Characterization of Blockade Antibody Responses in GII.2.1976 Snow Mountain Virus-Infected Subjects

Snow Mountain virus (GII.2.1976) is the prototype strain of GII.2 noroviruses (NoVs), which cause an estimated 8% of norovirus outbreaks, yet little is known about the immunobiology of these viruses. To define the human immune response induced by SMV infection and the antigenic relationship between different GII.2 strains that have circulated between 1976 and 2010, we developed a panel of four GII.2 variant virus-like particles (VLPs) and compared their antigenicities by enzyme immunoassay (EIA) and surrogate antibody neutralization (blockade) assays. Volunteers infected with GII.2.1976 developed a mean 167-fold increase in blockade response against the homotypic VLP by day 8 postchallenge. Blockade extended cross-genotype activity in some individuals but not cross-genogroup activity. Polyclonal sera from GII.2.1976-infected volunteers blocked GII.2.1976 significantly better than they blocked GII.2.2002, GII.2.2008, and GII.2.2010, suggesting that blockade epitopes within the GII.2 strains have evolved in the past decade. To potentially map these epitope changes, we developed mouse monoclonal antibodies (MAbs) against GII.2.1976 VLPs and compared their reactivities to a panel of norovirus VLPs. One MAb had broad cross-genogroup EIA reactivity to a nonblockade, linear, conserved epitope. Six MAbs recognized conformational epitopes exclusive to the GII.2 strains. Two MAbs recognized GII.2 blockade epitopes, and both blocked the entire panel of GII.2 variants. These data indicate that the GII.2 strains, unlike the predominant GII.4 strains, have undergone only a limited amount of evolution in blockade epitopes between 1976 and 2010 and indicate that the GII.2-protective component of a multivalent norovirus vaccine may not require frequent reformulation.     

The selection of the ‘wild’: A combined molecular approach for the identification of pure indigenous fish from hybridised populations

The identification of pure indigenous fish from hybridised populations represents a key issue in fisheries management and conservation biology. In the present study an approach for selection of purebred marble trout (Salmo trutta marmoratus C.) individuals out of admixed populations was set up and assessed. In a first step, baseline data sets of pure marble trout and pure brown trout specimens based on twelve microsatellite loci were used to simulate five consecutive generations of admixture. The baseline and the resulting simulation data sets were then combined with data of a ‘real’ hybridised marble trout population to perform a single individual assignment test as implemented in STRUCTURE. By this procedure the assignment approach was calibrated and it was possible to compare admixture coefficients obtained for individuals from different populations. The ranking of individual admixture coefficients on a plot and comparison with simulated data revealed that the test population was composed of pure marble trout individuals, first generation hybrids between marble trout and brown trout, and hybrid backcross specimens between both groups. However, by defining a critical q-value of 0.1 and additionally integrating individual sequence data of the mtDNA control region, it was possible to indicate individuals, which could be selected for the establishment of a pure marble trout strain.     

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.     

The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome encodes several smaller open reading frames (ORFs) located in the 3′ region of the genome that are predicted to express eight novel proteins termed accessory proteins. The accessory proteins are designated ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b and range in size from 39 to 274 amino acids (35, 50). These SARS-CoV-specific ORFs are not present in other coronaviruses and do not display significant homology with any known proteins in the NCBI database. Five of these are predicted to code for polypeptides of greater than 50 amino acids (35, 50). Antibodies reactive against all of the SARS-CoV proteins have been detected in sera isolated from SARS patients, indicating that these proteins are expressed by the virus in vivo (7, 9, 17-19, 45, 59). Expression of three of the ORF proteins has been demonstrated during infection using protein-specific antibodies and include the ORFs 3a, 6, and 7a (12, 37, 41, 60). Six of the eight group-specific ORFs, including ORFs 3a, 3b, 6, 7a, 7b, and 9b, were deleted from recombinant SARS-CoV and shown to be dispensable for in vitro and in vivo replication (66).Related coronaviruses also encode unique accessory proteins in the 3′ region of the genome, often referred to as group-specific ORFs. Similar to SARS-CoV, several of these proteins are dispensable for viral replication. Murine hepatitis virus (MHV) expresses accessory proteins ORFs 2a, 4, and 5a. A recombinant virus in which ORF 2a was deleted replicated normally in vitro but caused attenuated disease in vivo (55). Deletion of the group-specific ORF 7 in porcine coronavirus TGEV also results in reduced replication and virulence in vivo despite normal replication in vitro (38). Similarly, in feline infectious peritonitis virus (FIPV), group-specific proteins are dispensable for replication in cell culture but contribute to pathogenesis in vivo (20). Thus, while the SARS-CoV group specific proteins are unnecessary for in vitro and in vivo replication, their expression may underlie the devastating pathology associated with SARS disease. Detailed characterization of these novel proteins may contribute to a better understanding of SARS pathogenesis and host-virus interactions.The ORF 3a protein is expressed from subgenomic RNA3, which contains the 3a and 3b ORFs (35, 50). The 3a protein, which is the largest group-specific SARS-CoV accessory protein at 274 amino acids, has been reported to localize to the Golgi apparatus, the plasma membrane, and intracellular vesicles of unknown origin (67, 68). The protein is efficiently transported to the cell surface and is also internalized during the process of endocytosis (60).The mechanism of SARS-CoV-induced cell death has been investigated by several groups. Studies to date have used overexpression of individual SARS-CoV ORFs to evaluate their intrinsic cytotoxicity. Using this approach, the following proteins have been reported to cause apoptosis: the 3CL-like protease; spike; ORFs 3a, 3b, and 7a; and the envelope (E), membrane (M), and nucleocapsid (N) proteins (23, 31, 32, 36, 46, 58, 61, 65, 69). However, since all of these reports utilize overexpression of individual proteins, it is unclear whether these effects may be attributable to high, nonphysiological levels of protein and whether they occur during infection. Analysis of recombinant viruses with specific mutations or deletions is necessary to determine the relative contribution of these proteins to the cytotoxicity of SARS-CoV during infection (63). Therefore, the cytotoxic component(s) of SARS-CoV have not been fully defined.Here, we have investigated the function of the ORF 3a protein in the context of SARS-CoV infection and by overexpression. We confirm that ORF 3a contributes to SARS-CoV cytotoxicity using a recombinant strain deficient for expression of ORF 3a. While characterizing this deficient strain, we observed that SARS-CoV-induced vesicle formation, a feature that has been documented in cells from infected SARS patients, is dependent on ORF 3a. Furthermore, we observed that SARS-CoV infection causes Golgi fragmentation by ORF 3a. Additional characterization of 3a in transfected cells revealed that the protein colocalizes with markers of the trans-Golgi network (TGN) and late endosomal pathways and causes an accumulation of these vesicles. Finally, we report that Arf1 overexpression rescued SARS-CoV or 3a-induced Golgi fragmentation, suggesting that the ORF 3a protein may perturb Arf1-mediated vesicle trafficking.     

