhibit the cellular immune response to viral infection. Using a reverse genetics approach, four influenza virus strains were constructed featuring mutations that have different effects on RNA secondary structure. Growth curve experiments and ELISA data show that, at least in the first viral replication cycle, mutations G123A and A132G affecting RNA structure in the (82–148) NS RNA region influence NS1 protein expression.

  • EOS, an Ikaros family zinc finger transcription factor, interacts with the HTLV-1 oncoprotein Tax and is downregulated in peripheral blood mononuclear cells of HTLV-1-infected individuals, irrespective of clinical statuses
    Virol. J. (IF 2.464) Pub Date : 2019-12-19
    Tadasuke Naito; Hiroshi Ushirogawa; Takuya Fukushima; Yuetsu Tanaka; Mineki Saito

    EOS plays an important role in maintaining the suppressive function of regulatory T cells (Tregs), and induces a regulated transformation of Tregs into T helper-like cells, which are capable of secreting proinflammatory cytokines in response to specific inflammatory signals. Meanwhile, significant reduction in Treg activity along with production of proinflammatory cytokines has been reported in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In this study, to examine whether there is an alteration in EOS expression in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals especially HAM/TSP, we investigated the expression of HTLV-1 tax genotype, proviral load (PVL), and the mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals including adult T-cell leukemia/lymphoma (ATL), HAM/TSP, or asymptomatic carriers. The expression levels of EOS mRNA and protein in various HTLV-1-infected or uninfected human T-cell lines were also investigated. EOS was highly expressed at the protein level in most HTLV-1 infected T-cell lines, and was augmented after the HTLV-1 regulatory factor Tax was induced in a Tax-inducible JPX-9 cell line. Immunoprecipitation experiments demonstrated a physical interaction between EOS and the viral regulatory protein Tax, but not HBZ. Meanwhile, there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals irrespective of their clinical statuses. We found an inverse correlation between EOS mRNA levels and HTLV-1 PVL in ATL patients, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP patients, whereas this correlation was not observed in other clinical statuses. These findings suggest that both Tax and HBZ can alter the expression of EOS through undetermined mechanisms, and dysregulated expression of EOS in PBMCs of HTLV-1 infected individuals may contribute to the pathological progression of HTLV-1-associated diseases, such as ATL and HAM/TSP.

  • A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures
    Virol. J. (IF 2.464) Pub Date : 2019-12-19
    Yuki Takamatsu; Olga Dolnik; Takeshi Noda; Stephan Becker

    Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

  • Evaluation of novel inactivated vaccines for the SAT 1, SAT 2 and SAT 3 serotypes of foot-and-mouth disease in pigs
    Virol. J. (IF 2.464) Pub Date : 2019-12-16
    Hye-Eun Jo; Su-Hwa You; Joo-Hyung Choi; Mi-Kyeong Ko; Sung Ho Shin; Jisoo Song; Hyundong Jo; Min Ja Lee; Su-Mi Kim; Byounghan Kim; Jong-Hyeon Park

    The foot-and-mouth disease (FMD) virus is classified into seven serotypes, of which the South African types have South African Territories (SAT)1, SAT2, and SAT3 that are prevalent in Africa. Especially SAT2 have spread to Arabian Peninsula and the Palestinian Autonomous Territories. Of these viruses, the incidence of SAT2 is the highest. It is important to prepare for the spread of the virus to other continents, even though most FMD viruses are bovine-derived. In particular, due to the high breeding density of pigs in Asia, more attention is usually paid to the immunity and protection of pigs than cattle. For this reason, this study investigated the immunity and protection of pigs against the SAT viruses. Specific vaccines were developed for SAT1, SAT2, and SAT3 serotypes. These vaccine viruses were designed to be distinguished from the wild-type strain. An immunogenicity test was conducted using these vaccines in both cattle (n = 5/group) and pigs (n = 20/group). High virus-neutralizing titer of antibodies (> 1:100) was induced in only 2 weeks after the immunization of cattle with the individual vaccine for SAT1, SAT2 or SAT3, and a clear immune response was induced after the second immunization in pigs. When the vaccinated pigs (n = 4–5/group) were challenged by the homologous wild-type virus strain 4 weeks after immunization, all the pigs were protected from the challenge. This study confirmed that these vaccines can be used against SAT1, SAT2, and SAT3 viruses in cattle and pigs. The vaccine strains developed in this study are expected to be used as vaccines that can protect against FMD in the event of a future FMD outbreak in pigs in consideration of the situation in Asia.

  • Mapping of CD4+ T-cell epitopes in bovine leukemia virus from five cattle with differential susceptibilities to bovine leukemia virus disease progression
    Virol. J. (IF 2.464) Pub Date : 2019-12-16
    Lanlan Bai; Shin-nosuke Takeshima; Masaaki Sato; William C. Davis; Satoshi Wada; Junko Kohara; Yoko Aida

    Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia virus, is the etiological agent of enzootic bovine leukosis, a disease characterized by a highly prolonged course involving persistent lymphocytosis and B-cell lymphoma. The bovine major histocompatibility complex class II region plays a key role in the subclinical progression of BLV infection. In this study, we aimed to evaluate the roles of CD4+ T-cell epitopes in disease progression in cattle. We examined five Japanese Black cattle, including three disease-susceptible animals, one disease-resistant animal, and one normal animal, classified according to genotyping of bovine leukocyte antigen (BoLA)-DRB3 and BoLA-DQA1 alleles using polymerase chain reaction sequence-based typing methods. All cattle were inoculated with BLV-infected blood collected from BLV experimentally infected cattle and then subjected to CD4+ T-cell epitope mapping by cell proliferation assays. Five Japanese Black cattle were successfully infected with BLV, and CD4+ T-cell epitope mapping was then conducted. Disease-resistant and normal cattle showed low and moderate proviral loads and harbored six or five types of CD4+ T-cell epitopes, respectively. In contrast, the one of three disease-susceptible cattle with the highest proviral load did not harbor CD4+ T-cell epitopes, and two of three other cattle with high proviral loads each had only one epitope. Thus, the CD4+ T-cell epitope repertoire was less frequent in disease-susceptible cattle than in other cattle. Although only a few cattle were included in this study, our results showed that CD4+ T-cell epitopes may be associated with BoLA-DRB3-DQA1 haplotypes, which conferred differential susceptibilities to BLV proviral loads. These CD4+ T-cell epitopes could be useful for the design of anti-BLV vaccines targeting disease-susceptible Japanese Black cattle. Further studies of CD4+ T-cell epitopes in other breeds and using larger numbers of cattle with differential susceptibilities are required to confirm these findings.

  • In-depth analysis of the replication cycle of Orpheovirus
    Virol. J. (IF 2.464) Pub Date : 2019-12-16
    Fernanda Souza; Rodrigo Rodrigues; Erik Reis; Maurício Lima; Bernard La Scola; Jônatas Abrahão

    After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

  • Endothelial IL-8 induced by porcine circovirus type 2 affects dendritic cell maturation and antigen-presenting function
    Virol. J. (IF 2.464) Pub Date : 2019-12-12
    Shiyu Liu; Qiuming Li; Jinzeng Qiao; Jianfang Wang; Defeng Cui; Kewei Gu; Shuanghai Zhou; Huanrong Li

    Porcine circovirus (PCV) disease caused by PCV type 2 (PCV2) is mainly attributed to immunosuppression and immune damage. PCV2 can infect vascular endothelial cells and induce high expression of endothelial IL-8. Dendritic cells (DCs), as professional antigen-presenting cells, can not only present antigens but also activate naïve T-cells, causing an immune response. To demonstrate whether endothelial IL-8 is the main factor inhibiting the maturation and related functions of dendritic cells during PCV2 infection, monocyte-derived DCs (MoDCs) and porcine iliac artery endothelial cells (PIECs) processed by different methods were co-cultured in two ways. Flow cytometry, molecular probe labeling, fluorescence quantitative PCR, and the MTS assay were used to detect the changes in related functions and molecules of MoDCs. Compared to those in the PIEC-DC group, the endothelial IL-8 upregulation co-culture group showed significantly lower double-positive rates for CD80/86 and MHC-II of MoDCs and significantly increased endocytosis of MoDCs. Meanwhile, the adhesion rate and average fluorescence intensity of MoDCs were significantly downregulated in migration and adhesion experiments. Furthermore, the MHC-I and LAMP7 mRNA levels in MoDCs and the proliferation of MoDC-stimulated T-cells were markedly reduced. However, the changes in MoDCs of the endothelial IL-8 downregulation co-culture group were the opposite. PCV2-induced endothelial IL-8 reduces the adhesion and migration ability of MoDCs, resulting in a decreased maturation rate of MoDCs, and further inhibits antigen presentation by DCs. These results may explain the immunosuppressive mechanism of PCV2 from the perspective of the interaction between endothelial cells and DCs in vitro.

