Search

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 short peptide involved in many biological processes such as apoptosis, cell survival, inflammatory response, and reaction to stressors like oxidative stress, between others. In the ovary, a correct balance between pro- and anti-apoptotic factors is crucial for folliculogenesis. In the follicular atresia, survival or death of granulosa cells is a critical process. The goal of this study was to evaluate the action of HN on granulosa cell fate. To explore endogenous HN function in the ovary, we used a recombinant baculovirus (BV) encoding a short-hairpin RNA targeted to silence HN (shHN). HN downregulation modified ovarian histoarchitecture and increased apoptosis of granulosa cells. HN was also detected in a granulosa tumor cell line (KGN). Transduction of KGN cells with BV-shHN resulted in HN downregulation and increased apoptosis. On the other hand, treatment of KGN cells with exogenous HN increased cell viability and decreased apoptosis. In summary, these findings indicate that HN is a cytoprotective factor in granulosa cells of antral follicles, suggesting that this peptide would be involved in the regulation of folliculogenesis. Also, this peptide is a cytoprotective factor in KGN cells, and therefore, it could be involved in granulosa tumor cell behavior.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Agrarias y Forestales, Centro de Investigaciones en Fitopatología, Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

Breast cancer is the most common cancer as well as the first cause of death by cancer in women worldwide. BIRC6 (baculoviral IAP repeat-containing protein 6) is a member of the inhibitors of apoptosis protein family thought to play an important role in the progression or chemoresistance of many cancers. The aim of the present work was to investigate the role of apoptosis inhibitor BIRC6 in breast cancer, focusing particularly on its involvement in the metastatic cascade. We analyzed BIRC6 mRNA expression levels and Copy Number Variations (CNV) in three breast cancer databases from The Cancer Genome Atlas (TCGA) comparing clinical and molecular attributes. Genomic analysis was performed using CBioportal platform while transcriptomic studies (mRNA expression levels, correlation heatmaps, survival plots and Gene Ontology) were performed with USC Xena and R. Statistical significance was set at p-values less than 0.05. Our analyses showed that there was a differential expression of BIRC6 in cancer samples when compared to normal samples. CNV 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, in conditions of high BIRC6 mRNA levels, there are differential expression patterns in apoptotic, proliferation, and metastatic pathways. In summary, our in silico analyses suggest that BIRC6 exhibits an antiapoptotic, pro-proliferative and an apparent pro-metastatic role and could be a relevant molecular target for treatment of Breast Cancer tumors., Instituto de Biotecnologia y Biologia Molecular

Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

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

Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) represents a strong candidate to develop environmental-friendly pesticides against the fall armyworm (Spodoptera frugiperda), a widespread pest that poses a severe threat to different crops around the world. However, little is known regarding the genomic diversity present inside SfMNPV isolates and how it shapes the interactions between virus and host. Here, the genomic diversity present inside an isolate of SfMNPV was explored using high-throughput sequencing for the first time. We identified 704 intrahost single nucleotide variants, from which 184 are nonsynonymous mutations distributed among 82 different coding sequences. We detected several structural variants affecting SfMNPV genome, including two previously reported deletions inside the egt region. A comparative analysis between polymorphisms present in different SfMNPV isolates and our intraisolate diversity data suggests that coding regions with higher genetic diversity are associated with oral infectivity or unknown functions. In this context, through molecular evolution studies we provide evidence of diversifying selection acting on sf29, a putative collagenase which could contribute to the oral infectivity of SfMNPV. Overall, our results contribute to deepen our understanding of the coevolution between SfMNPV and the fall armyworm and will be useful to improve the applicability of this virus as a biological control agent., Instituto de Biotecnologia y Biologia Molecular

