Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.

BackgroundSystems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion.ResultsWe have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway® entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics.ConclusionsThe E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.

Erythromelagia is a condition characterized by attacks of burning pain and inflammation in the extremeties. An epidemic form of this syndrome occurs in secondary students in rural China and a virus referred to as erythromelalgia-associated poxvirus (ERPV) was reported to have been recovered from throat swabs in 1987. Studies performed at the time suggested that ERPV belongs to the orthopoxvirus genus and has similarities with ectromelia virus, the causative agent of mousepox. We have determined the complete genome sequence of ERPV and demonstrated that it has 99.8% identity to the Naval strain of ectromelia virus and a slighly lower identity to the Moscow strain. Small DNA deletions in the Naval genome that are absent from ERPV may suggest that the sequenced strain of Naval was not the immediate progenitor of ERPV.

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.

While high-throughput protein-protein interaction screens were first published approximately 10 years ago, systematic attempts to map interactions among viruses and hosts started only a few years ago. HIV-human interactions dominate host-pathogen interaction databases (with approximately 2000 interactions) despite the fact that probably none of these interactions have been identified in systematic interaction screens. Recently, combinations of protein interaction data with RNAi and other functional genomics data allowed researchers to model more complex interaction networks. The rapid progress in this area promises a flood of new data in the near future, with clinical applications as soon as structural and functional genomics catches up with next-generation sequencing of human variation and structure-based drug design. Keywordshepatitis C virus; herpes viruses; HIV; mass spectrometry; protein interaction networks; protein purification; RNAi; yeast two-hybrid screens Host-virus interactions have been studied since the discovery of viruses in 1898, when Martinus Beijerinck described tobacco mosaic virus. However, only with the invention of the electron microscope, protein biochemistry and eventually nucleic acid sequencing has their systematic molecular characterization become possible. Considering their small size, it was no surprise that the first completely sequenced genome was a phage, namely MS2 [1]. To date, more than 5000 viruses have been described [2,101], although many have not been completely sequenced nor classified into any of the major virus families. The National Center for Biotechnology Information (NCBI) genome database lists 3398 complete sequences for 2319 viral genomes [102]. In fact, there may be up to a billion virus particles present in a milliliter of seawater, at least in certain areas [3], and most bacteria and higher organisms appear to be infected by phage or other viruses (e.g., [4]). If there are millions of species in our biosphere there is likely to be a similar number of viral species that infect them. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. NIH Public Access Experimental methods to study host-pathogen interactionsIn contrast to viruses that enter their host cells completely and must express all their proteins in the host cell, many bacteria inject only a few effector proteins into their host cells. For example, pathogenic Escherichia coli strains such as O157 encode in the order of 50 effector proteins that are injected into host cells [6]. The challenge here is to identify the bacterial effector proteins. For viruses, we can safely assume that all viral proteins enter the cell. Thus, an important goal of virus studies is to analyze the activity of each protein inside the cell. Initially this ca...

Molluscum contagiosum virus (MOCV), IMPORTANCEThe inability to propagate molluscum contagiosum virus, which causes benign skin lesions in young children and more extensive infections in immunosuppressed adults, has constrained our understanding of the biology of this human-specific virus. In the present study, we characterized the RNAs synthesized in abortively infected cultured cells and a human skin lesion by nextgeneration sequencing. These studies provided an initial transcription map of the MOCV genome, suggested temporal regulation of gene expression, and indicated that the in vitro replication block occurs prior to intermediate and late gene expression. RNA-seq and reporter assays, as described here, may help to further evaluate MOCV gene expression and define conditions that could enable MOCV replication in vitro. M olluscum contagiosum virus (MOCV) is the sole member of the Molluscipoxvirus genus of the Chordopoxvirinae subfamily of the Poxviridae (1). Although many poxviruses cause zoonoses, variola virus (the causative agent of smallpox) and MOCV are the only known human-specific poxviruses (2, 3). MOCV has a worldwide distribution and commonly infects young healthy children, where it causes papular skin lesions that may persist for many months before spontaneous resolution (4). However, widespread disfiguring skin lesions may occur in individuals with immunodeficiencies. For the latter, the most successful therapy is treatment of the underlying immunodeficiency. Although several MOCV variants have been recognized by restriction endonuclease analysis and limited DNA sequencing, they produce indistinguishable lesions (4).Knowledge of MOCV is limited because of the lack of either a cell culture system or useful animal model. The inoculation of primate cells with MOCV produces an abortive infection with cell rounding and related cytopathic effects (CPE) (5). However, the cells regain a more normal appearance after 48 h (6, 7). Evidence that MOCV gene expression is necessary for CPE was supported by the ability of inhibitors of RNA and protein synthesis to prevent this phenomenon (7,8). Following infection, electron microscopy revealed MOCV cores within the cytoplasm, consistent with early gene expression, but the disassembly of the cores or assembly of new virus particles was not observed (7). More direct evidence for early gene expression in human fibroblasts was obtained by RNA-DNA hybridization and reverse transcription-PCR (RT-PCR) (7,

<p>Resumen<br />El propósito de esta revisión es presentar al equipo de salud latinoamericano y de la región del caribe un panorama de la situación actual con el virus Zika (ZIKAV), y al mismo tiempo, proveer conocimiento clínica y molecular relevante para enfrentar este problema emergente. Esperamos que esta revisión tenga un impacto positivo en el diagnóstico, vigilancia, y tratamiento de esta enfermedad viral, especialmente en comunidades endémicas, como parte de un esfuerzo colectivo para enfrentar este virus. Este manuscrito será distribuido electrónicamente y físicamente como una iniciativa de salud pública y epidemiologica.<br /><br /><br />Abstract<br />The purpose of this review is to provide Latin America's and the region's healthcare professionals with an overview of the current situation related to Zika virus (ZIKAV), and at the same time, to provide relevant clinical and molecular knowledge against this emerging problem. We expect to have a positive impact in diagnostic, surveillance and treatment of this viral disease, specially in those endemic communities, as part of a collective effort against the virus. This review will be distributed as hard-copy and online as a public health and epidemiological initiative.</p><p> </p><p>Palabras claves:<br />ZIKAV, control de vector, brote, vigilancia epidemiológica, microcefalia<br /><br /><br />Keywords: <br />Zika virus, vector control, outbreak, survillance, microcephaly</p>

Neuroendocrine tumors (NETs) comprise a heterogenous group of rare malignancies, which are increasing in incidence worldwide. To further understand the epidemiology of NETs in the Republic of Panama, the present study used two study groups, which included patients from several hospitals and clinics throughout the country, who were referred to the three largest national reference centers: The Complejo Hospitalario Metropolitano, Hospital Santo Tomas and Instituto Oncologico Nacional. These two groups comprised a retrospective cohort, which included cases reported between 2016 and 2017, and a second cohort, which was retrospective, but data were continuously collected from patients diagnosed with NETs between 2018 and 2019. Data from 157 patients with NETs reported that 83% of patients were in the 40-80 years old age group. The majority of cases (46%) presented as grade G1 tumors, while 29% were G3. Computerized tomography scans with contrast, and analysis of the Ki-67 biomarker and immunohistology markers (chromogranin A and synaptophysin) was performed in the majority of the cases. The results revealed that the most frequent anatomical sites for the primary tumor were the colorectum (17.2%), pancreas (12.7%) and stomach (12.1%), and the most frequent organ with metastasis was the liver, accounting for 34% of all cases. In conclusion, the present study is the first comprehensive study of NET in Panama to the best of our knowledge, which provides evidence of the demographic characteristics of the population, clinical features and overall survival for the affected population in this Central American country.