Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment

Coinfection of a cell by two different strains of a segmented virus can give rise to a “reassortant” with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous “backbone.” To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34⁺ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.     

A Chemokine-Like Viral Protein Enhances Alpha Interferon Production by Plasmacytoid Dendritic Cells but Delays CD8+ T Cell Activation and Impairs Viral Clearance

Murine cytomegalovirus encodes numerous proteins that act on a variety of pathways to modulate the innate and adaptive immune responses. Here, we demonstrate that a chemokine-like protein encoded by murine cytomegalovirus activates the early innate immune response and delays adaptive immunity, thereby impairing viral clearance. The protein, m131/129 (also known as MCK-2), is not required to establish infection in the spleen; however, a mutant virus lacking m131/129 was cleared more rapidly from this organ. In the absence of m131/129 expression, there was enhanced activation of dendritic cells (DC), and virus-specific CD8⁺ T cells were recruited into the immune response earlier. Viral mutants lacking m131/129 elicited weaker production of alpha interferon (IFN-α) at 40 h postinfection, indicating that this protein exerts its effects during early rounds of viral replication in the spleen. Furthermore, while wild-type and mutant viruses activated plasmacytoid dendritic cells (pDC) equally at this time, as measured by the upregulation of costimulatory molecules, the presence of m131/129 stimulated more pDC to secrete IFN-α, accounting for the stronger IFN-α response than from the wild-type virus. These data provide evidence for a novel immunomodulatory function of a viral chemokine and expose the multifunctionality of immune evasion proteins. In addition, these results broaden our understanding of the interplay between innate and adaptive immunity.     

Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.     

Genome Scale Evolution of Myxoma Virus Reveals Host-Pathogen Adaptation and Rapid Geographic Spread

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.     

Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10⁹ and 3.0 × 10⁹ mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques'' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.     

The effects of predator removal on mallard production and population change in northeastern North Dakota

In 1994, Delta Waterfowl Foundation began trapping mammalian meso-predators in North Dakota during the breeding season in an attempt to increase waterfowl nest success and enhance recruitment into the fall flight and subsequent breeding population. Multiple studies on these sites demonstrated that removing predators results in near doubling of nest success, which previous simulation modeling suggests is the most influential vital rate influencing the population growth rate of mid-continent mallards (Anas platyrhynchos). We present an assessment of the impact of predator removal on mallard production using population models. We conducted this study on 9 township-sized (93.2 km²) sites (4–8 sites annually per vital rate) in northeastern North Dakota from 2006–2008. Trappers removed mammalian meso-predators on 5 sites and the other 4 served as unmanaged reference sites. To estimate recruitment, we used derived estimates and process variance of pair numbers, hen success (nest survival corrected for renesting), initial brood size, pre-fledging survival, and post-fledging survival, along with previously published estimates of breeding propensity and adult female survival rates. Trapped sites had greater hen success (H = 0.69, = 0.03) than reference sites (H = 0.53, = 0.06), but similar indicated breeding pairs, initial brood size, and pre-fledging survival. We estimated that females on trapped sites added 140 more mallards of both sexes to the fall flight than females on reference sites, at an approximate cost of $74.29 per incremental mallard. Additionally, trapping predators provided a marginal increase (0.04) in finite population growth. We found that predator removal targeted at mammalian nest predators did not produce as many incremental mallards as previously thought and may not be a viable strategy for increasing mallard productivity under conditions similar to those observed during this study. We conducted a sensitivity analysis and determined that pre-fledging survival was the most influential factor regulating mallard population growth. Although hen success increased as a result of trapping, duckling survival became a limiting factor. We suggest that waterfowl managers assess multiple vital rates to determine the likelihood that management actions focused on a single parameter, such as nest success, will yield desired population level effects. © 2012 The Wildlife Society.