The fall armyworm, Spodoptera frugiperda (JE Smith) is a key pest in the Americas. Control strategies are mainly carried out by use of chemical insecticides and transgenic crops expressing Bacillus thuringiensis toxins. In the last years, resistance of S. frugiperda populations to transgenic corn was reported in different Latin American countries. The baculovirus Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) is a pathogenic agent for the fall armyworm and a potential alternative for its control in integrated pest management strategies. In this work, we analyze some characteristics of two baculovirus isolates collected from maize (SfMNPV-M) and cotton (SfMNPV-C) fields from Argentina. The isolates were compared by restriction enzymes patterns and the analysis reveals the presence of genotypic variants in the SfMNPV-M isolate. We confirmed a deletion by sequencing fragments encompassing egt gene and most part of its contiguous gene (orf A) in a SfMNVP-M genotypic variant. Additionally, we estimated the 50% lethal dose and median survival time of each isolate in bioassays with S. frugiperda larvae., Instituto de Biotecnología y Biología Molecular

Baculoviruses are arthropod-specific, enveloped viruses with circular, supercoiled double-stranded deoxyribonucleic acid genomes. While many viruses are studied to seek solutions for their adverse impact on human, veterinary, and plant health, the study of baculoviruses was stimulated initially by their potential utility to control insect pests. Later, the utility of baculovirus as gene expression vectors was evidenced leading to numerous applications. Several strategies are employed to obtain recombinant viruses that express large quantities of heterologous proteins. A major step forward was the development of bacmid technology (the construction of bacterial artificial chromosomes containing the genome of the baculovirus) which allows the manipulation of the baculovirus genome in bacteria. With this technology, foreign genes can be introduced into the bacmid by homologous and site-directed recombination or by transposition. Baculoviruses have been used to explore fundamental questions in molecular biology such as the nature of programmed cell-death. Moreover, the ability of baculoviruses to transduce mammalian cells led to the consideration of their use as gene-therapy and vaccine vectors. Strategies for genetic engineering of baculoviruses have been developed to meet the requirements of new application areas. Display of foreign proteins on the surface of virions or in nucleocapsid structures, the assembly of expressed proteins to form virus-like particles or protein complexes have been explored and validated as vaccines. The aim of this chapter is to update the areas of application of the baculoviruses in protein expression, alternative vaccine designs and gene therapy of infectious diseases and genetic disorders. Finally, we review the baculovirus-derived products on the market and in the pipeline for biomedical and veterinary use.

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.

MicroRNAs are regulatory RNAs that are scarcely described in Baculoviruses. In this work we predicted a microRNA in silico, denominated agmnpv-miR-4, encoded in the genome of Anticarsia gemmatalis Nucleopolyhedrovirus (AgMNPV), which is homologous to the already validated bmnpv-miR-4 from Bombyx mori Nucleopolyhedrovirus (BmNPV). Considering information known for bmnpv-miR-4 such as seed sequence, coding location in the genome and putative target binding, we searched for the coding sequence of agmnpv-miR-4 in AgMNPV genome. A precursor sequence of agmnpv-miR-4 was predicted, and we identified a putative 23 nt mature microRNA, agmnpv-miR-4, coded in the complementary strand of AgMNPV-2D between positions 49,450 and 49,472. We validated agmnpv-miR-4 by Northern blot from HighFive cells and A. gemmatalis larve extracts infected with AgMNPV.

Polyhedron envelope protein (PEP) is the major component of the calyx that surrounds the baculovirus occlusion body (OB). PEP has been associated with the stabilization and resistance of polyhedra in the environment. Due to the abundant levels of PEP in OBs, we decided to use this protein as a fusion partner to redirect foreign proteins to baculovirus polyhedra. In this study we developed a strategy that involves the generation of a monoclonal transformed insect cell line expressing a protein of interest fused to the the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) N-terminus of PEP that enables the packaging of foreign proteins into the OBs without generating a recombinant baculovirus. This proved to be an efficient platform that could be exploited to improve wild type baculovirus for their use as bioinsecticides without facing the concerns of releasing genetically modified DNA to the environment and bypassing the associated regulatory issues. We demonstrated, using immunological, proteomic and microscopy techniques, that the envelope of AgMNPV OBs can effectively trap chimeric proteins in an infected insect cell line expressing AgMNPV PEP fused to the enhanced green fluorescent protein (eGFP). Furthermore, packaging of chimeric PEP also took place with heterologous OBs such as those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), another group I alphabaculovirus., 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

