Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein.

Dengue virus (DENV) is the most prevalent and burdensome arbovirus transmitted by Aedes mosquitoes, against which there is only a limited licensed vaccine and no approved drug treatment. A Chromobacterium species, C. sp. Panama, isolated from the midgut of A. aegypti is able to inhibit DENV replication within the mosquito and in vitro. Here we show that C. sp. Panama mediates its anti-DENV activity through secreted factors that are proteinous in nature. The inhibitory effect occurs prior to virus attachment to cells, and is attributed to a factor that destabilizes the virion by promoting the degradation of the viral envelope protein. Bioassay-guided fractionation, coupled with mass spectrometry, allowed for the identification of a C. sp. Panama-secreted neutral protease and an aminopeptidase that are co-expressed and appear to act synergistically to degrade the viral envelope (E) protein and thus prevent viral attachment and subsequent infection of cells. This is the first study characterizing the anti-DENV activity of a common soil and mosquito-associated bacterium, thereby contributing towards understanding how such bacteria may limit disease transmission, and providing new tools for dengue prevention and therapeutics.

Detection of white spot syndrome virus (WSSV) in the Pacific oyster Crassostrea gigas.

Oysters Crassostrea gigas were placed at water supply canals of three shrimp farms in Guasave, Mexico where WSSV outbreaks occur. Animals were sampled through April-August and September-December to detect WSSV DNA. By using three different PCR protocols, only oysters from a farm undergoing a WSSV outbreak were found WSSV-positive in gills and digestive gland. Two WSSV amplicons were sequenced and they corresponded over 99% to WSSV genome segments. Results showed that oysters can capture WSSV particles suspended in water. Susceptibility of oysters to WSSV infection and their role as a carrier remain to be determined.

Venezuelan equine encephalitis virus in the mosquito vector Aedes taeniorhynchus: infection initiated by a small number of susceptible epithelial cells and a population bottleneck.

We evaluated infection of Aedes taeniorhynchus mosquitoes, vectors of Venezuelan equine encephalitis virus (VEEV), using radiolabeled virus and replicon particles expressing green (GFP) or cherry fluorescent protein (CFP). More epidemic VEEV bound to and infected mosquito midguts compared to an enzootic strain, and a small number of midgut cells was preferentially infected. Chimeric replicons infected midgut cells at rates comparable to those of the structural gene donor. The numbers of midgut cells infected averaged 28, and many infections were initiated in only 1-5 cells. Infection by a mixture of GFP- and CFP-expressing replicons indicated that only about 100 midgut cells were susceptible. Intrathoracic injections yielded similar patterns of replication with both VEEV strains, suggesting that midgut infection is the primary limitation to transmission. These results indicate that the structural proteins determine initial infection of a small number of midgut cells, and that VEEV undergoes population bottlenecks during vector infection.

Susceptibility/resistance of Anticarsia gemmatalis larvae to its nucleopolyhedrovirus (AgMNPV): Structural study of the peritrophic membrane.

This investigation compares the peritrophic membrane (PM) morphology along the midgut of susceptible (SL) and resistant (RL) Anticarsia gemmatalis larvae to the AgMNPV. The PM increased the thickness from the anterior to the posterior midgut region in both insects strain; however, the intensity of FITC-WGA reaction of the PM in the RL were greater than in SL. The PM in RL was ultrastructurally constituted by several layers of fibrous/vesicular materials in comparison with the few ones in SL. Our results showed that the structure of PM in the RL could be one of the resistance barriers to AgMNPV.

Clinical, pathological and antigenic aspects of bovine viral diarrhea virus (BVDV) type 2 isolates identified in Brazil.

Nucleotide sequencing and phylogenetic analysis of Brazilian bovine viral diarrhea virus (BVDV) field isolates identified four viruses belonging to the genotype 2. Comparison of 5' UTR sequences from these isolates to those of North American BVDV type 2 revealed genomic variations that correlated with the geographic origins of the isolates. Two of the Brazilian type 2 viruses were isolated from clinical cases of gastroenteric/respiratory disease and two were isolated from healthy bovine fetuses. The clinical cases affected young animals (8- and 18-months-old) and were characterized by diarrhea, respiratory signs, extensive oral and digestive tract erosions, conjunctival and vulvar congestion, occasional digestive bleeding and vulvar and heart petechial hemorrhage. Antigenic analysis of these isolates with a panel of 10 monoclonal antibodies revealed marked antigenic differences in the major envelope glycoprotein, gp53/E2, compared to standard laboratory and vaccine BVDV strains. In addition, virus-specific antisera raised to Brazilian BVDV type 2 viruses displayed very low serological cross-reactivity with standard BVDV type 1 strains. Differences up to 64-fold in cross-neutralization titers were observed between BVDV type 1 and Brazilian BVDV type 2 isolates. The identification of BVDV type 2 among Brazilian cattle may have important implications for epidemiological studies, diagnostic and immunization strategies. Furthermore, the low neutralizing activity of BVDV type 1 antisera against the recently identified Brazilian BVDV type 2 isolates raises the question about the degree of protection conferred by BVDV vaccines, most of them based on a single type 1 strain.

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.

Distribution of cytopathic and noncytopathic bovine viral diarrhea virus antigens in tissues of calves following acute experimental infection.

The distribution of cytopathic and noncytopathic biotypes of bovine viral diarrhea virus (BVDV) in the tissues of colostrum-fed and colostrum-deprived calves was investigated. Colostrum-fed (group A) and colostrum-deprived (group B) calves were experimentally infected with the BVDV isolate 80/1, which contains both BVDV biotypes. Colostrum-deprived calves were also experimentally infected with a noncytopathic BVDV (group C) or with a cytopathic BVDV (group D) cloned from the 80/1 isolate. All calves were sequentially euthanized, and a wide range of tissue samples were processed for immunofluorescent and virus isolation studies. In group A, consistent immunofluorescent staining for BVDV was detected in vascular smooth muscle of numerous blood vessels in the tissues examined, mainly at 11 and 13 days postinoculation. A predominance of samples containing cytopathic BVDV was observed in the calves of this group, following virus isolation studies. Both cytopathic and noncytopathic BVDV were detected/recovered from a larger range of specimens in the calves in group B than from the calves in group A. In the calves in all the experimental groups, large amounts of BVDV antigen were detected mainly in tissue samples from the lymphoid and gastrointestinal systems, whereas only minimal amounts of BVDV were detected in the respiratory tract. Abundant noncytopathic BVDV antigen was also detected in pituitary gland and in Langerhans islets in pancreases of colostrum-deprived calves infected with the cloned noncytopathic BVDV. Noncytopathic BVDV was isolated from a wider range of tissues from calves in group C than in the colostrum-deprived calves infected with both BVDV biotypes. A cytopathic BVDV was isolated/detected in retropharyngeal, mesenteric, and abomasal lymph nodes and in thymus of 2 calves in group C. Cytopathic BVDV was detected/isolated mainly from mesenteric lymph nodes and Peyer's patches of the calves in group D.

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells.

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.

Gastrointestinal immune responses in HIV infected subjects.

The gut associated lymphoid tissue is responsible for specific responses to intestinal antigens. During HIV infection, mucosal immune deficiency may account for the gastrointestinal infections. In this review we describe the humoral and cellular mucosal immune responses in normal and HIV-infected subjects.