  • Veterinary use of bacteriophage therapy in intensively-reared livestock
    Virol. J. (IF 2.464) Pub Date : 2019-12-12
    Adriano Gigante; Robert J Atterbury

    Zoonoses are infectious diseases transmitted directly or indirectly between animals and humans. Several important zoonotic pathogens colonize farm animals asymptomatically, which may lead to contamination of the food chain and public health hazards. Moreover, routine sampling of carcasses at retail by government authorities over the past 20 years suggests the prevalence of antibiotic resistance in foodborne pathogens has increased. If this continues, antibiotics may be ineffective against such pathogens in the future and alternative approaches, such as phage therapy, may be necessary. Intensive livestock farming is the only realistic way of meeting the demand for meat from an increasing global population and growth in middle class consumers in developing countries, particularly in Asia. This review elaborates on the use of phages to control zoonotic pathogens in intensively-reared livestock (poultry and pigs).

  • PCR-based reverse genetics strategy for bluetongue virus recovery
    Virol. J. (IF 2.464) Pub Date : 2019-12-05
    Qingyuan Xu; Jinying Ge; Maolin Li; Encheng Sun; Yawei Zhou; Yunze Guo; Donglai Wu; Zhigao Bu

    Bluetongue virus (BTV), an emerging insect vector mediated pathogen affecting both wild ruminants and livestock, has a genome consisting of 10 linear double-stranded RNA genome segments. BTV has a severe economic impact on agriculture in many parts of the world. Current reverse genetics (RG) strategy to rescue BTV mainly rely on in vitro synthesis of RNA transcripts from cloned complimentary DNA (cDNA) corresponding to viral genome segments with the aid of helper plasmids. RNA synthesis is a laborious job which is further complicated with a need for expensive reagents and a meticulous operational procedure. Additionally, the target genes must be cloned into a specific vector to prepare templates for RNA transcription. In this study, we have developed a PCR based BTV RG system with easy two-step transfection. Viable viruses were recovered following a first transfection with the seven helper plasmids and a second transfection with the 10 PCR products on the BSR cells. Further, recovered viruses were characterized with indirect immunofluorescence assays (IFA) and gene sequencing. And the proliferation properties of these viruses were also compared with wild type BTV. Interestingly, we have identified that viruses containing the segment 2 of the genome from reassortant BTV, grew slightly slower than the others. In this study, a convenient PCR based RG platform for BTV is established, and this strategy could be an effective alternative to the original available BTV rescue methods. Furthermore, this RG strategy is likely applicable for other Orbiviruses.

  • The tyrosine 73 and serine 83 dephosphorylation of H1N1 swine influenza virus NS1 protein attenuates virus replication and induces high levels of beta interferon
    Virol. J. (IF 2.464) Pub Date : 2019-12-05
    Jinghua Cheng; Jie Tao; Benqiang Li; Ying Shi; Huili Liu

    Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated. In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway. We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-β expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-β and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-β response upon rSIV NS1 Y73F and rSIV NS1 S83A infection. We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.

  • Antiviral activities of extremophilic actinomycetes extracts from Kazakhstan’s unique ecosystems against influenza viruses and paramyxoviruses
    Virol. J. (IF 2.464) Pub Date : 2019-12-02
    Vladimir Berezin; Diyora Abdukhakimova; Lyudmila Trenozhnikova; Andrey Bogoyavlenskiy; Aizhan Turmagambetova; Alpamys Issanov; Azliyati Azizan

    Commercially available antiviral drugs, when used in the treatment of viral infections, do not always result in success. This is an urgent problem currently that needs to be addressed because several viruses including influenza and paramyxoviruses are acquiring multi-drug resistance. A potential solution for this emerging issue is to create new antiviral drugs from available compounds of natural products. It is known that the majority of drugs have been developed using compounds derived from actinomycetes, which are naturally occurring gram-positive bacteria. The purpose of this study was to investigate the antiviral properties of extremophilic actinomycetes extracts from strains that were isolated from extreme environments in Kazakhstan. Five strains of extremophilic actinomycetes isolated from the unique ecosystems of Kazakhstan were extracted and tested for antiviral activity against influenza viruses (strains H7N1, H5N3, H1N1 and H3N2) and paramyxoviruses (Sendai Virus and Newcastle Disease Virus). The antiviral activity of these selected extracts was tested by checking their effect on hemagglutination and neuraminidase activities of the studied viruses. Additionally, actinomycetes extracts were compared with commercially available antiviral drugs and some plant preparations that have been shown to exhibit antiviral properties. The main findings show that extracts from strains K-192, K-340, K-362, K-522 and K525 showed antiviral activities when tested using influenza viruses, Sendai Virus, and Newcastle Disease Virus. These activities were comparable to those shown by Rimantadine and Tamiflu drugs, and “Virospan” and “Flavovir” plant preparations. We identified several extracts with antiviral activities against several strains of influenza viruses and paramyxoviruses. Our research findings can be applied towards characterization and development of new antiviral drugs from the active actinomycetes extracts.

  • Influenza A virus hemagglutinin mutations associated with use of neuraminidase inhibitors correlate with decreased inhibition by anti-influenza antibodies
    Virol. J. (IF 2.464) Pub Date : 2019-11-29
    Natalia A. Ilyushina; Takashi E. Komatsu; William L. Ince; Eric F. Donaldson; Nicolette Lee; Julian J. O’Rear; Raymond P. Donnelly

    Vaccination and the use of neuraminidase inhibitors (NAIs) are currently the front lines of defense against seasonal influenza. The activity of influenza vaccines and antivirals drugs such as the NAIs can be affected by mutations in the influenza hemagglutinin (HA) protein. Numerous HA substitutions have been identified in nonclinical NAI resistance-selection experiments as well as in clinical specimens from NAI treatment or surveillance studies. These mutations are listed in the prescribing information (package inserts) for FDA-approved NAIs, including oseltamivir, zanamivir, and peramivir. NAI treatment-emergent H1 HA mutations were mapped onto the H1N1 HA1 trimeric crystal structure and most of them localized to the HA antigenic sites predicted to be important for anti-influenza immunity. Recombinant A/California/04/09 (H1N1)-like viruses carrying HA V152I, G155E, S162 N, S183P, and D222G mutations were generated. We then evaluated the impact of these mutations on the immune reactivity and replication potential of the recombinant viruses in a human respiratory epithelial cell line, Calu− 3. We found that the G155E and D222G mutations significantly increased viral titers ~ 13-fold compared to the wild-type virus. The hemagglutination and microneutralization activity of goat and ferret antisera, monoclonal antibodies, and human serum samples raised against pandemic A(H1N1)pdm09 viruses was ~ 100-fold lower against mutants carrying G155E or D222G compared to the wild-type virus. Although the mechanism by which HA mutations emerge during NAI treatment is uncertain, some NAI treatment-emergent HA mutations correlate with decreased immunity to influenza virus.

  • Directional entry and release of Zika virus from polarized epithelial cells
    Virol. J. (IF 2.464) Pub Date : 2019-08-08
    Manasi Tamhankar; Jean L. Patterson

    Both vector borne and sexual transmission of Zika virus (ZIKV) involve infection of epithelial cells in the initial stages of infection. Epithelial cells are unique in their ability to form polarized monolayers and their barrier function. Cell polarity induces an asymmetry in the epithelial monolayer, which is maintained by tight junctions and specialized sorting machinery. This differential localization can have a potential impact of virus infection. Asymmetrical distribution of a viral receptor can restrict virus entry to a particular membrane while polarized sorting can lead to a directional release of virions. The present study examined the impact of cell polarity on ZIKV infection and release. A polarized Caco-2 cell model we described previously was used to assess ZIKV infection. Transepithelial resistance (TEER) was used to assess epithelial cell polarity, and virus infection was measured by immunofluorescence microscopy and qRT-PCR. Cell permeability was measured using a fluorescein leakage assay. Statistical significance was calculated using one-way ANOVA and significance was set at p < 0.05. Using the Caco-2 cell model for polarized epithelial cells, we report that Zika virus preferentially infects polarized cells from the apical route and is released vectorially through the basolateral route. Our data also indicates that release occurs without disruption of cell permeability. Our results show that ZIKV has directional infection and egress in a polarized cell system. This mechanism of directional infection may be one of the mechanisms that enables the cross the epithelial barrier effectively without a disruption in cell monolayer integrity. Elucidation of entry and release characteristics of Zika virus in polarized epithelial cells can lead to better understanding of virus dissemination in the host, and can help in developing effective therapeutic interventions.

  • Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
    Virol. J. (IF 2.464) Pub Date : 2019-08-09
    Franziska Kugler; Ingo Drexler; Ulrike Protzer; Dieter Hoffmann; Hassan Moeini

    Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied. We found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently. Comparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs.

  • Hepatitis C virus genotyping based on Core and NS5B regions in Cameroonian patients
    Virol. J. (IF 2.464) Pub Date : 2019-08-09
    Paul Alain Tagnouokam-Ngoupo; Marie Nicole Ngoufack; Sebastien Kenmoe; Simon Frédéric Lissock; Marie Amougou-Atsama; Robert Banai; Laure Ngono; Richard Njouom

    Current HCV treatments are genotype specific although potential pan-genotype treatments have recently been described. Therefore, genotyping is an essential tool for the therapeutic management of HCV infection and a variety of technologies have been developed for HCV genotypes determination. Sequences analysis of HCV sub-genomic regions is considered as gold standard and is widely used for HCV genotyping. Here, we compared HCV genotyping using core and NS5B regions in routine practice in HCV-positive Cameroonian patients. All plasma samples received at Centre Pasteur of Cameroon (CPC) in 2016 for HCV genotyping were included. Viral loads were determined using the Abbott Real Time assay. Further, genotyping was based on the amplification and sequencing of core and NS5B regions following by phylogenetic analysis of corresponding sequences. A total of 369 samples were received during the study period with high viral load values (median: 930,952 IU/ml; IQR: 281,833-2,861,179). Positive amplification was obtained in at least one genomic region (core or NS5B) for all the samples with similar amplification rate in the two genomic regions (p = 0.34). Phylogenetic analysis showed that among the 369 samples, 146 (39.6%) were classified as genotype 4, 132 (35.8%) as genotype 1, 89 (24.1%) as genotype 2, in both core and NS5B regions. Interestingly, for two samples (0.54%) discordant genotypes were obtained in both regions with the core region classified as genotype 4 while the NS5B was identified as genotype 1 indicating the presence of putative HCV recombinant virus or multiple infections in these samples. Discrimination of HCV subtypes was most likely possible with NS5B compared to core region. We found high amplification rates of HCV in both core and NS5B regions, and a good concordance was obtained at genotype level using both regions except for two samples where putative 1–4 recombinants/multiple infections were detected. Therefore, HCV genotyping based on at least two genomic regions could help to identify putative recombinants and improve therapeutic management of HCV infection.

  • Evaluation of novel rapid detection kits for dengue virus NS1 antigen in Dhaka, Bangladesh, in 2017
    Virol. J. (IF 2.464) Pub Date : 2019-08-15
    Keita Suzuki; Emi E. Nakayama; Akatsuki Saito; Akio Egawa; Tairyu Sato; Juthamas Phadungsombat; Rummana Rahim; Abu Hasan; Hisahiko Iwamoto; Mizanur Rahman; Tatsuo Shioda

    Dengue virus (DENV) infection is one of the biggest challenges for human health in the world. In addition, a secondary DENV infection sometimes causes dengue hemorrhagic fever (DHF), which frequently leads to death. For this reason, accurate diagnosis record management is useful for prediction of DHF. Therefore, the demand for DENV rapid diagnosis tests (RDTs) is increasing because these tests are easy and rapid to use. However, commercially available RDTs often show low sensitivity for DENV and cross-reactivity against other flaviviruses, especially Zika virus (ZIKV). We developed two types of novel DENV non-structural protein 1 (NS1) detection RDTs, designated TKK-1st and TKK-2nd kits. Specificities of the monoclonal antibodies (MAbs) used in these kits were confirmed by enzyme-linked immuno-sorbent assay (ELISA), dot blot, and western blot using recombinant NS1 proteins and synthetic peptides. For evaluation of sensitivity, specificity, and cross-reactivity of the novel DENV NS1 RDTs, we first used cultured DENV and other flaviviruses, ZIKV and Japanese encephalitis virus (JEV). We then used clinical specimens obtained in Bangladesh in 2017 for further evaluation of kit sensitivity and specificity in comparison with commercially available RDTs. In addition, RNA extracted from sera were used for viral genome sequencing and genotyping. Epitopes of three out of four MAbs used in the two novel RDTs were located in amino acid positions 100 to 122 in the NS1 protein, a region that shows low levels of homology with other flaviviruses. Our new kits showed high levels of sensitivity against various serotypes and genotypes of DENV and exhibited high levels of specificity without cross-reactivity against ZIKV and JEV. In clinical specimens, our RDTs showed sensitivities of 96.0% (145/151, TKK-1st kit) and 96.7% (146/151, TKK-2nd kit), and specificities of 98.0% (98/100, TKK-1st kit and TKK-2nd kit). On the other hand, in the case of the commercially available SD Bioline RDT, sensitivity was 83.4% (126/151) and specificity was 99.0% (99/100) against the same clinical specimens. Our novel DENV NS1-targeting RDTs demonstrated high levels of sensitivity and lacked cross-reactivity against ZIKV and JEV compared with commercially available RDTs.

  • Human immunodeficiency virus type 1 ((HIV-1) subtypes in the northwest region, Cameroon
    Virol. J. (IF 2.464) Pub Date : 2019-08-15
    Lem Edith Abongwa; Anthony Kebira Nyamache; Judith Ndongo Torimiro; Paul Okemo; Fokunang Charles

    The high genetic diversity of HIV-1 has been shown to influence the global distribution, disease progression, treatment success, and the development of an effective vaccine. Despite the low HIV prevalence in Cameroon, all the major HIV subtypes alongside several circulating recombinant forms (CRFs) and unique recombinant forms (URFs) have been reported in Cameroon. To date, HIV-1 diversity in some parts of Cameroon has been largely studied however, information on circulating HIV-1 subtypes in the Northwest region (NWR) of Cameroon is dearth. Therefore the aim of this study was to determine the current circulating HIV-1 subtypes among adults in the NWR of Cameroon. The genetic analysis of the reverse transcriptase region of the pol gene was performed on 81 samples. The samples were collected from drug naïve patients aged between 18 and 61 years residing within the rural and urban towns in the NWR during the period between February and April 2016. Viral RNA was extracted from plasma, reverse-transcribed, further amplified by nested-PCR before sequencing using an in-house protocol. Generated sequences were then phylogenetically analyzed together with references using MEGA 7. Phylogenetic analysis revealed a broad viral diversity including CRF02 _AG (74.1%), F2 (7.4%), D (7.4%), G (3.7%), A1 (1.2%), CRF22_01A1 (2.5%), CRF06_cpx (1.2%), CRF09_cpx (1.2%), CRF11_cpx (1.2%). Three close epidemic clusters were found among F2 (1) and CRF02_AG (2) variants. For the first time we are reporting the CRF22_01A1 subtype in this region. Our findings update HIV-1 subtypes information in Cameroon and uphold previous studies that CRF02_AG is the most prevalent subtype. This CRF02_AG subtype may have important public health, research, and clinical consequences.

  • Rapid detection of human herpes virus by next-generation sequencing in a patient with encephalitis
    Virol. J. (IF 2.464) Pub Date : 2019-08-17
    Yu Zhang; Kun Hong; Yueli Zou; Hui Bu

    The inflammatory or non-inflammatory changes caused by the virus entering the nervous system and related tissues are central nervous system virus infections. Viral infection is a common infectious disease of the central nervous system, of which herpes simplex virus encephalitis is the most common. However, conventional laboratory techniques to detect an infectious agent are difficult to achieve etiological diagnosis. Here we present a patient with severe and progressive encephalitis, requiring diagnosis of the specific pathogen to guide clinical treatments. Application of next-generation sequencing provided a quick and definite diagnosis of the etiology of encephalitis and enabled our patient to be treated appropriately.

