Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMVKSEGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n = 3 per time point) and infected (EGFP+) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP+ cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.
The global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation. In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare the in vivo tropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despite in vitro differences, attenuated and pathogenic MV show highly similar in vivo tropism in the lungs. However, systemic spread of attenuated MV is restricted.Measles virus (MV) is one of the most contagious human viruses and is transmitted via aerosols or by direct contact with contaminated respiratory secretions. Clinical symptoms appear approximately 2 weeks after infection and include fever, rash, cough, coryza, and conjunctivitis (20). Measles is associated with immunosuppression, resulting in increased susceptibility to opportunistic infections. While significant progress has been made in global control programs, 164,000 deaths were attributed to measles in 2008 (46).MV was first isolated in cell culture in 1954 (16). This Edmonston wild-type MV strain was passaged multiple times in primary human kidney and amnion cells and adapted to eggs and chicken embryo fibroblasts to produce the live-attenuated Edmonston-B vaccine virus (15), which was later replaced by the more attenuated MV strains (Edmonston-Zagreb, Moraten, and Schwarz) (34). These vaccines have been shown to be safe and effective, and high coverage in two-dose regimens has successfully interrupted endemic MV transmission in large geographic areas (6).For many years, laboratory-adapted MV-Edmonston strains were used as the prototype virus and were shown to display a wide cellular tropism in vitro. The virus efficiently infected epithelial cells, which were considered the target cells for primary MV infection in vivo (22). In 1993,...
Bovine Respiratory Disease (BRD) is the leading cause of mortality in calves. The objective of this study was to examine the response of the host’s bronchial lymph node transcriptome to Bovine Respiratory Syncytial Virus (BRSV) in a controlled viral challenge. Holstein-Friesian calves were either inoculated with virus (103.5 TCID50/ml × 15 ml) (n = 12) or mock challenged with phosphate buffered saline (n = 6). Clinical signs were scored daily and blood was collected for haematology counts, until euthanasia at day 7 post-challenge. RNA was extracted and sequenced (75 bp paired-end) from bronchial lymph nodes. Sequence reads were aligned to the UMD3.1 bovine reference genome and differential gene expression analysis was performed using EdgeR. There was a clear separation between BRSV challenged and control calves based on gene expression changes, despite an observed mild clinical manifestation of the disease. Therefore, measuring host gene expression levels may be beneficial for the diagnosis of subclinical BRD. There were 934 differentially expressed genes (DEG) (p < 0.05, FDR <0.1, fold change >2) between the BRSV challenged and control calves. Over-represented gene ontology terms, pathways and molecular functions, among the DEG, were associated with immune responses. The top enriched pathways included interferon signaling, granzyme B signaling and pathogen pattern recognition receptors, which are responsible for the cytotoxic responses necessary to eliminate the virus.
The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adenoassociated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (CAU) or guanine to adenine (GAA) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, AAG and UAC transitions, with preference for next neighbour-nucleotides U5A.C.G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells. INTRODUCTIONThe cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) is a potent inhibitor of retroviruses, hepatitis B virus, adenoassociated virus and transposable elements, and is able to deaminate cytidines in ssDNA replication intermediates leading to cytidine to uridine [CAU(T)] and guanine to adenine (GAA) nucleotide exchanges, a process also referred to as DNA editing or hypermutation (Harris et al., 2003;Mangeat et al., 2003; Zhang et al., 2003;Turelli et al., 2004;Chen et al., 2006;Aguiar & Peterlin, 2008). As an effector molecule of the innate immune response A3G is induced by certain cytokines and detected in human tissues including lung, liver, tonsils, spleen and lymph nodes, where it is expressed predominantly by lymphoid and myeloid cells (Bonvin et al., 2006;Peng et al., 2006;Sarkis et al., 2006;Tanaka et al., 2006;Stopak et al., 2007;Koning et al., 2009;Chen et al., 2010).The antiviral activity of A3G against human immunodeficiency virus (HIV)-1 was extensively investigated (Sheehy et al., 2002Stopak et al., 2003;Yu et al., 2003). In addition to its deaminase activity, A3G exerts antiviral activity in another manner, probably depending on its RNA-binding capacity (Svarovskaia et al., 2004;Zennou et al., 2004; Khan et al., 2005;Newman et al., 2005;Bishop et al., 2006;Wedekind et al., 2006;Burnett & Spearman, 2007;Holmes et al., 2007;Huthoff & Malim, 2007; Iwatani et al., 2007;Bishop et al., 2008;Chelico et al., 2008). It contains two canonical cytidine deaminase domains, of which the C-terminal one is kn...
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