Mosquito Defensins Enhance Japanese Encephalitis Virus Infection by Facilitating Virus Adsorption and Entry within the Mosquito.

Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death, and disability. Although the Culex mosquito is the primary vector of JEV, little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via the defensin/lipoprotein receptor-related protein 2 (LRP2) axis. Mosquito defensin bound the ED III domain of the viral envelope (E) protein and directly mediated efficient virus adsorption on the target cell surface; the receptor LRP2, which is expressed on the cell surface, affected defensin-dependent adsorption. As a result, mosquito defensin enhanced JEV infection in the salivary gland, increasing the possibility of viral transmission by mosquitoes. These findings demonstrate the novel role of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.IMPORTANCE In this study, we observed the complex roles of mosquito defensin in JEV infection; mosquito defensin exhibited a weak antiviral effect but strongly enhanced binding. In the latter, defensin directly binds the ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. Together, our results indicate that mosquito defensin plays an important role in facilitating JEV infection and potential transmission.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

FDG PET/CT Findings in Multiple Splenic Amebomas (Amebic Granulomas).

Splenic ameboma (amebic granuloma) is rare. A 38-year-old man with a severe pneumonia due to inhalation of swimming pool water 18 months ago complained of night sweat, tiredness, and anorexia for 1 month. Abdominal ultrasound showed multiple nodules in the spleen. These nodules showed slight enhancement on enhanced CT and intense FDG uptake on PET/CT. Splenectomy was performed. Multiple splenic amebomas were confirmed by pathology. Free-living amoebae were also detected in samples of pleural effusion and blood. Splenic ameboma, although rare, should be included in the differential diagnosis with isolated focally increased FDG activity in the spleen.

[Morphological and Molecular Identification of Acanthamoeba sp., A New Pathogen for Human Respiratory Tract Infection].

Objective: To identify the species of a morphologically Acanthamoeba-like pathogen in sputum from a patient with repeated cough. Methods: Protozoa were isolated from the sputum and cultured for morphological observation of the trophozoites and cysts. DNA was extracted from the cultivated sample, and PCR was performed using primers as follows: 18S universal primers for amoeba family（Ami6F1 and Ami9R） and for amoeba genus（JDP1 and JDP2）, and primers for 18S full-length sequence of S-7 ATCC reference strain of Acanthamoeba griffini （AacGF and AscGR）. The 18S rRNA was sequenced, followed by homology analysis. The maximum likelihood method was used to construct phylogenetic tree. Results: Microscopic examination showed that the trophozites had spine and irregular-shape pseudopodia bulge. The cysts were encapsulated by double membrane layer with the inner membrane having star-like processes. As expected, PCR amplification resulted in bands of 830, 479 and 1 957 bp, respectively, which were blasted to be 99%, 99% and 100% homologous to those of A. griffini（U07412.1）. Phylogenetic tree indicated that this acanthamobe in the patient's sample was 91.4%, 99.6%, 94.5% and 91.8% homologous to keratitis-associated A. castellanii, A. polyphage, A. cullbertsoni and A. rhysodes. Conclusion: The parasite in sputum of the patient with respiratory tract infection is Acanthamoeba griffini.

A systematic study on hemocyte identification and plasma prophenoloxidase from Culex pipiens quinquefasciatus at different developmental stages.

Culexpipiens quinquefasciatus (C. quinquefasciatus) is an important vector that can transmit human diseases such as West Nile virus, lymphatic filariasis, Japanese encephalitis and St. Louis encephalitis. However, very limited research concerning the humoral and cellular immune defenses of C. quinquefasciatus has been done. Here we present the research on hemocyte identification and plasma including hemocyte prophenoloxidase from C. quinquefasciatus at all developmental stages in order to obtain a complete picture of C. quinquefasciatus innate immunity. We identified hemocytes into four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes. Prophenoloxidase (PPO) is an essential enzyme to induce melanization after encapsulation. PPO-positive hemocytes and plasma PPO were observed at all developmental stages. As for specific hemocyte types, prophenoloxidase was found in the plasmatocytes at larval stage alone and in the smallest prohemocytes during almost all developmental stages. Moreover, the granulocytes were PPO-positive from blood-fed female mosquitoes and oenocytoids were observed PPO-positive in pupae and in adult females after blood-feeding. As for plasma, there were different patterns of PPO in C. quinquefasciatus at different developmental stages. These results are forming a basis for further studies on the function of C. quinquefasciatus hemocytes and prophenoloxidase as well as their involvement in fighting against mosquito-borne pathogens.

[Preparation and identification of monoclonal antibodies against the Region II+ motif in circumsporozoite protein of Plasmodium falciparum].

OBJECTIVE: To develop and identify the monoclonal antibodies (McAbs) against Region II+ motif in circumsporozoite protein of Plasmodium falciparum. METHODS: BALB/c mice were immunized with 12 peptides within Region II+ in circumsporozoite protein of P. falciparum. Spleen cells isolated from the immunized mice were fused with myeloma cell. After three times screening with ELISA, 3 positive hybridoma cell lines were obtained. RESULTS: ELISA test indicated that the McAbs reacted with recombinant circumsporozoite protein fragment containing tandemly repeat region and conserved Region II+. IFA test showed that the McAbs recognized not only the sporozoites of P. falciparum, but also the sporozoifes of P. yoelii. CONCLUSION: McAbs obtained can probe the Region II+ motif in circumsporozoite protein of P. falciparum, which might also recognize that of other Plasmodium species.