  • The altered gut virome community in rhesus monkeys is correlated with the gut bacterial microbiome and associated metabolites
    Virol. J. (IF 2.464) Pub Date : 2019-08-19
    Heng Li; Hongzhe Li; Jingjing Wang; Lei Guo; Haitao Fan; Huiwen Zheng; Zening Yang; Xing Huang; Manman Chu; Fengmei Yang; Zhanlong He; Nan Li; Jinxi Yang; Qiongwen Wu; Haijing Shi; Longding Liu

    The gut microbiome is closely associated with the health of the host; although the interaction between the bacterial microbiome and the whole virome has rarely been studied, it is likely of medical importance. Examination of the interactions between the gut bacterial microbiome and virome of rhesus monkey would significantly contribute to revealing the gut microbiome composition. Here, we conducted a metagenomic analysis of the gut microbiome of rhesus monkeys in a longitudinal cohort treated with an antibiotic cocktail, and we documented the interactions between the bacterial microbiome and virome. The depletion of viral populations was confirmed at the species level by real-time PCR. We also detected changes in the gut metabolome by GC-MS and LC-MS. A majority of bacteria were depleted after treatment with antibiotics, and the Shannon diversity index decreased from 2.95 to 0.22. Furthermore, the abundance-based coverage estimator (ACE) decreased from 104.47 to 33.84, and the abundance of eukaryotic viruses also changed substantially. In the annotation, 6 families of DNA viruses and 1 bacteriophage family were present in the normal monkeys but absent after gut bacterial microbiome depletion. Intriguingly, we discovered that changes in the gut bacterial microbiome composition may promote changes in the gut virome composition, and tryptophan, arginine, and quinone may play roles in the interaction between the bacterial microbiome and virome. Our results indicated that the clearly altered composition of the virome was correlated with depletion in the bacterial community and that metabolites produced by bacteria possibly play important roles in the interaction.

  • Changes in Bemisia tabaci feeding behaviors caused directly and indirectly by cucurbit chlorotic yellows virus
    Virol. J. (IF 2.464) Pub Date : 2019-08-22
    Shaohua Lu; Mingshun Chen; Jingjing Li; Yan Shi; Qinsheng Gu; Fengming Yan

    Plant viruses can affect vector’s behaviors in order to enhance viral transmission. Cucurbit chlorotic yellows virus (CCYV) (genus Crinivirus) is an emergent RNA plant virus and is transmitted specifically by biotypes B and Q of tobacco whitefly, Bemisia tabaci (Gennadius), in a semipersistent manner. We used the electrical penetration graph (EPG) to investigate the effect of CCYV on the feeding behaviors of B. tabaci biotypes B and Q. CCYV could affect, both directly and indirectly, the feeding behaviors of B. tabaci to various degrees, depending on biotypes and sexes of the insect. CCYV showed stronger direct effects on biotype Q than on biotype B in terms of increased non-phloem probing and phloem salivation. CCYV increased non-phloem probing and phloem salivation more on females than on males of biotype Q, and increased phloem salivation more on females than on males of biotype B. CCYV had stronger indirect effects, via virus-infested plants, on biotype B than on biotype Q by enhancing phloem sap ingestion and feeding bouts. CCYV increased non-phloem probing and feeding bouts more on males than on females of biotype B, and decreased phloem sap ingestion more on males than on females on biotype Q indirectly. The results clearly indicated that CCYV affects the feeding behaviors of B. tabaci, which may lead to increased ability of the B. tabaci for CCYV transmission.

  • Clinical validation of the HPVIR high-risk HPV test on cervical samples according to the international guidelines for human papillomavirus DNA test requirements for cervical cancer screening
    Virol. J. (IF 2.464) Pub Date : 2019-08-22
    Inger Gustavsson; Riina Aarnio; Mattias Myrnäs; Julia Hedlund-Lindberg; Ongeziwe Taku; Tracy Meiring; Ingrid Wikström; Stefan Enroth; Anna-Lise Williamson; Matts Olovsson; Ulf Gyllensten

    The indicating FTA card is a dry medium used for collection of cervical samples. HPVIR is a multiplex real-time PCR test that detects 12 high-risk human papillomavirus types (hrHPV) and provides single genotype information for HPV16, − 31, − 35, − 39, − 51, − 56, and − 59 and pooled type information for HPV18/45 and HPV33/52/58. The aim of this study was to evaluate whether a strategy with cervical samples collected on the FTA card and subsequently analysed with the HPVIR test complies with the criteria of the international guidelines for a clinically validated method for cervical screening. We performed a non-inferiority test comparing the clinical sensitivity and specificity of the candidate test (FTA card and HPVIR) with a clinically validated reference test (Cobas® HPV test) based on liquid-based cytology (LBC) samples. Two clinical samples (LBC and FTA) were collected from 896 participants in population-based screening. For evaluation of the specificity we used 799 women without ≥ CIN2, and for clinical sensitivity we used 67 women with histologically confirmed ≥ CIN2. The reproducibility was studied by performing inter- and intra-laboratory tests of 558 additional clinical samples. The clinical sensitivity and specificity for samples collected on the FTA card and analysed using the HPVIR test were non-inferior to samples analysed with the Cobas® HPV test based on LBC samples (non-inferiority test score, p = 1.0 × 10− 2 and p = 1.89 × 10− 9, respectively). Adequate agreement of > 87% was seen in both the intra- and inter-laboratory comparisons. Samples collected on the indicating FTA card and analysed with HPVIR test fulfil the requirements of the international guidelines and can therefore be used in primary cervical cancer screening.

  • Molecular characterization of bovine leukemia virus reveals existence of genotype 4 in Chinese dairy cattle
    Virol. J. (IF 2.464) Pub Date : 2019-08-27
    Yi Yang; Lina Chen; Maoli Dong; Wenjiang Huang; Xiaoli Hao; Yalan Peng; Zaicheng Gong; Aijian Qin; Shaobin Shang; Zhangping Yang

    Bovine leukemia virus (BLV) causes enzootic bovine leucosis and is widely spread worldwide, except several European countries, Australia and New Zealand. Although BLV is highly prevalent in China, information about the genetic diversity and evolutionary dynamics of BLV among Chinese dairy herds is still lacking. To determine the genetic variability of BLV, 219 cows from four cities of Ningxia province of China were screened for BLV infection by fluorescence resonance energy transfer (FRET)-PCR and sequencing, 16 selected positive samples were subjected to molecular characterization. Phylogenetic analysis using the neighbor-joining (NJ) method on complete sequences of envelope (env) gene of BLV obtained from China and those available in GenBank (representing BLV genotypes 1–10) revealed that those Chinese strains belonged to genotypes 4 and 6. Totally, 23 mutations were identified and 16 of them were determined to be unique mutations among Chinese strains. Alignment of the deduced amino acid sequences demonstrated six mutations in glycoprotein 51 (gp51) and three mutations in glycoprotein 30 (gp30) located in the identified neutralizing domain (ND), CD8+ T cell epitope, E-epitope, B-epitope, gp51N12 and cytoplasmic domain of transmembrane protein. This study reported for the first time the BLV genotype 4 in China, and further studies are warranted to compare its immunogenicity and pathogenicity with other BLV genotypes.

  • Virology Downunder, a meeting commentary from the 2019 Lorne Infection and Immunity Conference, Australia
    Virol. J. (IF 2.464) Pub Date : 2019-09-02
    Gregor Ebert; Prasad N. Paradkar; Sarah L. Londrigan

    The aim of this article is to summarise the virology content presented at the 9th Lorne Infection and Immunity Conference, Australia, in February 2019. The broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting.