Until recently, members of the monogeneric family Arenaviridae (arenaviruses) have been known to infect only muroid rodents and, in one case, possibly phyllostomid bats. The paradigm of arenaviruses exclusively infecting small mammals shifted dramatically when several groups independently published the detection and isolation of a divergent group of arenaviruses in captive alethinophidian snakes. Preliminary phylogenetic analyses suggest that these reptilian arenaviruses constitute a sister clade to mammalian arenaviruses. Here, the members of the International Committee on Taxonomy of Viruses (ICTV) Arenaviridae Study Group, together with other experts, outline the taxonomic reorganization of the family Arenaviridae to accommodate reptilian arenaviruses and other recently discovered mammalian arenaviruses and to improve compliance with the Rules of the International Code of Virus Classification and Nomenclature (ICVCN). PAirwise Sequence Comparison (PASC) of arenavirus genomes and NP amino acid pairwise distances support the modification of the present classification. As a result, the current genus Arenavirus is replaced by two genera, Mammarenavirus and Reptarenavirus, which are established to accommodate mammalian and reptilian arenaviruses, respectively, in the same family. The current species landscape among mammalian arenaviruses is upheld, with two new species added for Lunk and Merino Walk viruses and minor corrections to the spelling of some names. The published snake arenaviruses are distributed among three new separate reptarenavirus species. Finally, a non-Latinized binomial species name scheme is adopted for all arenavirus species. In addition, the current virus abbreviations have been evaluated, and some changes are introduced to unequivocally identify each virus in electronic databases, manuscripts, and oral proceedings. Fil: Radoshitzky, Sheli R.. United States Army Medical Research. Institute of Infectious Diseases; Estados Unidos Fil: Bào, Yīmíng. National Institutes of Health; Estados Unidos Fil: Buchmeier, Michael J.. University of California at Irvine; Estados Unidos Fil: Charrel, Rémi N.. École des Hautes Etudes en Santé Publique; Francia. Aix Marseille Université; Francia Fil: Clawson, Anna N.. National Institutes of Health; Estados Unidos Fil: Clegg, Christopher S.. Les Mandinaux; Francia Fil: DeRisi, Joseph L.. University of California; Estados Unidos Fil: Emonet, Sébastien. Echelon Recherche de Lyon. Institut de Recherche Biomedicale de Armees. Unité de Virologie; Francia Fil: Gonzalez, Jean Paul. Metabiota Inc.; Estados Unidos Fil: Kuhn, Jens H.. National Institutes of Health; Estados Unidos Fil: Lukashevich, Igor S.. University of Louisville; Estados Unidos Fil: Peters, Clarence J.. University of Texas; Estados Unidos 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: Salvato, Maria S.. University of Maryland; Estados Unidos Fil: Stenglein, Mark D.. State University of Colorado - Fort Collins; Estados Unidos Fil: de la Torre, Juan Carlos. La Jolla Institute for Allergy and Immunology; Estados Unidos

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.

Arenaviruses are enveloped viruses that have a bi-segmented negative-stranded RNA genome. The genomic RNA segments, large (L) and small (S), use an ambisense coding strategy to encode two open reading frames in opposite orientation, separated by a noncoding intergenic region. Several arenaviruses are etiological agents of emerging diseases. At present, they are responsible for up to 500,000 zoonotic infections per year in endemic areas of Africa and South America and can lead to severe and lethal hemorrhagic fever as well as neurological symptoms. Arenaviridae represents the largest group of hemorrhagic fever (HF)-causing viruses: five of the South American arenaviruses (CHPV, GTOV, JUNV, MACV, and SABV) are associated with HF in humans.

