Dengue Virus Inoculation Research Articles

Aedes aegypti Mosquito Probing Enhances Dengue Virus Infection of Resident Myeloid Cells in Human Skin.

The most prevalent arthropod-borne viruses, including the dengue viruses, are primarily transmitted by infected mosquitoes. However, the dynamics of dengue virus (DENV) infection and dissemination in human skin following Aedes aegypti probing remain poorly understood. We exposed human skin explants to adult female Ae. aegypti mosquitoes following their infection with DENV-2 by intrathoracic injection. Skin explants inoculated with a similar quantity of DENV-2 by a bifurcated needle were used as controls. Quantitative in situ imaging revealed that DENV replication was greatest in keratinocytes in the base of the epidermis, accounting for 50-60% of all infected cells regardless of the route of inoculation. However, DENV inoculation by Ae. aegypti probing resulted in an earlier and increased viral replication in the dermis, infecting twice as many cells at 24 h when compared to needle inoculation. Within the dermis, enhanced replication of DENV by Ae. aegypti infected mosquitoes was mediated by increased local recruitment of skin-resident macrophages, dermal dendritic cells, and epidermal Langerhans cells relative to needle inoculation. An enhanced but less pronounced influx of resident myeloid cells to the site of mosquito probing was also observed in the absence of infection. Ae. aegypti probing also increased recruitment and infection of dermal mast cells. Our findings reveal for the first time that keratinocytes are the primary targets of DENV infection following Ae. aegypti inoculation, even though most of the virus is inoculated into the dermis during probing. The data also show that mosquito probing promotes the local recruitment and infection of skin-resident myeloid cells in the absence of an intact vasculature, indicating that influx of blood-derived neutrophils is not an essential requirement for DENV spread within and out of skin.

Robust Plasma Cell Response to Skin-Inoculated Dengue Virus in Mice.

Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG+ PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM+ PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67+) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model.

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells.

Lymphocyte activation and hepatic cellular infiltration in immunocompetent mice infected by dengue virus.

Activation and expansion of dengue virus-specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus-induced T cell activation. Mice were inoculated with 10(8) PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT-PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O-glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O-glycosylated CD43 and IFN-gamma production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8(+) T cell to CD4(+) T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8(+) T cell to CD4(+) T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes.

デング熱病原体に関する研究 (XII)

In this communication a report is made on inoculation of dengue virus (Hawaiian strain) in one-day-old hen's egg.The results are summarized as follows.1) The Hawaiian strain of dengue virus has been propagated in one-day-old chick embryos. The titer of embryos harvested within a period of up to 6 days after inoculation, remained at levels of 10-3.6 (mouse adapted strain) to 10-4.5 mouse LD50 (egg successive strain).2) The highest concentration of virus was found in one-day-old chick embryos, and five-days-old chick embryos contained the virus 10 to 23 times less per 0.02g of tissue than the former.But, there was no difference between one-day-old chick embryos and five-days-old chick embryos in the content of virus in whole chicke mbryas.

STUDIES UPON THE ETIOLOGY OF DENGUE FEVER

The dengue cases studied during this epidemic were, as a whole, typical examples of the disease. The onset was sudden, often of a violent character, ushered in with severe headache and backache, vague pains throughout the body, and fever ranging from 101-105 degrees F. On the 2nd or 3rd day a maculopapular eruption appeared, generally on the neck, chest, and arms, but sometimes more widely disseminated. In most instances the fever remained for 3 or 4 days, after which it dropped to normal to rise again in 24 to 48 hours. The secondary rise, while occasionally higher than the primary one, was as a rule of a milder character. A few cases occurred in which marked jaundice existed. As reported for previous epidemics, no fatalities were recorded and hence no human material for histological study was available. Those cases caught in the fastigium of the disease were selected when possible as a source of material. The leucocytic count in all of the observed human cases was below normal. The experiments herein reported upon the transmission of dengue fever to the guinea pig are based upon the use of material secured from sixteen typical human cases of the disease, and upon the inoculation of many animals. Of the 143 animals used for the initial transmission, 42, representing eleven human cases out of the sixteen studied, reacted in a characteristic manner. The reaction occasioned in these animals by the inoculations closely resembles the symptoms seen in human dengue, differing only in the absence of exanthem. The primary pyrexia following regularly after a definite incubation period of 2 to 5 days, the secondary rise in temperature after a 24 to 48 hour remission ("saddleback"curve), and the concomitant fall in the circulating leucocytes present a syndrome identical with that of the human disease. Dark-field and special tinctorial studies of the dengue material, both human and experimental, have failed to reveal any visible spirochetal microorganism. It seems unlikely that an organism of any proportion other than a minute body is the excitant of dengue fever. In the experimentally induced infection of the guinea pigs by the inoculation of dengue virus there occurs a rather constant and characteristic gross alteration of the lymphoid tissues, and of the spleen especially, which latter organ often attains a size three to four times that of the normal. Enlarged lymph glands in the peritoneal areas are also a fairly constant finding. The adrenal glands occasionally show marked enlargement. The histopathology consists of an endothelial proliferation occurring chiefly in connection with the Malpighian bodies of the spleen and the germinal centers of the lymph nodes. Experiments performed with a view of demonstrating the protection afforded the recovered guinea pig were somewhat confused by delayed fatalities or later intercurrent infections to which the dengue-inoculated animal seems highly susceptible. The constant and uniform results for all the positive cases indicate that the virus of dengue fever is not only transmissible to the guinea pig but is susceptible to propagation in this species through an undetermined number of successive generations. After certain of the virus strains had been propagated through four or more generations they were discontinued. Two strains, however, have been continued for 36 and 45 generations respectively. The equally successful results with Berkefeld filtered material and whole defibrinated blood indicate that the excitant of dengue fever belongs in the group of filterable viruses.