  • Identification of virion-associated transcriptional transactivator (VATT) of SGIV ICP46 promoter and their binding site on promoter
    Virol. J. (IF 2.464) Pub Date : 2019-09-03
    Li-Qun Xia; Jian-Lin Chen; Hong-Lian Zhang; Jia Cai; Sheng Zhou; Yi-Shan Lu

    Iridoviruses are large DNA viruses that cause diseases in fish, amphibians and insects. Singapore grouper iridovirus (SGIV) is isolated from cultured grouper and characterized as a ranavirus. ICP46 is defined to be a core gene of the family Iridoviridae and SGIV ICP46 was demonstrated to be an immediate-early (IE) gene associated with cell growth control and could contribute to virus replication in previous research. The transcription start site (TSS) and 5′-untranslated region (5′-UTR) of SGIV ICP46 were determined using 5′ RACE. The core promoter elements of ICP46s were analyzed by bioinformatics analysis. The core promoter region and the regulation model of SGIV ICP46 promoter were revealed by the construction of serially deleted promoter plasmids, transfections, drug treat and luciferase reporter assays. The identification of virion-associated transcriptional transactivator (VATT) that interact with SGIV ICP46 promoter and their binding site on promoter were performed by electrophoretic mobility shift assays (EMSA), DNA pull-down assays and mass spectrometry (MS). SGIV ICP46 was found to have short 5′-UTR and a presumptive downstream promoter element (DPE), AGACA, which locates at + 36 to + 39 nt downstream of the TSS. The core promoter region of SGIV ICP46 located from − 22 to + 42 nt relative to the TSS. VATTs were involved in the promoter activation of SGIV ICP46 and further identified to be VP12, VP39, VP57 and MCP. A 10-base DNA sequence “ATGGCTTTCG” between the TSS and presumptive DPE was determined to be the binding site of the VATTs. Our study showed that four VAATs (VP12, VP39, VP57 and MCP) might bind with the SGIV ICP46 promoter and be involved in the promoter activation. Further, the binding site of the VATTs on promoter was a 10-base DNA sequence between the TSS and presumptive DPE.

  • Viral respiratory tract infections in young children with cystic fibrosis: a prospective full-year seasonal study
    Virol. J. (IF 2.464) Pub Date : 2019-09-03
    Mathilde Eymery; Florence Morfin; Anne Doleans-Jordheim; Marie Perceval; Camille Ohlmann; Catherine Mainguy; Philippe Reix

    Viral respiratory tract infections are common during early childhood. How they impact cystic fibrosis lung disease history in young children is poorly known. The principal aim of our study was to determinate respiratory tract infections frequency in this cystic fibrosis young population. Secondary outcomes were nature of viral agents recovered and impact of such infections. We conducted a prospective cohort study of 25 children affected by cystic fibrosis and aged less than 2 years. Nasal samplings were taken systematically monthly or bimonthly with additional samples taken during respiratory tract infections episodes. Ten pathogens were tested by a combination of five duplex RT-PCRs or PCRs: influenza A and B, respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus/enterovirus (RV/EV)), coronavirus (HKU1, NL63, 229E and OC43), parainfluenza virus (1–4), adenovirus and bocavirus (Respiratory Multi-Well System MWS r-gene®, BioMérieux, Marcy l’Étoile, France). Cycle thresholds (CTs) were reported for all positive samples and considered positive for values below 40. Quantitative variables were compared using a nonparametric statistical test (Wilcoxon signed rank for paired comparisons). Pearson’s correlation coefficient (r) was used to assess relationships between two variables. Statistical analyses were performed using SAS v9.4 (SAS Institute, Cary, NC, USA) or GraphPad Prism V6.00 (GraphPad Software, La Jolla, CA, USA). The significance level was set at 0.05. The mean age at inclusion was 9.6 ± 6.7 months. The patients had 3.4 ± 1.7 respiratory tract infections episodes per child per year. Forty-four respiratory tract infections (69%) were associated with virus: rhinovirus and enterovirus (RV/EV) were implied in 61% of them and respiratory syncytial virus (RSV) in 14%. Only one patient required hospitalization for lower respiratory tract infections. 86% of the patients were treated by antibiotics for a mean of 13.8 ± 6.2 days. RSV infections (n = 6) were usually of mild severity. Respiratory tract infections in young children with cystic fibrosis were of mild severity, rarely requiring hospitalization. Unsurprisingly, RV/EV were the most frequent agents. RSV-related morbidity seems low in this population. This raises the question of the usefulness of RSV preventive medication in this young population.

  • A novel method to rescue and culture duck Astrovirus type 1 in vitro
    Virol. J. (IF 2.464) Pub Date : 2019-09-05
    Ruihua Zhang; Jingjing Lan; Haie Li; Junhao Chen; Yupeng Yang; Shaoli Lin; Zhijing Xie; Shijin Jiang

    Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 μg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.

  • A novel double-stranded RNA mycovirus isolated from Trichoderma harzianum
    Virol. J. (IF 2.464) Pub Date : 2019-09-11
    Chenchen Liu; Mei Li; Estifanos Tsegaye Redda; Jie Mei; Jiantai Zhang; Beilei Wu; Xiliang Jiang

    Trichoderma spp. are used extensively in agriculture as biological control agents to prevent soil-borne plant diseases. In recent years, mycoviruses from fungi have attracted increasing attention due to their effects on their hosts, but Trichoderma mycoviruses have not been the subject of extensive study. We sought to discover novel mycoviruses from Trichoderma spp. and to determine the effects of the biocontrol function of Trichoderma spp. Mycoviruses were screened by dsRNA extraction and metagenomic analysis. RT-PCR, 5′ RACE, and 3′ RACE were used to obtain the genome sequence. MEGA software was used to classify the new mycovirus. The effects of the identified mycovirus on the biological properties of the host strain 525 were evaluated using cucumber plants and Fusarium oxysporum f. sp. cucumerinum. A novel mycovirus, Trichoderma harzianum mycovirus 1 (ThMV1) (accession number MH155602), was discovered in Trichoderma harzianum strain 525, a soil-borne fungus collected from Inner Mongolia, China. The mycovirus exhibited a double-stranded RNA (dsRNA) genome with a complete genome sequence of 3160 base pairs and two open reading frames (ORFs) on the negative strand. Phylogenetic analysis indicated that it belongs to an unclassified family of dsRNA mycoviruses. The removal of ThMV1 from the host 525 strain reduced host biomass production and improved the biocontrol capability of the host for Fusarium oxysporum f. sp. cucumerinum. At same time, the presence of ThMV1 improved the growth of cucumber. ThMV1 is a new unclassified mycovirus found in T. harzianum. It not only affects the phenotype of the host strain but also reduces its biocontrol function, which sheds light on the interaction between the mycovirus and Trichoderma spp.

  • E6 and E7 gene polymorphisms in human papillomavirus Type-6 identified in Southwest China
    Virol. J. (IF 2.464) Pub Date : 2019-09-12
    Zuyi Chen; Qiongyao Li; Jian Huang; Jin Li; Feng Yang; Xun Min; Zehui Chen

    Human papillomavirus type-6 (HPV6) is the major etiological agent of anogenital warts both men and women. The present study aimed to characterize the genetic diversity among HPV6 in Southwest China, and to investigate the origin of, selective pressure experienced by, and impact of the resultantly identified genetic variants on the HPV6 secondary structure. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by Molecular Evolutionary Genetics Analysis version 6.0. The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by Phylogenetic Analyses by Maximum Likelihood version 4.8 software. HPV6 was the most prevalent low risk HPV type in southwest China. In total, 143 E6 and E7 gene sequences of HPV6 isolated from patients were sequenced and compared to GenBank HPV6 reference sequence X00203. The results of these analyses revealed that both the HPV6 E6 and E7 were highly conserved within the analyzed patient samples, and comprised only 3 types of variant sequence, respectively. Furthermore, the analysis of HPV6 E6 and E7 sequences revealed seven/five single-nucleotide mutations, two/four and five/one of which were non-synonymous and synonymous, respectively. The phylogenetic analyses of the E6 and E7 sequences indicated that they belonged to sub-lineage A1 and sub-lineage B1, whereas the selective pressure analyses showed that only the E7 mutation sites 4R, 34E, and 52F were positive selection. HPV6 (detection rate = 13.10%) was very prevalent in southwest China, both the HPV6 E6 and E7 sequences were highly conserved within the analyzed patient samples in southwest China, indicating that the low risk HPV6 can adapt to the environment well without much evolution.