Identification of Cross-Reactive Norovirus CD4+ T Cell Epitopes

Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4⁺ T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans.Norovirus infection is characterized by the induction of both humoral and cellular immune responses. Humoral immunity in humans following norovirus infection has been described in detail for a limited number of norovirus strains (8, 10, 12, 17, 18, 29). Humans mount specific antibody responses to the infecting strain, which bear complex patterns of unique and cross-reactive, yet undefined, epitopes to other strains within or across genogroups (23, 29). Short-term immunity following homologous norovirus challenge has been documented, but long-term immunity remains controversial (16, 25). Furthermore, no studies to date have demonstrated cross-protection following heterologous norovirus challenge (30). While some susceptible individuals can become reinfected with multiple norovirus strains throughout their lifetimes, the mechanism of short-term protection and the impact of previous exposures on susceptibility to reinfection remain largely unknown.The role of T cells in controlling norovirus infection also remains largely undefined. A single comprehensive study detailing immune responses in genogroup II Snow Mountain virus-infected individuals revealed that CD4⁺ TH1 cells can be stimulated by virus-like particles (VLPs) to secrete gamma interferon (IFN-γ) and interleukin-2 (IL-2) (17). Furthermore, heterologous stimulation from VLPs derived from different norovirus strains within but not across genogroups also induced significant IFN-γ secretion compared to that for uninfected individuals (17). A follow-up study with genogroup I Norwalk virus (NV)-infected individuals confirmed high T cell cross-reactivity within a genogroup as measured by IFN-γ secretion (18). Further, vaccination of humans with VLPs also results in short-term IFN-γ production (27).Because norovirus infection studies in humans are confounded by previous exposure histories, the use of inbred mice maintained in pathogen-free environments allows for the study of norovirus immune responses in a naive background. While mice cannot be infected with human norovirus strains, VLP vaccines expressing norovirus structural proteins induce immune responses that can be measured and studied (14, 20). Mice immunized orally or intranasally with VLP vaccines in the presence of adjuvant similarly induced CD4⁺ IFN-γ responses in Peyer''s patches and spleen (22, 26). Induction of CD8⁺ T cells and secretion of the TH2 cytokine IL-4 were separately noted; however, it is unclear if these responses were influenced by VLPs or the coadministered vaccine adjuvants (22, 26). Further, coadministration of alphavirus adjuvant particles with multivalent norovirus VLP vaccine, including or excluding mouse norovirus (MNV) VLPs, resulted in significantly reduced MNV loads following MNV challenge (21). Multivalent VLP vaccines induced robust receptor-blocking antibody responses to heterologous human strains not included in the vaccine composition (20, 21). Moreover, natural infection with MNV supports a role for T cell immunity in viral clearance and protection (5).To advance our understanding of the scope of the cellular immune response within and between strains, we immunized mice with Venezuelan equine encephalitis (VEE) virus replicon particles (VRPs) expressing norovirus VLPs derived from the Norwalk virus (GI.1-1968) (1) or Farmington Hills virus (FH) (GII.4-2002) (19) strains and analyzed splenocytes for cytokine secretion, epitope identification, and heterologous stimulation. The data presented here indicate that the major capsid proteins of genogroup I and II noroviruses contain robust T cell epitopes that cross-react with related strains in the mouse yet also occur within regions of known variation, especially among the GII.4 noroviruses.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.