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

Humanin (HN) is a mitochondrial-derived peptide with potent cytoprotective effect in many cell types. Administration of HN has been proposed as a therapeutic approach for several chronic diseases, such as diabetes, neurodegenerative conditions and cardiovascular diseases. Although HN has been shown to protect normal tissues against the toxic effects of chemotherapy, its role in tumor chemoresistance is poorly understood. Here, we aimed to elucidate the role of HN in the progression and cytotoxic response of experimental triple negative breast cancer (TNBC). We detected HN expression in murine and human TNBC cells, which was upregulated in the presence of chemotherapeutic drugs, i.e. Doxorubicin (DOXO) and Cisplatin, as assessed by flow cytometry. Addition of HN protected TNBC cells from various cytotoxic insults, such as serum deprivation, proapoptotic cytokine TNF-α or chemotherapy with DOXO. We next evaluated the role of endogenous HN in the apoptotic response of TNBC cells, using a plasmid encoding a short hairpin RNA to inhibit HN expression (pUC.shHN). To readily assess transduction efficiency, the plasmid also encodes the red fluorescent protein dTomato as a reporter gene. Transfection of TNBC 4T1 tumor cells with pUC.shHN increased the percentage of apoptotic cells when compared to cells transfected with control plasmid, as assessed by flow cytometry after staining with propidium iodide. When transfected 4T1 cells were incubated with different concentrations of DOXO, pUC.shHN reduced their clonogenic capacity. We also evaluated the effect of HN in the progression of experimental mouse 4T1 TNBC treated with DOXO. HN was readily detected in tumor samples and lung metastases. Systemic administration of HN reduced tumor apoptotic rate, accelerated tumor progression and increased the development of lung metastases. In addition, administration of HN impaired the antitumor and antimetastatic effect of chemotherapy. In summary, our findings suggest that HN inhibits the response of TNBC cells to cytotoxic stimuli, facilitating tumor progression and chemoresistance. Thus, blockade of HN could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer. Citation Format: Camila Zuccato, Mariela A. Moreno Ayala, Maria F. Gottardo, Matias Luis Pidre, Antonela S. Asad, Alejandro J. Nicola Candia, Victor Romanowski, Adriana Seilicovich, Marianela Candolfi. Role of mitochondrial peptide Humanin in the response of experimental breast cancer to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1951.

In a previous study of experimental murine encephalitis induced by Junín virus (JV), an arenavirus, we showed increased expression of iNOS by unidentified cells, concomitant with the astrocyte reaction. The specific inhibition of iNOS was associated with greater mortality but lower astrocytosis, suggesting that the protective role of nitric oxide (NO) synthesized by iNOS was related to enhanced astrocyte activation, representing a beneficial cellular response to virus-induced central nervous system damage. In the present work, cultured astrocytes were used to study whether JV infection could trigger iNOS expression and assess its eventual relationship with viral replication, glial fibrilary acidic protein (GFAP) expression levels and the presence of apoptosis. We found that JV infection of astrocytes did not induce apoptosis but produced both increased iNOS synthesis, detected by immunocytochemistry and fluorescence activated cell sorting (FACS) analysis, and increased NO, which was indirectly measured by nitrite/nitrate levels. These changes occurred early relative to the increases in GFAP expression, as detected by immunocytochemistry, FACS analysis and RT-PCR. The fact that iNOS inhibition abolished enhanced GFAP expression in infected monolayers suggests that NO was directly involved. In addition, iNOS inhibition enhanced virus replication. Together with data from confocal microscopy, these results suggest that JV induces iNOS expression in infected astrocytes and that the resulting NO has an important role both in reducing viral replication and in enhancing subsequent astrocyte activation. © 2008 Elsevier Ireland Ltd. All rights reserved. Fil: Pozner, Roberto Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Collado, Maria Soledad. 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: Jaquenod de Giusti, Carolina. 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: Ure, Agustin Enrique. 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: Biedma, Marina Elizabeth. 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: 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: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; 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