  • Plasma diamine oxidase level predicts 6-month readmission for patients with hepatitis B virus-related decompensated cirrhosis
    Virol. J. (IF 2.464) Pub Date : 2019-09-18
    Feng-Cai Li; Yu-Chen Fan; Yue-Kai Li; Kai Wang

    Hepatitis B virus-related decompensated cirrhosis is difficult to cure but has a high readmission rate due to multiple complications. Our aim was to investigate the diagnostic potential value of plasma diamine oxidase (DAO) for 6-month readmission of patients with HBV-related decompensated cirrhosis. A total of 135 patients with HBV-related decompensated cirrhosis were prospectively collected at the onset of discharge of hospital, and then were followed up for at least 6 months with the readmission as the primary outcome. The plasma DAO level was measured using enzyme linked immunosorbent assay. In addition, 120 age and sex matched patients with HBV-related compensated cirrhosis were included as controls. A total of 36 patients (36.7%) with decompensated cirrhosis admitted to hospital during the 6-month follow up. The plasma DAO level of readmission group [21.1 (14.5; 29.0) ng/ml] was significantly higher than that in the non-readmission group [12.7 (9.3; 18.0) ng/mL, P < 0.001]. Multivariate analysis showed that the plasma DAO level (HR = 1.102, P < 0.05) and hepatic encephalopathy (HE) (HR = 5.018, P < 0.05) were independent factors for 6-month readmission of decompensated cirrhosis. DAO level showed higher area under the curve of receiver operating characteristic (AUROC) than HE (0.769 vs. 0.598, P < 0.05) and Child-Pugh-Turcotte (CPT) score (0.769 vs. 0.652, P < 0.05) for predicting 6-month readmission rate, with the best cut-off value as 19.7 ng/mL. Furthermore, plasma DAO level (HR = 1.184, P < 0.05) was an independent factor and has the higher AUROC than CPT score for the onset of recurrent HE (0.905 vs. 0.738, P < 0.05) during the 6-month follow up. Plasma DAO level > 19.7 ng/mL predicts high rate of 6-month readmission in patients with HBV-related decompensated cirrhosis.

  • Serum and tissue miRNAs: potential biomarkers for the diagnosis of cervical cancer
    Virol. J. (IF 2.464) Pub Date : 2019-10-07
    Mahdieh Farzanehpour; Sayed-Hamidreza Mozhgani; Somayeh Jalilvand; Ebrahim Faghihloo; Setareh Akhavan; Vahid Salimi; Talat Mokhtari Azad

    Finding new biomarkers for the early detection of cervical cancer is an essential requirement in this field. In this study, we aimed to evaluate the expression level of potential biomarkers in progression of cervical cancer in patients with cervical cancer compared to normal subjects. The expression levels of tissue and serum miRNAs, including miR-9, miR-192 and miR-205, were investigated in 36 normal, 18 precancer, and 18 cervical cancer samples using real-time polymerase chain reaction. The results showed the higher significant expressions of miR-9, miR-192 and miR-205 in the tissue of cancer samples than those in the normal samples. Moreover, the miR-192 and miR-205 expression were significantly increased in the cancer group in comparison with the precancer group. Examination of serum samples revealed the increase in the expression level in the cancer groups than in the normal samples, for miR-9, miR-192 and miR-205 and the expressions of miR-9, miR-192 and miR-205 were significantly up-regulated in the precancer group in comparison with the normal group. Also the expression of miR-205 was remarkably increased in the cancer group in comparison with the precancer group. The receiver operating characteristic (ROC) analyses showed the highest area under the curve value for miR-192. Given the increased expression level of miR-192 in cancer and in precancerous tissue and serum compared with the normal tissue and serum validated by analysing the ROC curve, miR-192 can be used as potential biomarker for the early detection of cervical cancer.

  • Combining HPV DNA load with p16/Ki-67 staining to detect cervical precancerous lesions and predict the progression of CIN1–2 lesions
    Virol. J. (IF 2.464) Pub Date : 2019-10-16
    Yuejie Li; Jie Liu; Li Gong; Xingwang Sun; Wenbo Long

    Human Papilloma Virus (HPV) DNA tests are highly sensitive and can triage women with mild lesions, improving the prognosis and diagnosis of cervical lesions. However, additional efficient strategies should be developed to improve the specificity of these tests. This study aimed to evaluate the clinical value of HPV DNA load in improving the diagnosis and prognosis of cervical lesions by p16/Ki-67 testing. Histological samples were collected from 350 women with HR-HPV genotyping and analyzed by qRT-PCR. Immunohistochemical staining was used to assess p16 and Ki-67 expression and clinical performance characteristics were calculated. Of the cases, 271 had detectable HR-HPV infection, in which HPV-16 was most prevalent (52.0%), followed by HPV-58 (22.5%). P16/Ki-67-positivity increased with histological severity but not for HR-HPV infection. Amongst the 13 HR-HPV genotypes, only HPV-16 (P = 0.016) and HPV-58 (P = 0.004) viral loads significantly correlated with lesion severity. The P16/Ki-67/HPV DNA load co-test indicated an increased sensitivity for the detection of cervical intraepithelial neoplasia (CIN) lesions compared to p16/Ki-67 staining in HPV-16 and/or 58 positive cases. Viral load did not improve the sensitivity of p16/Ki-67 co-test in non-HPV-16 or 58 positive cases. The clinical performance of the p16/Ki-67/HPV DNA load co-test was limited for the prediction of the outcome of CIN1 lesions. However, amongst the 12 HPV-16 and/or 58 positive CIN2 cases in which return visit results were obtained, the behavior of the lesions could be predicted, with a sensitivity, specificity, positive prediction rate (PPV), and negative prediction rate (NPV) of 0.667, 1, 1 and 0.5, respectively. Combination of the assessment of HPV DNA load with the intensity of p16 and Ki-67 staining could increase the sensitivity of CIN lesion diagnosis and predict the outcome of CIN2 in patients with a HPV-16 and/or 58 infection.

  • Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence
    Virol. J. (IF 2.464) Pub Date : 2019-10-17
    Jun-Jun Liu; Yu Xiang; Richard A. Sniezko; Anna W. Schoettle; Holly Williams; Arezoo Zamany

    Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1–5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

  • Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses
    Virol. J. (IF 2.464) Pub Date : 2019-10-22
    Lin Liu; Ying Zhang; Pengfei Cui; Congcong Wang; Xianying Zeng; Guohua Deng; Xiurong Wang

    In 2017–2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans. In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV. The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry. The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

  • Tumor necrosis factor-α-induced protein 8-like 2 mRNA in peripheral blood mononuclear cells is associated with the disease progression of chronic hepatitis B virus infection
    Virol. J. (IF 2.464) Pub Date : 2019-10-28
    Yi Liu; Jia Jin; Jian Ji; Xi-Mei Gao; Yu-Chen Fan

    Tumor necrosis factor-alpha-induced protein 8-like 2 (TIPE2) is a novel target and molecule in the negative regulation of immune homeostasis. The present study aimed to investigate the dynamic expression of TIPE2 mRNA during the progression of chronic hepatitis B virus (HBV) infection. A total of 193 patients with chronic HBV infection were retrospectively recruited into this cross-sectional study, including 97 patients with chronic hepatitis B (CHB), 55 with liver cirrhosis and 41 with HBV-related hepatocellular carcinoma (HCC). TIPE2 mRNA was determined using real-time quantitative polymerase chain reaction. The expression of TIPE2 levels in patients with HCC was significantly decreased compared with expression in patients with liver cirrhosis, CHB and healthy controls (P < 0.05); meanwhile, the TIPE2 mRNA levels in patients with CHB and liver cirrhosis were significantly increased compared with levels in healthy controls (P < 0.01). In liver cirrhosis, the TIPE2 mRNA level in the decompensated state was significantly higher than that in the compensated state (P < 0.05). In HCC patients, TIPE2 mRNA was significantly associated with venous invasion, tumor size and tumor node metastasis stage. Furthermore, the optimal cutoff of 0.78 for the level of TIPE2 mRNA has a sensitivity of 97.56% and a specificity of 88.16% for discriminating HCC from patients with CHB and liver cirrhosis. TIPE2 mRNA was associated with various stages of chronic HBV infection, ranging from CHB to liver cirrhosis and HCC. Furthermore, TIPE2 mRNA with an optional cutoff value of 0.78 might serve as a promising biomarker to discriminate HBV-associated HCC from CHB and LC patients.

