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.

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.

Respiratory syncytial virus (RSV) vaccines - Two steps back for one leap forward

Respiratory viruses are among the most important causes of morbidity and mortality worldwide. From a vaccine viewpoint, such viruses may be divided into two principle groups-those where infection results in long-term immunity and whose continued survival requires constant mutation, and those where infection induces incomplete immunity and repeated infections are common, even with little or no mutation. Influenza virus and respiratory syncytial virus (RSV) typify the former and latter groups, respectively. Importantly, successful vaccines have been developed against influenza virus. However, this is not the case for RSV, despite many decades of research and several vaccine approaches. Similar to natural infection, the principle limitation of candidate RSV vaccines in humans is limited immunogenicity, characterised in part by short-term RSV-specific adaptive immunity. The specific reasons why natural RSV infection is insufficiently immunogenic in humans are unknown but circumvention of innate and adaptive immune responses are likely causes. Fundamental questions concerning RSV/host interactions remain to be addressed at both the innate and adaptive immune levels in humans in order to elucidate mechanisms of immune response circumvention. Taking the necessary steps back to generate such knowledge will provide the means to leap forward in our quest for a successful RSV vaccine. Recent developments relating to some of these questions are discussed. (C) 2007 Elsevier B.V. All rights reserved.

Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells.

Background Human respiratory syncytial virus (RSV) causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs). Methods To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. Results RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. Conclusions The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

Early target cells of measles virus after aerosol infection of non-human primates

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 rMV(KS)EGFP 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.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.

Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs

Objective—To determine whether genogroup 1 porcine torque teno virus (g1-TTV) can potentiate clinical disease associated with porcine circovirus type 2 (PCV2).

Sample population—33 gnotobiotic baby pigs.

Procedures—Pigs were allocated into 7 groups: group A, 5 uninoculated control pigs from 3 litters; group B, 4 pigs oronasally inoculated with PCV2 alone; group C, 4 pigs inoculated IP with first-passage g1-TTV alone; group D, 4 pigs inoculated IP with fourth-passage g1-TTV alone; group E, 6 pigs inoculated IP with first-passage g1-TTV and then oronasally inoculated with PCV2 7 days later; group F, 6 pigs inoculated IP with fourth-passage g1-TTV and then inoculated oronasally with PCV2 7 days later; and group G, 4 pigs inoculated oro-nasally with PCV2 and then inoculated IP with fourth-passage g1-TTV 7 days later.

Results—6 of 12 pigs inoculated with g1-TTV prior to PCV2 developed acute onset of postweaning multisystemic wasting syndrome (PMWS). None of the pigs inoculated with g1-TTV alone or PCV2 alone or that were challenge exposed to g1-TTV after establishment of infection with PCV2 developed clinical illness. Uninoculated control pigs remained healthy.

Conclusions and Clinical Relevance—These data implicated g1-TTV as another viral infection that facilitates PCV2-induced PMWS. This raises the possibility that torque teno viruses in swine may contribute to disease expression currently associated with only a single infectious agent.

Pathobiology of rabies virus and the European bat lyssaviruses in experimentally infected mice

A comparison of the clinicopathology of European bat lyssavirus (EBLV) types-1 and -2 and of rabies virus was undertaken. Following inoculation of mice at a peripheral site with these viruses, clinical signs of rabies and distribution of virus antigen in the mouse brain were examined. The appearance of clinical signs of disease varied both within and across the different virus species, with variation in incubation periods and weight loss throughout disease progression. The distribution of viral antigen throughout the regions of the brain examined was similar for each of the isolates during the different stages of disease progression, suggesting that antigen distribution was not associated with clinical presentation. However, specific regions of the brain including the cerebellum, caudal medulla, hypothalamus and thalamus, showed notable differences in the proportion of virus antigen positive cells present in comparison to other brain regions suggesting that these areas are important in disease development irrespective of virus species.

Respiratory syncytial virus interaction with human airway epithelium

Although respiratory syncytial virus (RSV) is a major human respiratory pathogen, our knowledge of how it causes disease in humans is limited. Airway epithelial cells are the primary targets of RSV infection in vivo, so the generation and exploitation of RSV infection models based on morphologically and physiologically authentic well-differentiated primary human airway epithelial cells cultured at an air-liquid interface (WD-PAECs) provide timely developments that will help to bridge this gap. Here we review the interaction of RSV with WD-PAEC cultures, the authenticity of the RSV-WD-PAEC models relative to RSV infection of human airway epithelium in vivo, and future directions for their exploitation in our quest to understand RSV pathogenesis in humans.

Transmission Potential of Rift Valley Fever virus over the course of the 2010 epidemic in South Africa

A Rift Valley fever (RVF) epidemic affecting animals on domestic livestock farms was reported in South Africa during January-August 2010. The first cases occurred after heavy rainfall, and the virus subsequently spread countrywide. To determine the possible effect of environmental conditions and vaccination on RVF virus transmissibility, we estimated the effective reproduction number (R) for the virus over the course of the epidemic by extending the Wallinga and Teunis algorithm with spatial information. Re reached its highest value in mid-February and fell below unity around mid-March, when vaccination coverage was 7.5%-45.7% and vector-suitable environmental conditions were maintained. The epidemic fade-out likely resulted first from the immunization of animals following natural infection or vaccination. The decline in vector-suitable environmental conditions from April onwards and further vaccination helped maintain R below unity. Increased availability of vaccine use data would enable evaluation of the effect of RVF vaccination campaigns.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype

The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses.