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50 Pág., In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratifcation vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV, This work was supported in part through Laulima Govern ment Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Gov ernment Solutions, LLC under Contract No. HHSN272201800013C. This work was also funded in part by Contract No. HSHQDC 15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges support from the Mississippi Agri cultural and Forestry Experiment Station (MAFES), USDA-ARS pro ject 58-6066-9-033 and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project, under Accession Num ber 1021494.

PURPOSE The aim of the present work was to investigate the role of apoptosis inhibitor BIRC6 (baculoviral IAP repeat-containing protein 6) in breast cancer (BC), focusing particularly on its involvement in the metastatic cascade. METHODS We analyzed BIRC6 mRNA expression levels and copy number variations in three BC databases from The Cancer Genome Atlas comparing clinical and molecular attributes. Genomic analysis was performed using the cBioPortal platform, whereas transcriptomic studies (mRNA expression levels, correlation heatmaps, survival plots, and gene ontology) were performed using USC Xena and R. Statistical significance was set at P < .05. RESULTS Our bioinformatic analyses showed that there was a differential expression of BIRC6 in cancer samples when compared with normal samples. Copy number variations that involve amplification and gain of BIRC6 gene were correlated with negative hormone receptor tumors, higher prognostic indexes, younger age at diagnosis, and both chemotherapy and radiotherapy administration. Transcriptomic and gene ontology analyses showed that, under conditions of high BIRC6 mRNA levels, there are differential expression patterns in apoptotic, proliferation, and metastatic pathways. CONCLUSION In summary, our in silico data suggest that BIRC6 plays an antiapoptotic, pro-proliferative, and apparent prometastatic role and could be a relevant molecular target for treatment of BC tumors.

Humanin (HN) is a mitochondrial-derived peptide with cytoprotective efect in many tissues. Administration of HN analogs has been proposed as therapeutic approach for degenerative diseases. Although HN has been shown to protect normal tissues from chemotherapy, its role in tumor pathogenesis is poorly understood. Here, we evaluated the efect of HN on the progression of experimental triple negative breast cancer (TNBC). The meta-analysis of transcriptomic data from The Cancer Genome Atlas indicated that HN and its receptors are expressed in breast cancer specimens. By immunohistochemistry we observed up-regulation of HN in TNBC biopsies when compared to mammary gland sections from healthy donors. Addition of exogenous HN protected TNBC cells from apoptotic stimuli whereas shRNA-mediated HN silencing reduced their viability and enhanced their chemo-sensitivity. Systemic administration of HN in TNBC-bearing mice reduced tumor apoptotic rate, impaired the antitumor and anti-metastatic efect of chemotherapy and stimulated tumor progression, accelerating tumor growth and development of spontaneous lung metastases. These fndings suggest that HN may exert pro-tumoral efects and thus, caution should be taken when using exogenous HN to treat degenerative diseases. In addition, our study suggests that HN blockade could constitute a therapeutic strategy to improve the efcacy of chemotherapy in breast cancer., Instituto de Biotecnologia y Biologia Molecular

The New World arenavirus Junin (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF). Previous studies of human macrophage infection by the Old-World arenaviruses Mopeia and Lassa showed that while the non-pathogenic Mopeia virus replicates and activates human macrophages, the pathogenic Lassa virus replicates but fails to activate human macrophages. Less is known in regard to the impact of New World arenavirus infection on the human macrophage immune response. Macrophage activation is critical for controlling infections but could also be usurped favoring immune evasion. Therefore, it is crucial to understand how the JUNV infection modulates macrophage plasticity to clarify its role in AHF pathogenesis. With this aim in mind, we compared infection with the attenuated Candid 1 (C#1) or the pathogenic P strains of the JUNV virus in human macrophage cultures. The results showed that both JUNV strains similarly replicated and induced morphological changes as early as 1 day post-infection. However, both strains differentially induced the expression of CD71, the receptor for cell entry, the activation and maturation molecules CD80, CD86, and HLA-DR and selectively modulated cytokine production. Higher levels of TNF-α, IL-10, and IL-12 were detected with C#1 strain, while the P strain induced only higher levels of IL-6. We also found that C#1 strain infection skewed macrophage polarization to M1, whereas the P strain shifted the response to an M2 phenotype. Interestingly, the MERTK receptor, that negatively regulates the immune response, was down-regulated by C#1 strain and up-regulated by P strain infection. Similarly, the target genes of MERTK activation, the cytokine suppressors SOCS1 and SOCS3, were also increased after P strain infection, in addition to IRF-1, that regulates type I IFN levels, which were higher with C#1 compared with P strain infection. Together, this differential activation/polarization pattern of macrophages elicited by P strain suggests a more evasive immune response and may have important implications in the pathogenesis of AHF and underpinning the development of new potential therapeutic strategies, Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular, Centro de Investigación y Desarrollo en Fermentaciones Industriales

Members of the family Arenaviridae produce enveloped virions containing genomes consisting of two or three single-stranded RNA segments totalling about 10.5 kb. Arenaviruses can infect mammals, including humans and other primates, snakes, and fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae.

Autophagy, a highly regulated degradative process that promotes cellular homeostasis, is increasingly recognised as a fundamental component of the cellular response against viral infection. In this study, we investigated the role of autophagy during Junı´n virus (JUNV) multiplication using human A549 cells. We found that JUNV infection induces an increment of the LC3-II/LC3-I ratio, an accumulation of punctate pattern in RFP-LC3-transfected cells and the colocalisation of viral nucleoprotein and LC3 protein, suggesting autophagosome formation. JUNV infection also induced the degradation of the autophagy receptor p62, suggesting that complete autophagic flux was triggered. In addition, we showed that inhibition of autophagy with bafilomycin A1 or 3-methyladenine significantly reduces viral multiplication. Moreover, viral yield was increased when autophagy was induced using rapamycin. Furthermore, JUNV infection induced the colocalisation of p62, ATG16, RAB5, RAB7A and LAMP1 with the autophagosomal LC3 protein. That suggests that phagosomes undergo the maturation process during viral infection. Finally, we demonstrated that siRNA experiments targeting essential autophagy genes (ATG5, ATG7 and Beclin 1) reduce viral protein synthesis and viral yield. Overall, our results indicate that JUNV activates host autophagy machinery enhancing its multiplication., Instituto de Biotecnologia y Biologia Molecular

This work was supported in part through Battelle Memorial Institute?s prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I (J.H.K.). This work was also funded in part by National Institutes of Health (NIH) contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (R.B.T.) Rega Institute. Infectious Diseases unit. Leuven, Leuven, Belgium. United States Department of Agriculture. Agricultural Research Service. US Horticultural Research Laboratory. Fort Pierce, FL, USA. Ministry of Health of the Russian Federation. N. F. Gamaleya Federal Research Center for Epidemiology and Microbiology. D. I. Ivanovsky Institute of Virology. Moscow, Russia. University of Ljubljana. Ljubljana Faculty of Medicine. Ljubljana, Slovenia. Mississippi State University. Department of Biological Sciences. Mississippi State, MS, USA. University of Texas Medical Branch. Galveston, TX, USA. Institute of Diagnostic Virology. Friedrich-Loefer-Institut. Greifswald-Insel Riems, Germany. Centers for Disease Control and Prevention. Division of High-Consequence Pathogens and Pathology. Viral Special Pathogens Branch. Atlanta, GA, USA. Colorado State University. Department of Microbiology. Immunology & Pathology, Arthropod-borne and Infectious Diseases Laboratory. Fort Collins, CO, USA. Columbia University. Center for Infection and Immunity. Department of Epidemiology, Mailman School of Public Health. New York, NY, USA. University of California. Department of Molecular Biology and Biochemistry. Irvine, CA, USA. National Health Laboratory Service. Division of Virology. Bloemfontein. Republic of South Africa / University of the Free State. Division of Virology. Bloemfontein, Republic of South Africa. Colorado State University. Department of Microbiology. Immunology & Pathology, Arthropod-borne and Infectious Diseases Laboratory. Fort Collins, CO, USA. Unit? des Virus Emergents (Aix-Marseille Univ?IRD 190? Inserm 1207?IHU M?diterran?e Infection). Marseille, France. International Rice Research Institute. Plant Breeding Genetics and Biotechnology Division. Los Ba?os, Philippines. Les MandinauxLe Grand Madieu. France. The Scripps Research Institute. Department of Immunology and Microbiology IMM-6. La Jolla, USA. Unit? des Virus Emergents (Aix-Marseille Univ?IRD 190?Inserm 1207?IHU M?diterran?e Infection). Marseille, France. University of California. Department of Medicine. San Francisco, USA / University of California. Department of Biochemistry and Biophysics. San Francisco, USA / University of California. Department of Microbiology. San Francisco, USA. Istituto Agronomico Mediterraneo di Bari. Valenzano, Italy. Public Health Agency of Canada. National Microbiology Laboratory. Zoonotic Diseases and Special Pathogens. Winnipeg, Canada. Mayo Clinic. Department of Molecular Medicine. Rochester, USA. Istituto Agronomico Mediterraneo di Bari. Valenzano, Italy. Hacettepe University. Faculty of Medicine. Department of Medical Microbiology. Virology Unit. Ankara, Turkey. University of Texas Medical Branch. Galveston, TX, USA. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. Chinese Center for Disease Control and Prevention. National Institute for Viral Disease Control and Prevention. Beijing, China. Kansas State University. Center of Excellence for Emerging and Zoonotic Animal Disease. Manhattan, USA. Chinese Center for Disease Control and Prevention. National Institute for Communicable Disease Control and Prevention. Beijing, China / Fudan University. Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences. Shanghai, China. WHO Collaborating Centre for Arboviruses and Hemorrhagic Fever Reference and Research. Bernhard-Nocht Institute for Tropical Medicine. Department of Virology. Hamburg, Germany. CNRS- Paris-Diderot. Institut Jacques Monod. Paris, France. The University of Queensland. School of Chemistry and Molecular Biosciences. Australian Infectious Diseases Research Centre. Brisbane, Australia. Public Health England. Salisbury, UK. Centers for Disease Control and Prevention. Fort Collins, USA. Indian Agricultural Research Institute. Division of Plant Pathology. New Delhi, India. Seoul National University. College of Agriculture and Life Sciences. Department of Agricultural Biotechnology, Center for Fungal Pathogenesis. Seoul, Korea. Humboldt-University Berlin, and Berlin Institute of Health. corporate member of Free University Berlin. Institute of Virology. Charit?-Universit?tsmedizin Berlin. Berlin, Germany / German Centre for Infection Research. Berlin, Germany. Humboldt-University Berlin, and Berlin Institute of Health. corporate member of Free University Berlin. Institute of Virology. Charit?-Universit?tsmedizin Berlin. Berlin, Germany / Slovak Academy of Sciences. Biomedical Research Center. Bratislava, Slovakia. Karolinska University Hospital. Center for Infectious Medicine, Karolinska Institutet. Department of Medicine Huddinge. Stockholm, Sweden. Wageningen University. Department of Plant Sciences. Laboratory of Virology. Wageningen, The Netherlands. Centers for Disease Control and Prevention. Fort Collins, USA. University of Washington. Department of Microbiology. Washington, USA. University of Louisville. School of Medicine. The Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases. Department of Pharmacology and Toxicology. Louisville, USA. Humboldt-University Berlin, and Berlin Institute of Health. corporate member of Free University Berlin. Institute of Virology. Charit?-Universit?tsmedizin Berlin. Berlin, Germany / German Centre for Infection Research. Berlin, Germany. University of Bari Aldo Moro. Department of Plant, Soil and Food Sciences. Bari, Italy. University of Hamburg. Biocentre Klein Flottbek. Hamburg, Germany. Folkhalsomyndigheten. Stockholm, Sweden. University of Hamburg. Biocentre Klein Flottbek. Hamburg, Germany. Washington State University. Irrigated Agricultural Research and Extension Center. Department of Plant Pathology. Prosser, USA. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Centro de Inova??es Tecnol?gicas. Ananindeua, PA, Brasil. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. Aristotle University of Thessaloniki. National Reference Centre for Arboviruses and Haemorrhagic Fever Viruses. Department of Microbiology, Medical School. Thessaloniki, Greece. National Health Laboratory Service. National Institute for Communicable Diseases. Centre for Emerging Zoonotic and Parasitic Diseases. Sandringham, South Africa / University of Pretoria. Centre for Viral Zoonoses, Faculty of Health Sciences. Department of Medical Virology. Pretoria South Africa. University of Texas Medical Branch. Galveston, TX, USA. University of Helsinki. Department of Virology. Medicum, Helsinki, Finland. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. Universidade de Bras?lia. Departamento de Biologia Celular. Bras?lia , DF, Brazil. Universidad Nacional de La Plata - Consejo Nacional de Investigaciones Cient?ficas y T?cnicas. Centro Cientifico Technol?gico-La Plata. Instituto de Biotecnolog?a y Biolog?a Molecular. La Plata, Argentina. Institut Pasteur de Dakar. Dakar, Senegal. University of Maryland School of Medicine. Institute of Human Virology. Baltimore, USA. National Agriculture and Food Research Organization. Department of Planning and Coordination. Tsukuba, Japan. United States Army Medical Research Institute of Infectious Diseases. Fort Detrick, Frederick, USA. MRC-University of Glasgow Centre for Virus Research. Glasgow, UK. University of Tokyo. Asian Center for Bioresources and Environmental Sciences. Tokyo, Japan. University of Oxford. Department of Medicine. Oxford, UK. Bioinformatics Scientific Institute IRCCS E. MEDEA. Bosisio Parini, Italy. Korea University. College of Medicine. Department of Microbiology. Seoul. Republic of Korea. Centers for Disease Control and Prevention. Division of High-Consequence Pathogens and Pathology. Viral Special Pathogens Branch. Atlanta, GA, USA. Colorado State University. Immunology and Pathology. Department of Microbiology. Fort Collins, USA. University of Texas Medical Branch. Galveston, TX, USA. CNR. Institute for Sustainable Plant Protection. Torino, Italy. Fujian Agriculture and Forestry University. Institute of Plant Virology. Fujian Province Key Laboratory of Plant Virology. Fuzhou, China. North Carolina State University. Department of Entomology and Plant Pathology. Raleigh, USA. National Chung Hsing University. Department of Plant Pathology. Taichung, Taiwan. Universidade Federal de Vi?osa. Departamento de Fitopatologia/BIOAGRO. Vi?osa, MG, Brazil. Chinese Center for Disease Control and Prevention. National Institute for Communicable Disease Control and Prevention. Beijing, China / Fudan University. Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences. Shanghai, China. Chinese Academy of Agricultural Sciences. Institute of Plant Protection. State Key Laboratory for Biology of Plant Diseases and Insect Pests. Beijing, China. National Institutes of Health (NIH). National Institute of Allergy and Infectious Diseases (NIAID). Division of Clinical Research (DCR). Integrated Research Facility at Fort Detrick (IRF-Frederick). Frederick, USA. In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Pituitary tumors are the most common primary intracranial neoplasms. Humanin (HN) and Rattin (HNr), a rat homolog of HN, are short peptides with a cytoprotective action. In the present study, we aimed to evaluate whether endogenous HNr plays an antiapoptotic role in pituitary tumor cells. Thus, we used RNA interference based on short-hairpin RNA (shRNA) targeted to HNr (shHNr). A plasmid including the coding sequences for shHNr and dTomato fluorescent reporter gene was developed (pUC-shHNr). Transfection of somatolactotrope GH3 cells with pUC-shHNr increased apoptosis, suggesting that endogenous HNr plays a cytoprotective role in pituitary tumor cells. In order to evaluate the effect of blockade of endogenous HNr expression in vivo, we constructed a recombinant baculovirus (BV) encoding shHNr (BV-shHNr). In vitro, BV-shRNA was capable of transducing more than 80% of GH3 cells and decreased HNr mRNA. Also, BV-shHNr increased apoptosis in transduced GH3 cells. Intratumor injection of BV-shHNr to nude mice bearing s.c. GH3 tumors increased the number of apoptotic cells, delayed tumor growth and enhanced survival rate, suggesting that endogenous HNr may be involved in pituitary tumor progression. These preclinical data suggests that the silencing of HN expression could have a therapeutic impact on the treatment of pituitary tumors., Instituto de Biotecnología y Biología Molecular