  • Characterization and pathogenicity of Vero cell-attenuated porcine epidemic diarrhea virus CT strain
    Virol. J. (IF 2.464) Pub Date : 2019-10-28
    Yu Wu; Wei Li; Qingfeng Zhou; Qunhui Li; Zhichao Xu; Hanqin Shen; Feng Chen

    Porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global pig industry. Currently available PEDV vaccine strains have limited protective effects against PEDV variant strains. In this study, the highly virulent epidemic virus strain CT was serially passaged in Vero cells for up to 120 generations (P120). Characterization of the different passages revealed that compared with P10 and P64, P120 had a higher viral titer and more obvious cytopathic effects, thereby demonstrating better cell adaptability. Pathogenicity experiments using P120 in piglets revealed significant reductions in clinical symptoms, histopathological lesions, and intestinal PEDV antigen distribution; the piglet survival rate in the P120 group was 100%. Furthermore, whole-genome sequencing identified 13 amino acid changes in P120, which might be responsible for the attenuated virulence of P120. Thus, an attenuated strain was obtained via cell passaging and that this strain could be used in preparing attenuated vaccines.

  • Multiplex PCR-based titration (MPBT) assay for determination of infectious titers of the three Sabin strains of live poliovirus vaccine
    Virol. J. (IF 2.464) Pub Date : 2019-10-28
    Hasmik Manukyan; Elvira Rodionova; Tatiana Zagorodnyaya; Tsai-Lien Lin; Konstantin Chumakov; Majid Laassri

    Conventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge. A new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1–5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay. The multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

  • Characteristics of human papillomaviruses distribution in Guizhou Province, China
    Virol. J. (IF 2.464) Pub Date : 2019-10-29
    Zuyi Chen; Qiongyao Li; Qiong Huang; Huaqing Liu; Hongwu Jiang; Zehui Chen; Zhengyuan An; Qingfang Luo

    Human papillomavirus (HPV) is one of the most common sexually transmitted viruses. Data about HPV infection in Guizhou is limited. 56,768 cervical samples were collected and genotyped for 15 main high risk and 6 main low risk HPV types. 16.95% (9623/56768) of samples were HPV positive; 90.70% (8728/9623) of HPV positive women were infected by high risk HPV. High risk and high risk mix infection (1458; 70.85%) was the most common mix HPV infection type. The highest HPV detection rate was found in age group 41–45 years old (detection rate = 17.89%) (χ2 = 204.77; P < 0.001); the highest within-group HPV infection rates were found in the ≤20 (25.62%) and ≥ 61 (24.67%) years old age groups, the lowest within-group HPV infection rate was found in the 31–35 years old age group (15.02%). The highest mix infection proportions were found in the ≥61 (36.06%) and ≤ 20 (33.63%) years old age groups (χ2 = 111.21; P < 0.001), the lowest mix infection proportion was found in the 41–45 (17.42%) years old age group. The highest high risk infection proportions were found in the 26–30 (92.98%), ≥61 (92.68%), and 36–40 (92.16%) years old age groups (χ2 = 31.72; P < 0.001), the lowest high risk infection proportion was found in the ≤20 (84.96%) years old age group. HPV infection rates varied with seasons in Guizhou. Characteristics of HPV distribution in Guizhou were identified. There were significant differences in HPV distribution among age groups, prevention strategies should be adjusted according to the characteristics.

  • Identifying novel biomarkers of the pediatric influenza infection by weighted co-expression network analysis
    Virol. J. (IF 2.464) Pub Date : 2019-10-29
    Mohadeseh Zarei Ghobadi; Sayed-Hamidreza Mozhgani; Mahdieh Farzanehpour; Farida Behzadian

    Despite the high yearly prevalence of Influenza, the pathogenesis mechanism and involved genes have not been fully known. Finding the patterns and mapping the complex interactions between different genes help us to find the possible biomarkers and treatment targets. Herein, weighted gene co-expression network analysis (WGCNA) was employed to construct a co-expression network among genes identified by microarray analysis of the pediatric influenza-infected samples. Three of the 38 modules were found as the most related modules to influenza infection. At a functional level, we found that the genes in these modules regulate the immune responses, protein targeting, and defense to virus. Moreover, the analysis of differentially expressed genes disclosed 719 DEGs between the normal and infected subjects. The comprehensive investigation of genes in the module involved in immune system and viral defense (yellow module) revealed that SP110, HERC5, SAMD9L, RTP4, C19orf66, HELZ2, EPSTI1, and PHF11 which were also identified as DEGs (except C19orf66) have the potential to be as the biomarkers and also drug targeting for the treatment of pediatric influenza. The WGCN analysis revealed co-expressed genes which were involved in the innate immune system and defense to virus. The differentially expressed genes in the identified modules can be considered for designing drug targets. Moreover, modules can help to find pathogenesis routes in the future.

  • Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection
    Virol. J. (IF 2.464) Pub Date : 2019-10-29
    Chukiat Sirivichayakul; Kriengsak Limkittikul; Pornthep Chanthavanich; Sutee Yoksan; Anuttarasakdi Ratchatatat; Jacqueline Kyungah Lim; Watcharee Arunsodsai; Arunee Sabchareon

    Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand. The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3–11 years in Ratchaburi Province, Thailand. The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects. This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

  • Giant virus vs amoeba: fight for supremacy
    Virol. J. (IF 2.464) Pub Date : 2019-11-04
    Graziele Oliveira; Bernard La Scola; Jônatas Abrahão

    Since the discovery of mimivirus, numerous giant viruses associated with free-living amoebae have been described. The genome of giant viruses can be more than 2.5 megabases, and virus particles can exceed the size of many bacteria. The unexpected characteristics of these viruses have made them intriguing research targets and, as a result, studies focusing on their interactions with their amoeba host have gained increased attention. Studies have shown that giant viruses can establish host–pathogen interactions, which have not been previously demonstrated, including the unprecedented interaction with a new group of small viruses, called virophages, that parasitize their viral factories. In this brief review, we present recent advances in virophage–giant virus–host interactions and highlight selected studies involving interactions between giant viruses and amoebae. These unprecedented interactions involve the giant viruses mimivirus, marseillevirus, tupanviruses and faustovirus, all of which modulate the amoeba environment, affecting both their replication and their spread to new hosts.

  • Important roles of C-terminal residues in degradation of capsid protein of classical swine fever virus
    Virol. J. (IF 2.464) Pub Date : 2019-11-06
    Yuming Chen; Erpeng Zhu; Shuangqi Fan; Hongxing Ding; Shengming Ma; Mengjiao Zhu; Shaofeng Deng; Jinding Chen; Mingqiu Zhao

    Capsid (C) protein plays an important role in the replication of classical swine fever virus (CSFV). The ubiquitin proteasome system (UPS) involves in replication of many viruses via modulation of viral proteins. The relationship of CSFV with UPS is poorly understood and the impact of 26S proteasome on C protein has never been reported before. In this study, fused C protein with an EGFP tag is expressed in PK-15 and 3D4/2 cells. MG132 and 3-methyladenine (3-MA) are used to detect the roles of 26S proteasome and autophagolysosome in expression levels of C protein. Truncated and mutant C proteins are used to find the exact residues responsible for the degradation of C protein. Immunoprecipitaion is performed to find whether C protein is ubiquitinated or not. C-EGFP protein expresses in a cleaved form at a low level and is degraded by 26S proteasome which could be partly inhibited by MG132. C-terminal residues play more important roles in the degradation of C protein than N-terminal residues. Residues 260 to 267, especially M260 and L261, are crucial for the degradation. In addition, C-terminal residues 262 to 267 determine cleavage efficiency of C protein. CSFV C protein is degraded by 26S proteasome in a ubiquitin-independent manner. Last 8 residues at C-terminus of immature C protein play a major role in proteasomal degradation of CSFV C protein and determine the cleavage efficiency of C protein by signal peptide peptidase (SPP). Our findings provide valuable help for fully understanding degradation process of C protein and contribute to fully understanding the role of C protein in CSFV replication.

  • A newly isolated roseophage represents a distinct member of Siphoviridae family
    Virol. J. (IF 2.464) Pub Date : 2019-11-06
    Lanlan Cai; Ruijie Ma; Hong Chen; Yunlan Yang; Nianzhi Jiao; Rui Zhang

    Members of the Roseobacter lineage are a major group of marine heterotrophic bacteria because of their wide distribution, versatile lifestyles and important biogeochemical roles. Bacteriophages, the most abundant biological entities in the ocean, play important roles in shaping their hosts’ population structures and mediating genetic exchange between hosts. However, our knowledge of roseophages (bacteriophages that infect Roseobacter) is far behind that of their host counterparts, partly reflecting the need to isolate and analyze the phages associated with this ecologically important bacterial clade. vB_DshS-R4C (R4C), a novel virulent roseophage that infects Dinoroseobacter shibae DFL12T, was isolated with the double-layer agar method. The phage morphology was visualized with transmission electron microscopy. We characterized R4C in-depth with a genomic analysis and investigated the distribution of the R4C genome in different environments with a metagenomic recruitment analysis. The double-stranded DNA genome of R4C consists of 36,291 bp with a high GC content of 66.75%. It has 49 genes with low DNA and protein homologies to those of other known phages. Morphological and phylogenetic analyses suggested that R4C is a novel member of the family Siphoviridae and is most closely related to phages in the genus Cronusvirus. However, unlike the Cronusvirus phages, R4C encodes an integrase, implying its ability to establish a lysogenic life cycle. A terminal analysis shows that, like that of λ phage, the R4C genome utilize the ‘cohesive ends’ DNA-packaging mechanism. Significantly, homologues of the R4C genes are more prevalent in coastal areas than in the open ocean. Information about this newly discovered phage extends our understanding of bacteriophage diversity, evolution, and their roles in different environments.

  • Patient with severe fever with thrombocytopenia syndrome virus infection and central nervous system disturbance in Dongyang, Zhejiang Province, China, 2017
    Virol. J. (IF 2.464) Pub Date : 2019-11-07
    Yi Sun; Bin Guo; Hao Yan; Ai Lan Wu; Wen Wu Yao; Kang Chen; Jun Hang Pan; Zhao Xia Li; Hai Yan Mao; Yan Jun Zhang

    Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever that was first described in China in 2011. We report a patient who died of Severe fever with thrombocytopenia syndrome virus (SFTSV) infection, with a rapidly progressive central nervous system (CNS) disturbance, in Dongyang, Zhejiang Province, China, in 2017. A 64-year-old man was admitted to hospital after 4 days of fever. SFTSV was detected 1 day after the patient was admitted to hospital. The patient presented with CNS disturbance and died 4 days after admission. Detailed clinical and epidemiological investigations and laboratory tests were conducted. Reduced platelet, white blood cell, lymphocyte, and neutrophil counts, elevated lactate dehydrogenase, creatine kinase, aspartate aminotransferaseand alanine aminotransferase concentrations, and an increased activated partial thromboplastin time were observed. In a phylogenetic analysis, the isolate clustered close to a strain derived from South Korea. Conclusions: This is the first case of SFTSV infection with CNS disturbance in Dongyang, Zhejiang Province, China. The surveillance of suspected cases of SFTS is important in SFTSV endemic regions.

  • Identification of microRNAs regulated by tobacco curly shoot virus co-infection with its betasatellite in Nicotiana benthamiana
    Virol. J. (IF 2.464) Pub Date : 2019-11-07
    Jiang Du; Gentu Wu; Zhongpiao Zhou; Jiayuan Zhang; Mingjun Li; Miao Sun; Kairong Jiang; Ling Qing

    MicroRNAs (miRNAs) are a class of 21–24 nucleotide endogenous non-coding small RNAs that play important roles in plant development and defense responses to biotic and abiotic stresses. Tobacco curly shoot virus (TbCSV) is a monopartite begomovirus, cause leaf curling and plant stunting symptoms in many Solanaceae plants. The betasatellite of TbCSV (TbCSB) induces more severe symptoms and enhances virus accumulation when co-infect the plants with TbCSV. In this study, miRNAs regulated by TbCSV and TbCSB co-infection in Nicotiana benthamiana were characterized using high-throughput sequencing technology. Small RNA sequencing analysis revealed that a total of 13 known miRNAs and 42 novel miRNAs were differentially expressed in TbCSV and TbCSB co-infected N. benthamiana plants. Several potential miRNA-targeted genes were identified through data mining and were involved in both catalytic and metabolic processes, in addition to plant defense mechanisms against virus infections according to Gene Ontology (GO) analyses. In addition, the expressions of several differentially expressed miRNAs and their miRNA-targeted gene were validated through quantitative real time polymerase chain reaction (qRT-PCR) approach. A large number of miRNAs are identified, and their target genes, functional annotations also have been explored. Our results provide the information on N. benthamiana miRNAs and would be useful to further understand miRNA regulatory mechanisms after TbCSV and TbCSB co-infection.

  • Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman
    Virol. J. (IF 2.464) Pub Date : 2019-11-09
    Muhammad Shafiq Shahid; Muhammad Shafiq; Amir Raza; Abdullah M. Al-Sadi; Rob W. Briddon

    In Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection. Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation. The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites. The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

  • Whole-genome sequencing of human Pegivirus variant from an Egyptian patient co-infected with hepatitis C virus: a case report
    Virol. J. (IF 2.464) Pub Date : 2019-11-11
    Hany K. Soliman; Mohamed Abouelhoda; Mahmoud N. El Rouby; Ola S. Ahmed; G. Esmat; Zeinab K. Hassan; Mohammed M. Hafez; Dina Ahmed Mehaney; Manojkumar Selvaraju; Rania Kamal Darwish; Yehia A. Osman; Abdel-Rahman N. Zekri

    Human pegivirus (HPgV) is structurally similar to hepatitis C virus (HCV) and was discovered 20 years ago. Its distribution, natural history and exact rule of this viral group in human hosts remain unclear. Our aim was to determine, by deep next-generation sequencing (NGS), the entire genome sequence of HPgV that was discovered in an Egyptian patient while analyzing HCV sequence from the same patient. We also inspected whether the co-infection of HCV and HPgV will affect the patient response to HCV viral treatment. To the best of our knowledge, this is the first report for a newly isolated HPgV in an Egyptian patient who is co-infected with HCV. The deep Next Generation Sequencing (NGS) technique was used to detect HCV sequence in hepatitis C patient’s plasma. The results revealed the presence of HPgV with HCV. This co-infection was confirmed using conventional PCR of the HPgV 5′ untranslated region. The patient was then subjected to direct-acting-antiviral treatment (DAA). At the end of the treatment, the patient showed a good response to the HCV treatment (i.e., no HCV-RNA was detected in the plasma), while the HPgV-RNA was still detected. Sequence alignment and phylogenetic analyses demonstrated that the detected HPgV was a novel isolate and was not previously published. We report a new variant of HPgV in a patient suffering from hepatitis C viral infection.

  • Association of N6-methyladenosine with viruses and related diseases
    Virol. J. (IF 2.464) Pub Date : 2019-11-11
    Fang Wu; Wenzhao Cheng; Feiyuan Zhao; Mingqing Tang; Yong Diao; Ruian Xu

    N6-methyladenosine (m6A) modification is the most prevalent internal modification of eukaryotic mRNA modulating gene expression. m6A modification is a dynamic reversible process regulated by three protein groups: methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers). m6A modification is involved in all phases of RNA metabolism, including RNA folding, stability, splicing, nuclear exporting, translational modulation and degradation. In recent years, numerous studies have reported that abnormal m6A modification causes aberrant expression of important viral genes. Herein, we review the role of m6A in viral lifecycle and its contribution to the pathogenesis of human diseases. Particularly, we focus on the viruses associated with human diseases such as HIV-1, IAV, HBV, HCV, EBV and many others. A better understanding of m6A-virus relationship would provide new insights into the viral replication process and pathogenesis of human diseases caused by viruses. In addition, exploration of the role of m6A in disease-causing viruses will reveal novel approaches for the treatment of such diseases.

  • Heparin sulfate is the attachment factor of duck Tembus virus on both BHK21 and DEF cells
    Virol. J. (IF 2.464) Pub Date : 2019-11-12
    Shaoxiong Wu; Zhen Wu; Yuanyuan Wu; Tao Wang; Mingshu Wang; Renyong Jia; Dekang Zhu; Mafeng Liu; Xinxin Zhao; Qiao Yang; Ying Wu; Shaqiu Zhang; Yunya Liu; Ling Zhang; Yanling Yu; Leichang Pan; Shun Chen; Anchun Cheng

    Duck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure. In this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8. We found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step. Our results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV.

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