The human sodium iodide symporter as a reporter gene for studying Middle East respiratory syndrome coronavirus pathogenesis

Single photon emission computed tomography (SPECT) is frequently used in oncology and cardiology to evaluate disease progression and/or treatment efficacy. Such technology allows for real-time evaluation of disease progression and when applied to studying infectious diseases may provide insight into pathogenesis. Insertion of a SPECT-compatible reporter gene into a virus may provide insight into mechanisms of pathogenesis and viral tropism. The human sodium iodide symporter (hNIS), a SPECT and positron emission tomography reporter gene, was inserted into Middle East respiratory syndrome coronavirus (MERS-CoV), a recently emerged virus that can cause severe respiratory disease and death in afflicted humans to obtain a quantifiable and sensitive marker for viral replication to further MERS-CoV animal model development. The recombinant virus was evaluated for fitness, stability, and reporter gene functionality. The recombinant and parental viruses demonstrated equal fitness in terms of peak titer and replication kinetics, were stable for up to six in vitro passages, and were functional. Further in vivo evaluation indicated variable stability, but resolution limits hampered in vivo functional evaluation. These data support the further development of hNIS for monitoring infection in animal models of viral disease.IMPORTANCE Advanced medical imaging such as single photon emission computed tomography with computed tomography (SPECT/CT) enhances fields such as oncology and cardiology. Application of SPECT/CT, magnetic resonance imaging, and positron emission tomography to infectious disease may enhance pathogenesis studies and provide alternate biomarkers of disease progression. The experiments described in this article focus on insertion of a SPECT/CT-compatible reporter gene into MERS-CoV to demonstrate that a functional SPECT/CT reporter gene can be inserted into a virus

Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences

Virus nomenclature below the species level : a standardized nomenclature for filovirus strains and variants rescued from cDNA

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratoryadapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming,\virus name[(\strain[/)\isolation host-suffix[/ \country of sampling[/\year of sampling[/\genetic variant designation[-\isolate designation[, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to ‘‘Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1’’ (with the suffix ‘‘rec’’ identifying the recombinant nature of the virus and ‘‘abc1’’ being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as ‘‘EBOV H.sap/COD/95/ Kik-abc1’’) and abbreviations (such as ‘‘EBOV/Kik-abc1’’) could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. ‘‘EBOV’’ would suffice if only one EBOV strain/variant/isolate is addressed.http://link.springer.com/journal/705hb201

Virus nomenclature below the species level : a standardized nomenclature for laboratory animal-adapted strains and variants of viruses assigned to the family Filoviridae

The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming ( //<year of sampling>/-) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology “Vector” to cause disease in guinea pigs after seven passages would be akin to “Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga- GPA-P7”. As was proposed for the names of natural filovirus variants, we suggest using the fulllength designation in databases, as well as in the method section of publications. Shortened designations (such as “EBOV VECTOR/C.por/COD/76/May-GPA-P7”) and abbreviations (such as “EBOV/May-GPA-P7”) could be used in the remainder of the text depending on how critical it is to convey information contained in the full-length name. “EBOV” would suffice if only one EBOV strain/variant/isolate is addressed.This work was funded in part by the Joint Science and Technology Office for Chem Bio Defense (proposal #TMTI0048_09_RD_T to SB).http://www.springerlink.com/content/0304-8608/hb2013ab201

Simian Hemorrhagic Fever Virus Cell Entry Is Dependent on CD163 and Uses a Clathrin-Mediated Endocytosis-Like Pathway

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component

Loss in lung volume and changes in the immune response demonstrate disease progression in African green monkeys infected by small-particle aerosol and intratracheal exposure to Nipah virus

<div><p>Nipah virus (NiV) is a paramyxovirus (genus <i>Henipavirus</i>) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8–10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition.</p></div

CT analysis of the lungs of NiV infected AGMs.

<p>Examples of temporal analysis of changes within the lungs of AGMs infected by intratracheal or aerosol inoculation (A). In animals infected by the IT route, images shows a vascular pattern of infiltration extending to adjacent alveoli, manifested as radiating ground glass opacities (indicated by red arrows), progressing by day 8 to diffuse lobar consolidation (blue arrows) that tends to extend from cranial to caudal in severity. In animals infected by aerosolization of small particles, diffuse progressive thickening of airway walls and adjacent interstitium are typically observable within 6 days of infection, which ultimately extends into the alveoli, uniformly throughout the lungs. Where IT inoculation tends to induce some amount of consolidation, typically in caudal lobes, infection by small particle aerosol appears to cause disseminated pulmonary changes throughout all lobes. Quantification (B) of lung volume demonstrates that animals lost up to 45% of their total lung volume with all three of the animals infected by IT inoculation losing 18% or greater of their lung volume.</p

CBC on NiV infected AGMs.

<p>Temporal analysis of lymphocytes (A), neutrophils (B), monocytes (C) and platelets (D) demonstrating lymphopenia, neutrophilia and monocytosis developing over the course of disease. The rapid drop in platelet count occurs in the terminal phase of disease. IT inoculated animals are indicated by (●) and aerosol inoculated animals by (■). The mean for the group is indicated by a dash (-). The shaded area indicates the normal range for the specified value [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005532#pntd.0005532.ref044" target="_blank">44</a>].</p

Comparison of CT evaluation of lungs with gross pathology from NiV infected animals.

<p>Gross pathologic assessment was in agreement with CT images, indicating severity was often greater in the caudodorsal lung lobes of both models. Extensive lung congestion, hyperemia and multifocal areas of consolidation were more likely to be observed using the IT route of infection, while the aerosol model exhibited mildly congested areas with multifocal areas of hemorrhage. Tracheal mucosa was typically found to be congested, edematous and often hemorrhagic.</p