The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9–11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals., Instituto de Biotecnologia y Biologia Molecular

The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23–25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines., Highlights • The GVN is an international research network comprised of 38 Centers of Excellence and 6 Affiliates in 24 countries. • The 2016 Global Virus Network (GVN) Meeting was held in Sapporo, Japan from October 23–25. • New data were presented on all various aspects of medical virology, therapy, and vaccines were presented. • International collaboration is needed to develop effective viral vaccines and therapeutics. • The 2017 international GVN meeting will be held on September 25–28 in Melbourne, Australia.

Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzyme-linked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses., Instituto de Biotecnologia y Biologia Molecular

The arenavirus family contains several important human pathogens including Lassa fever virus (LASV), lymphocytic choriomeningitis virus (LCMV) and the New World clade B viruses Junin (JUNV) and Machupo (MACV). Previously, α-dystroglycan (α-DG) was identified as a receptor recognized by LASV and certain strains of LCMV. However, other studies have suggested that α-DG is probably not used by the clade B viruses, and the receptor(s) for these pathogens is currently unknown. Using pseudotyped retroviral vectors displaying arenavirus glycoproteins (GPs), we are able to explore the role played by the GP in viral entry in the absence of other viral proteins. By examining the ability of the vectors to transduce DG knockout murine embryonic stem (ES) cells, we have confirmed that LASV has an absolute requirement for α-DG in these cells. However, the LCMV GP can still direct substantial entry into murine ES cells in the absence of α-DG, even when the GP from the clone 13 variant is used that has previously been reported to be highly dependent on α-DG for entry. We also found that neither LASV or LCMV pseudotyped vectors were able to transduce human or murine lymphocytes, presumably due to the glycosylation state of α-DG in these cells. In contrast, the JUNV and MACV GPs displayed broad tropism on human, murine and avian cell types, including lymphocytes, and showed no requirement for α-DG in murine ES cells. These findings highlight the importance of molecules other than α-DG for arenavirus entry. An alternate receptor is present on murine ES cells that can be used by LCMV but not by LASV, and which is not available on human or murine lymphocytes, while a distinct and widely expressed receptor(s) is used by the clade B viruses., Facultad de Ciencias Exactas

The G2 fusion subunit of the Junín virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that α-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever., Instituto de Biotecnologia y Biologia Molecular

Significant progress has been made in the past 10 years in unraveling the molecular biology of highly pathogenic arenaviruses that are endemic in several West African countries (Lassa fever virus) and in some regions of South America (Argentine and Bolivian hemorrhagic fever viruses). While this has resulted in proof-of-concept studies of novel vaccine candidates in non-human primates and in the discovery of several novel antiviral small molecule drug candidates, none of them has been tested in the clinic to date. The recent Ebola outbreak in West Africa has demonstrated very clearly that there is an urgent need to develop the prophylactic and therapeutic armamentarium against viral hemorrhagic fever viruses as part of a global preparedness for future epidemics. As it pertains to this goal, the present article summarizes the current knowledge of highly pathogenic arenaviruses and identifies opportunities for translational research. Fil: Kerber, R.. Bernhard Nocht Institute for Tropical Medicine; Alemania Fil: Reindl, S.. Bernhard Nocht Institute for Tropical Medicine; Alemania Fil: Romanowski, Victor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Gomez, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina Fil: Ogbaini Emovon, E.. Irrua Specialist Teaching Hospital; Nigeria Fil: Günther, S.. Bernhard Nocht Institute for Tropical Medicine; Alemania Fil: ter Meulen, J.. Philipps University Marburg; Alemania

