Characterization of a Novel Bat Adenovirus Isolated from Straw-Colored Fruit Bat (Eidolon helvum).

Bats are important reservoirs for emerging zoonotic viruses. For extensive surveys of potential pathogens in straw-colored fruit bats (Eidolon helvum) in Zambia, a total of 107 spleen samples of E. helvum in 2006 were inoculated onto Vero E6 cells. The cell culture inoculated with one of the samples (ZFB06-106) exhibited remarkable cytopathic changes. Based on the ultrastructural property in negative staining and cross-reactivity in immunofluorescence assays, the virus was suspected to be an adenovirus, and tentatively named E. helvum adenovirus 06-106 (EhAdV 06-106). Analysis of the full-length genome of 30,134 bp, determined by next-generation sequencing, showed the presence of 28 open reading frames. Phylogenetic analyses confirmed that EhAdV 06-106 represented a novel bat adenovirus species in the genus Mastadenovirus. The virus shared similar characteristics of low G + C contents with recently isolated members of species Bat mastadenoviruses E, F and G, from which EhAdV 06-106 diverged by more than 15% based on the distance matrix analysis of DNA polymerase amino acid sequences. According to the taxonomic criteria, we propose the tentative new species name "Bat mastadenovirus H". Because EhAdV 06-106 exhibited a wide in vitro cell tropism, the virus might have a potential risk as an emerging virus through cross-species transmission

Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability

Putative endogenous filovirus VP35-like protein potentially functions as an IFN antagonist but not a polymerase cofactor

It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs) have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35) is found in the little brown bat (Myotis lucifugus) genome. Putative mlEFL35-derived protein (mlEFL35p) contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN) production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-beta promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor

Single-nucleotide polymorphisms in human NPC1 influence filovirus entry into cells

Niemann-Pick C1 (NPC1), a host receptor involved in the envelope glycoprotein (GP)-mediated entry of filoviruses into cells, is believed to be a major determinant of cell susceptibility to filovirus infection. It is known that proteolytically digested Ebola virus (EBOV) GP interacts with 2 protruding loops in domain C of NPC1. Using previously published structural data and the National Center for Biotechnology Information Single-Nucleotide Polymorphism (SNP) database, we identified 10 naturally occurring missense SNPs in human NPC1. To investigate whether these SNPs affect cell susceptibility to filovirus infection, we generated Vero E6 cell lines stably expressing NPC1 with SNP substitutions and compared their susceptibility to vesicular stomatitis virus pseudotyped with filovirus GPs and infectious EBOV. We found that some of the substitutions resulted in reduced susceptibility to filoviruses, as indicated by the lower titers and smaller plaque/focus sizes of the viruses. Our data suggest that human NPC1 SNPs may likely affect host susceptibility to filoviruses

Inhibition of the RIG-I-mediated signaling pathway by mlEFL35 and VP35s.

<p><b>(A)</b> The RIG-I-mediated signaling pathway is shown. The human IFN-β promoter is activated through RIG-I, IPS-1 and TBK1. The IFN-β promotor activity was measured by luciferase reporter assays. <b>(B)</b> HEK 293 cells were transfected with each plasmid expressing HA-tagged influenza A virus NS1 (IAVs-NS1-HA), EBOV VP35 (HA-ZVP35), RESTV VP35 (HA-RVP35) or mlEFL35p (HA-mlEFL35p) and the plasmids for the reporter gene expression along with the RIG-I CARD domain vector, IPS-1 or TBK1 expression vector. NS1 and VP35 are known as IFN antagonists. Western blotting was performed to examine the expression of NS1, VP35s, and mlEFL35p. Each HA-tagged protein (IAVs-NS1-HA, HA-ZVP35, HA-RVP35, and HA-mlEFL35p) was detected with an anti-HA-tag antibody. (<b>C</b>) Transfected cells were solubilized and luciferase assays were performed. Relative luciferase activities were calculated by setting the values given by the cells transfected with a control empty plasmid expressing the HA tag alone. Significantly lower values compared to control cells (Empty) are indicated by asterisks (*p < 0.05, **p < 0.01). (<b>D</b>) Concentrations of IFN-β in the supernatants of cells transfected with the indicated plasmids (1000 ng) were measured by ELISA. Means and standard deviations of five independent experiments are shown. Significantly lower values compared to control cells (Empty) are indicated by asterisks (<b>*</b><i>p</i> < 0.05, <b>**</b><i>p</i> < 0.01).</p

Expression of the mlEFL35p and VP35s in HEK 293T cells.

<p><b>(A)</b> Expression of each protein was confirmed by western blotting. HA-tagged mlEFL35p (HA-mlEFL35p), HA-tagged EBOV VP35 (HA-ZVP35) and HA-tagged RESTV VP35 (HA-RVP35) were detected as 30, 37, and 40 kDa proteins, respectively. <b>(B)</b> Distribution of each protein is visualized by an immunofluorescence assay with anti-HA antibodies. Cells were counterstained with DAPI.</p