There are two major strategies to genetically modify baculoviruses. One uses a bacmid-based system which replicates in Escherichia coli using a bacterial origin of replication. The other employs a transfer vector and viral DNA which are co-transfected into insect cells and utilise host enzyme-mediated homologous recombination. Putative recombinants are then typically screened by plaque assay. The bacmid system is more convenient, but it requires a number of complex construction and isolation steps to obtain the correct bacmid genome. Generally, the transfer vector method is preferable when only a small number of genetic modifications are required. In this study a rapid and reliable method was developed to extract baculovirus DNA for PCR analysis from cultured insect cells. Briefly, viral DNA was isolated in three steps: SDS lysis, chloroform extraction and ethanol precipitation. The method was tested for direct screening of recombinant viruses in plaque assays. Contrary to previous reports, baculovirus DNA was isolated directly from viral plaques and successfully analysed by PCR. No prior amplification of the virus by passage in tissue culture was necessary. The major advantage of this method was a reduction in assay times from a few days to a few hours. Moreover, this method is very convenient for detecting baculoviruses in cell culture: cross-contamination within viral stocks, monitoring mixed viral infection and confirmation of viral genomic integrity.

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 bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management. Fil: Biedma, Marina Elizabeth. 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: Salvador, Ricardo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. 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: Ferrelli, Maria Leticia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina. 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: Sciocco Cap, Alicia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; Argentina 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

The images correspond to a transgenic cell line derived from the lepidopteran cell line High FiveTM that was engineered to express GFP under the control of the AcMNPV (Autographa californica Multiple Nucleopolyhedrovirus) polyhedrin promoter., Instituto de Biotecnologia y Biologia Molecular

Anticarsia gemmatalis is an important pest in legume crops in South America and it has been successfully controlled using Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) in subtropical climate zones. Nevertheless, in temperate climates its speed of kill is too slow. Taking this into account, genetic modification of AgMNPV could lead to improvements of its biopesticidal properties. Here we report the generation of a two-component system that allows the production of recombinant AgMNPV. This system is based on a parental AgMNPV in which the polyhedrin gene (polh) was replaced by a bacterial β-galactosidase (lacZ) gene flanked by two target sites for the homing endonuclease I-PpoI. Co-transfection of insect cells with linearized (I-PpoI-digested) parental genome and a transfer vector allowed the restitution of polh and the expression of a heterologous gene upon homologous recombination, with a low background of non-recombinant AgMNPV. The system was validated by constructing a recombinant occlusion-positive (polh+) AgMNPV expressing the green fluorescent protein gene (gfp). This recombinant virus infected larvae normally per os and led to the expression of GFP in cell culture as well as in A. gemmatalis larvae. These results demonstrate that the system is an efficient method for the generation of recombinant AgMNPV expressing heterologous genes, which can be used for manifold purposes, including biotechnological and pharmaceutical applications and the production of orally infectious recombinants with improved biopesticidal properties., Facultad de Ciencias Exactas, 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

Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking., Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

The bean shoot borer, Epinotia aporema (Lep. Tortricidae), is an economically important pest of legume crops in South America. Recently, a granulovirus (EpapGV) was isolated from E. aporema larvae, and evaluated as a potential biological control agent. In order to generate a restriction map and to investigate the gene organisation of EpapGV genome, DNA isolated from occlusion bodies as well as a set of cloned genomic fragments were analysed using combinations of restriction endonucleases and Southern blot analyses that lead to a first version of the physical map. It was subsequently confirmed and refined by sequencing the termini of the cloned fragments and assessing their contiguity by comparing the sequences with databases to identify putative ORFs spanning neighbour fragments. This was also aided by PCR amplifications with primers that pointed outwards of the cloned viral DNA. The granulin gene was positioned on the physical map, cloned and sequenced. Its 747-nucleotide-long ORF encodes a predicted protein of 29 kDa and the core of the baculovirus very late promoter ATAAG was found 29 nucleotides upstream the initiation codon. In addition, 27 putative ORFs were located on the map and used to explore the genome organisation by GeneParityPlot against the fully sequenced granulovirus genomes. These data, taken together with the phylogenetic tree generated by alignment of the major occlusion proteins, indicate that EpapGV is closely related to CpGV, but has a distinct gene organisation.

The ecdysteroid UDP-glycosyltransferase (egt) gene of Epinotia aporema granulovirus (EpapGV) was cloned sequenced and its biological activity was assessed. It encodes a protein of 446 amino acids. Direct evidence that the cloned gene encodes an active EGT protein was obtained by transient expression assays in insect cells. The upstream untranslated region of the egt gene exhibits several consensus early promoter elements. Accordingly, the gene is expressed early upon infection of Epinotia aporema larvae and the EGT activity remains high until later times post infection. Sequence analyses indicate the presence of clusters of amino acid residues conserved among all the baculoviral EGTs, although their relation with proper protein folding, ligand binding and catalytic activity remain to be assessed. Phylogenetic trees consistently cluster the granulovirus EGTs separating them clearly from the nucleopolyhedroviruses.

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

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing β-galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh −/LacZ + plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of β-galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.

Arenaviridae is a worldwide distributed family, of enveloped, single stranded, RNA viruses. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as the geographic distribution. In this study the phylogenetic relationship among the members of the Arenaviridae was examined, using the reported genomic sequences. The comparison of the aligned nucleotide sequences of the S RNA and the predicted amino acid sequences of the GPC and N proteins, together with the phylogenetic analysis, strongly suggest a possible kinship of Pichinde and Oliveros viruses, with the Old World arenavirus group. This analysis points at the evolutive relationships between the arenaviruses of the Americas and can be used to evaluate the different hypotheses about their origin.

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.

The main function of baculoviral chitinase protein (V-CHIA) is to promote the final liquefaction of infected host larvae, facilitating the dispersion of occlusion bodies (OBs) in the environment. In this study, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and characterized. The 1,713 base pairs long open reading frame encodes a protein of 570 amino acids with a predicted molecular weight of 63 kDa. EpapGV V-CHIA sequence alignment resulted 62 % identical to Pieris rapae GV and Blastp search revealed a high conservation among all baculovirus chitinases. Amino acid sequence analysis indicated that the C-terminal KDEL present in most alphabaculovirus chitinases is absent in EpapGV V-CHIA, as well as in the rest of the betabaculoviruses. Phylogenetic analysis was performed with bacterial, lepidopteran, and baculoviral chitinase sequences available in databases. Using an AcMNPV bacmid (bApGOZA) a recombinant Ac-chiAEpapGV was obtained in order to overexpress EpapGV V-CHIA in cell culture. The presence of chitinase was detected in purified AcMNPV-chiAEpapGV OBs. Peritrophic membranes of Anticarsia gemmatalis larvae fed with recombinant OBs showed an altered structure. The results presented in this study show that EpapGV chitinase overexpression in recombinant baculovirus can cause association of this protein with OBs, and suggest that this could be used to evaluate the protein role in early stages of baculoviral infections., Instituto de Biotecnología y Biología Molecular

We used transient expression assays to assess the function of the baculovirus Spodoptera frugiperda M nucleopolyhedrovirus (SfMNPV) homologs of Autographa californica MNPV (AcMNPV) factors involved in late gene expression (lefs), in the Sf9 insect cell-line, which is permissive for both viruses. It is well-established that nineteen AcMNPV lefs support optimal levels of activity from a late promoter-reporter gene cassette in this assay. A subgroup of SfMNPV lefs predicted to function in transcription-specific events substituted the corresponding AcMNPV lefs very efficiently. When all SfMNPV lefs were assayed, including replication lefs, activity was low, but addition of two AcMNPV lefs not encoded in SfMNPV genome, resulted in augmented reporter activity. SfMNPV IE-1 was able to activate an early promoter cis-linked to an hr-derived element from SfMNPV but not from AcMNPV. However, the level of early promoter activation with SfMNPV IE-1 was lower compared to AcMNPV IE-1., Facultad de Ciencias Exactas, Instituto de Biotecnología y Biología Molecular