We report a fast and simple DNA isolation method from whole blood. It avoids cell separation and lysis steps and consists of three successive solvent extractions and an ethanol precipitation. All the steps are carried out at room temperature. The main advantage of this method is the immediate sample inactivation achieved by mixing the blood sample with Tris-HCl (pH 8.0) saturated phenol, thus minimizing the biohazard involved in the subsequent manipulation of the samples potentially contaminated with infectious agents (the procedure has been called SP for 'straight phenol'). In addition, extensive field sample collections are facilitated by the fact that the SP procedure can be stopped right after the simple manipulation of mixing the blood sample with the phenol; neither freezing nor refrigeration of the sample proved to be required. At this stage, the nucleases as well as infectious agent are inactivated and the rest of the protocol can wait to be carried out in the laboratory. In fact, the DNA preparation can be resumed after prolonged storage of the blood-phenol mix (up to 72 days has been checked in our laboratory) at room temperature without affecting the yield. The SP protocol may be scaled up, when large quantities of DNA are needed, or scaled down to smaller volumes, such as fingerprick blood samples.

To understand better how different genomic regions may confer pathogenicity for the coxsackievirus B (CVB), two intratypic CVB1 variants and a number of recombinant viruses were studied. Sequencing analysis showed 23 nucleotide changes between the parental non-pathogenic CVB1N and the pathogenic CVB1Nm. Mutations present in CVB1Nm were more conserved than those in CVB1N when compared to other CVB sequences. Inoculation in C3H/HeJ mice showed that the P1 region is critical for pathogenicity in murine pancreas and heart. The molecular determinants of disease for these organs partially overlap. Several P1 region amino acid differences appear to be located in the decay accelerating factor (DAF) footprint CVBs. CVB1N and CVB1Nm interacted with human CAR, but only CVB1N seemed to interact with human DAF, as determined using soluble receptors in a plaque reduction assay. However, the murine homologue Daf-1 did not interact with any virus assessed by haemagglutination. The results of this study suggest that an unknown receptor interaction with the virus play an important role in the pathogenicity of CVB1Nm. Further in vivo studies may clarify this issue., Instituto de Biotecnología y Biología Molecular

Argentine hemorrhagic fever (AHF) is an endemoepidemic disease with cardiovascular, renal and neurologic alterations acquired in the richest farming land in Argentina. It is caused by Junin virus, one of the few human pathogenic arenaviruses. The S RNA of Junin virus has been molecularly cloned and its nucleotide sequence determined in our laboratory. This information was used to develop a rapid nucleic acid-based diagnostic test commensurate with the low viraemia detected in AHF patients. Junin virus-specific cDNA probes labeled using various methods proved insensitive in dot-hybridizations. Therefore, a RT polymerase chain reaction (PCR) was developed using a pair of oligonucleotide primers to reverse-transcribe and amplify the viral S RNA. The amplification of the target sequences was measured by ethidium bromide staining of the DNA fragments after agarose gel electrophoresis. This type of assay allowed the specific detection of Junin virus RNA sequences present in a single infected BHK21 cell over a background of 10 4 uninfected cells. Control reactions were performed on RNA samples extracted from uninfected cells or cells infected with a high multiplicity of LCMV, another arenavirus present in the AHF endemic area. The PCR was first adapted to detect viral RNA in peripheral blood mononuclear cells, described to harbor most of the virus. A simplification of this assay allows the detection of Junin virus in RNA extracted from 100 μl of whole blood using guanidium thiocyanate disruption and acid phenol extraction. Under the conditions described in this paper, it is possible to detect up to 0.01 pfu of Junin virus in a blood sample. An early and rapid laboratory diagnostic test for AHF is important since the only effective therapy that reduces the mortality rate from 30% to less than 1% consists of early treatment with immune plasma.

