Cladocera from the Sudan: Red Sea Hills,Jebel Marra and valley of the main Nile

Twenty species of Cladocera are reported from the Nile, where lacustrine species dominate, and from Jebel Marra and the Red Sea Hills, where chydorids dominate. The community found in the Red Sea Hills is more typically desertic than that of Jebel Marra, which appears closely related to the fauna of the West and Central African Sahel.     

A revision of the Scapholeberinae (Crustacea: Cladocera)

The group of hyponeustonic daphniid cladocera previously known under the generic name Scapholeberis is raised to the rank of a subfamily (Scapholeberinae) and contains two genera, Megafenestra n.gen. (2 species), and Scapholeberis s.s. (7 species and one subspecies). The characters upon which this revision is based are: structure of the rostrum, structure of the first antennae, structure of trunk limbs 1, 2, and 5, presence and nature of headpore(s), structure of the ventral rim of the valves, armature of the distal rim of the valves, structure of the postabdomen and its end-claws. Males were examined in all but two species, and proved to be more primitive than females and much less diagnostic than in the Chydoridae. It also appeared that the shape and armature of the postabdomen are less important taxonomical tools than heretofore supposed. In more than half of the species, it is of generalised shape and hardly usable beyond the species-group level. The same, although less drastically, holds true for the P1 of males. Of greatest diagnostic value at the species level are the shape of the rostrum and the armature of the valve margins. A phylogeny of the subfamily is proposed, with Megafenestra as the more primitive genus, closely allied to Ceriodaphnia, and within Scapholeberis, S. mucronata as the most apomorphic species. In biogeographical respect, no species is cosmopolitan, although their ranges may be of continental dimensions. Species and populations appear to concentrate in the temperate and continental climate belts of the northern hemisphere, but more research in the southern hemisphere is needed. A key for the identification of all species is given.     

Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages

The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4 degrees C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP(5) and that the N protein bound to heparin as a homodimer. GP(3), which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP(5) complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.     

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.     

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.     

Standardized white spot syndrome virus (WSSV) inoculation procedures for intramuscular or oral routes

In the past, strategies to control white spot syndrome virus (WSSV) were mostly tested by infectivity trials in vivo using immersion or per os inoculation of undefined WSSV infectious doses, which complicated comparisons between experiments. In this study, the reproducibility of 3 defined doses (10, 30 and 90 shrimp infectious doses 50% endpoint [SID50]) of WSSV was determined in 3 experiments using intramuscular (i.m.) or oral inoculation in specific pathogen-free (SPF) Litopenaeus vannamei. Reproducibility was determined by the time of onset of disease, cumulative mortality, and median lethal time (LT50). By i.m. route, the 3 doses induced disease between 24 and 36 h post inoculation (hpi). Cumulative mortality was 100% at 84 hpi with doses of 30 and 90 SID50 and 108 hpi with a dose of 10 SID50. The LT50 of the doses 10, 30 and 90 SID50 were 52, 51 and 49 hpi and were not significantly different (p > 0.05). Shrimp orally inoculated with 10, 30 or 90 SID50 developed disease between 24 and 36 hpi. Cumulative mortality was 100% at 108 hpi with doses of 30 and 90 SID50 and 120 hpi with a dose of 10 SID50. The LT50 of 10, 30 and 90 SID50 were 65, 57 and 50 hpi; these were significantly different from each other (p < 0.05). A dose of 30 SID50 was selected as the standard for further WSSV challenges by i.m. or oral routes. These standardized inoculation procedures may be applied to other crustacea and WSSV strains in order to achieve comparable results among experiments.     

Identification of a Putative Receptor for Porcine Reproductive and Respiratory Syndrome Virus on Porcine Alveolar Macrophages

To identify the receptor which may determine the macrophage tropism of porcine reproductive and respiratory syndrome virus (PRRSV), monoclonal antibodies (MAbs) against porcine alveolar macrophages (PAM) were produced. Two MAbs (41D3 and 41D5) which completely blocked PRRSV infection of PAM were further characterized. It was found that they reduce the attachment of PRRSV to PAM and immunoprecipitate a 210-kDa membrane protein from PAM. This protein was detected on the cell membranes of PAM but not of PRRSV-nonpermissive cells. A colocalization was found between the reactive sites of MAb 41D3 and PRRSV on PAM membranes. All PRRSV-infected cells in tissues of experimentally infected pigs reacted with MAb 41D3. Taken together, all these data suggest that the identified 210-kDa membrane protein is a putative receptor for PRRSV on porcine macrophages.     

A tyrosine-based motif in the cytoplasmic tail of pseudorabies virus glycoprotein B is important for both antibody-induced internalization of viral glycoproteins and efficient cell-to-cell spread

Pseudorabies virus (PRV), a swine alphaherpesvirus, is capable of causing viremia in vaccinated animals. Two mechanisms that may help PRV avoid recognition by the host immune system during this viremia are direct cell-to-cell spread in tissue and antibody-induced internalization of viral cell surface glycoproteins in PRV-infected blood monocytes, the carrier cells of the virus in the blood. PRV glycoprotein B (gB) is crucial during both processes. Here we show that mutating a tyrosine residue located in a YXXPhi motif in the gB cytoplasmic tail results in decreased efficiency of cell-to-cell spread and a strong reduction in antibody-induced internalization of viral cell surface glycoproteins. Mutating the dileucine motif in the gB tail led to an increased cell-to-cell spread of the virus and the formation of large syncytia.     

Evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus OC43

The close genetic and antigenic relatedness among the group 2 coronaviruses human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and porcine hemagglutinating encephalomyelitis virus (PHEV) suggests that these three viruses with different host specificities diverged fairly recently. In this study, we determined the complete genomic sequence of PHEV (strain PHEV-VW572), revealing the presence of a truncated group 2-specific ns2 gene in PHEV in comparison to other group 2 coronaviruses. Using a relaxed molecular clock approach, we reconstructed the evolutionary relationships between PHEV, BCoV, and HCoV-OC43 in real-time units, which indicated relatively recent common ancestors for these species-specific coronaviruses.