Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Transcriptional upregulation of SOCS 1 and suppressors of cytokine signaling 3 mRNA in the absence of suppressors of cytokine signaling 2 mRNA after infection with West Nile virus or tick-borne encephalitis virus.

Suppressors of cytokine signaling (SOCS) proteins are a family of proteins that are able to act in a classic negative feedback loop to regulate cytokine signal transduction. The regulation of the immune response by SOCS proteins may contribute to persistent infection or even a fatal outcome. In this study, we have investigated the induction of SOCS 1-3 after peripheral infection with West Nile virus (WNV) or tick-borne encephalitis virus (TBEV) in the murine model. We have shown that the cytokine response after infection of mice with WNV or TBEV induces an upregulation in the brain of mRNA transcripts for SOCS 1 and SOCS 3, but not SOCS 2. We hypothesize that SOCS proteins may play a role in limiting cytokine responses in the brain as a neuroprotective mechanism, which may actually enhance the ability of neuroinvasive viruses such as WNV and TBEV to spread and cause disease.

Characterisation of virus transport and attenuation in epikarst using short pulse and prolonged injection multi-tracer testing

Attenuation processes controlling virus fate and transport in the vadose zone of karstified systems can strongly influence groundwater quality. This research compares the breakthrough of two bacteriophage tracers (H40/1 and T7), with contrasting properties, at subsurface monitoring points following application onto an overlying composite sequence of thin organic soil and weathered limestone (epikarst). Short pulse multi-tracer test results revealed that T7 (Source concentration, Co=1.8x106pfu/mL) and H40/1(Co=5.9x106pfu/mL) could reach sampling points 10m below ground less than 30 minutes after tracer application. Contrasting deposition rates, determined from simulated tracer responses, reflected the potential of the ground to differentially attenuate viruses. Prolonged application of both T7 (Co=2.3x104pfu/mL) and H40/1 (Co=1.3x105pfu/mL) over a five hour period during a subsequent test, in which ionic strength levels observed at monitoring points rose consistently, corresponded to a rapid rise in T7 levels, followed by a gradual decline before the end of tracer injection; this reflected reaction-limited deposition in the system. T7’s response contrasted with that of H40/1, whose concentration remained constant over a three hour period before declining dramatically prior to the end of tracer injection. Subsequent application of lower ionic strength tracer-free flush water generated a rapid rise in H40/1 levels and a more gradual release of T7. Results highlight the benefits of employing prolonged injection multi-tracer tests for identifying processes not apparent from conventional short pulse tests. Study findings demonstrate that despite rapid transport rates, the epikarst is capable of physicochemical filtration of viruses and their remobilization, depending on virus type and hydrochemical conditions.

Phocine distemper virus in seals, East Coast, United States, 2006

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.

De novo generation of a non-segmented negative strand RNA virus with a bicistronic gene

Reverse genetics has facilitated the use of non-segmented negative strand RNA viruses (NNSV) as vectors. Currently, heterologous gene expression necessitates insertion of extra-numeral transcription units (ENTUs), which may alter the NNSV polar transcription gradient and attenuate growth relative to wildtype (Wt). We hypothesized that rescuing recombinant Sendai Virus (rSeV) with a bicistronic gene might circumvent this attenuation but still allow heterologous open reading frame (ORF) expression. Therefore, we used a 9-nucleotide sequence previously described with internal ribosome entry site (IRES) activity, which, when constructed as several repeats, synergistically increased the level of expression of the second cistron [Chappell, S.A., Edelman, G.M., Mauro, V.P., 2000. A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity. Proc. Natl. Acad. Sci. U.S.A. 97, 1536-1541]. We inserted the Renilla luciferase (rLuc) ORF, preceded by 1, 3 or 7 IRES copies, downstream of the SeV N ORF in an infectious clone. Corresponding rSeVs were successfully rescued. Interestingly, bicistronic rSeVs grew as fast as or faster than Wt rSeV. Furthermore, SeV gene transcription downstream of the N/rLuc gene was either equivalent to, or slightly enhanced, compared to Wt rSeV. Importantly, all rSeV/rLuc viruses efficiently expressed rLuc. IRES repetition increased rLuc expression at a multiplicity of infection of 0.1, although without evidence of synergistic enhancement. In conclusion, our approach provides a novel way of insertion and expression of foreign genes in NNSVs. (C) 2008 Elsevier B.V. All rights reserved.