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus (JUNV), a member of the arenaviridae family. Although a recently introduced live attenuated vaccine has proven to be effective, AHF remains a potentially lethal infection. Like in other viral hemorrhagic fevers (VHF), AHF patients present with fever and hemorrhagic complications. Although the causes of the bleeding are poorly understood, impaired hemostasis, endothelial cell dysfunction and low platelet counts have been described. Thrombocytopenia is a common feature in VHF syndromes, and it is a major sign for its diagnosis. However, the underlying pathogenic mechanism has not yet been elucidated. We hypothesized that thrombocytopenia results from a viral-triggered alteration of the megakaryo/thrombopoiesis process. Therefore, we evaluated the impact of JUNV on megakaryopoiesis using an in vitro model of human CD34+ cells stimulated with thrombopoietin. Our results showed that CD34+ cells are infected with JUNV in a restricted fashion. Infection was transferrin receptor 1 (TfR1)-dependent and the surface expression of TfR1 was higher in infected cultures, suggesting a novel arenaviral dissemination strategy in hematopoietic progenitor cells. Although proliferation, survival, and commitment in JUNV-infected cultures were normal, viral infection impaired thrombopoiesis by decreasing in vitro proplatelet formation, platelet release, and P-selectin externalization via a bystander effect. The decrease in platelet release was also TfR1-dependent, mimicked by poly(I:C), and type I interferon (IFN α/ß) was implicated as a key paracrine mediator. Among the relevant molecules studied, only the transcription factor NF-E2 showed a moderate decrease in expression in megakaryocytes from either infected cultures or after type I IFN treatment. Moreover, type I IFN-treated megakaryocytes presented ultrastructural abnormalities resembling the reported thrombocytopenic NF-E2-/- mouse phenotype. Our study introduces a potential mechanism for thrombocytopenia in VHF and other diseases associated with increased bone marrow type I IFN levels., Facultad de Ciencias Exactas

Junin virus (JUNV), Machupo virus, Guanarito virus, Sabia virus, and Chapare virus are members of New World arenavirus clade B and are the etiological agents of viral hemorrhagic fevers that occur in South America. In this study, we produced three monoclonal antibodies (MAbs) to the recombinant nucleocapsid protein of JUNV, designated C6-9, C11-12, and E4-2. The specificity of these MAbs was examined by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, and an epitope-mapping method. Using these MAbs, we developed antigen (Ag) capture ELISA systems. We showed that by using MAb C6-9, JUNV Ag was specifically detected. On the other hand, by using MAb C11-12 or E-4-2, the Ags of all human pathogenic South American arenaviruses were detected. The combined use of these Ag capture ELISA systems in the present study may be useful for the diagnosis of acute-phase viral hemorrhagic fever due to infection by a South American arenavirus., Instituto de Biotecnologia y Biologia Molecular

Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper, we report the nucleotide sequences of L RNA of Candid #1 and examine the relationship to its more virulent ancestors Junin virus XJ#44 and XJ 13 (prototype) and other closely and distantly related arenaviruses. Comparisons of the nucleotide and amino acid sequences of L and Z genes of Candid #1 and its progenitor strains revealed twelve point mutations in the L polypeptide that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype. In contrast, Z ORF was completely conserved among all strains., Instituto de Biotecnología y Biología Molecular

A live attenuated virus vaccine has been recently developed to prevent Argentine hemorrhagic fever. In this paper, we report the nucleotide sequence of the glycoprotein precursor gene (GPC) of the Junin virus vaccine strain (Candid #1) and its flanking untranslated regions. The untranslated regions flanking the GPC genes of different arenaviruses are variable in length, sequence, and secondary structure. However, when this highly attenuated Junin virus strain is compared with the MC2 strain, which is of intermediate virulence, one nucleotide insertion and four nucleotide substitutions are found at positions that do not affect the predicted secondary structure. When Candid #1 and MC2 RNAs are compared, the nucleotide sequence changes in the GPC open reading frame are concentrated in the amino-proximal and the carboxy-proximal regions. The comparison of the amino acid residues shows that the major changes are located in the amino-proximal region of the GPC.

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future., La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular