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.

PGRP-LD mediates A. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis.

Peptidoglycan recognition proteins (PGRPs) and commensal microbes mediate pathogen infection outcomes in insect disease vectors. Although PGRP-LD is retained in multiple vectors, its role in host defense remains elusive. Here we report that Anopheles stephensi PGRP-LD protects the vector from malaria parasite infection by regulating gut homeostasis. Specifically, knock down of PGRP-LD (dsLD) increased susceptibility to Plasmodium berghei infection, decreased the abundance of gut microbiota and changed their spatial distribution. This outcome resulted from a change in the structural integrity of the peritrophic matrix (PM), which is a chitinous and proteinaceous barrier that lines the midgut lumen. Reduction of microbiota in dsLD mosquitoes due to the upregulation of immune effectors led to dysregulation of PM genes and PM fragmentation. Elimination of gut microbiota in antibiotic treated mosquitoes (Abx) led to PM loss and increased vectorial competence. Recolonization of Abx mosquitoes with indigenous Enterobacter sp. restored PM integrity and decreased mosquito vectorial capacity. Silencing PGRP-LD in mosquitoes without PM didn't influence their vector competence. Our results indicate that PGPR-LD protects the gut microbiota by preventing hyper-immunity, which in turn promotes PM structurally integrity. The intact PM plays a key role in limiting P. berghei infection.

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.

Chemokine receptor antagonist block inflammation and therapy Japanese encephalitis virus infection in mouse model.

Japanese encephalitis (JE) is a viral encephalitis disease caused by infection with the Japanese encephalitis virus (JEV). The virus can cross the blood-brain barrier and cause death or long-term sequela in infected humans or animals. In this study, we first investigated the distribution of JEV infection in brain and further analyzed the dynamic change in inflammation related genes, chemokines, as well as pathological characteristics. Results demonstrated that CCR2 and CCR5 antagonist could significantly inhibit the inflammation. The mice treated with CCR2 and CCR5 antagonists had a higher survival rate between 60% and 70%, respectively. In summary, our study thoroughly illustrated the characteristics of the dynamic change in inflammation related genes and chemokines induced by JEV infection. We further indicated that CCR5 and CCR2 are potential targets for treatment of JE.

[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.

[Research Progress on Opportunistic Parasitic Infections].

The incidence of opportunistic parasitic infections is increasing as a result of the growing population with immune deficiency. Currently, studies on opportunistic parasitic infections are limited by the lack of animal models, due to the limited biological knowledge on the opportunistic parasitic pathogens as well as the small number of studies on species identification and typing, epidemiologic status, as well as the source and route of infection. The prevalence of HIV has promoted the research and understanding of opportunistic parasitic infections, which, in turn, has greatly reduced the morbidity and mortality of opportunistic infections. However, there still exists a bottleneck for the control and prevention of infectious diseases, i.e., the lack of sensitive and specific diagnostic methods and effective therapy, due to the complicated clinical manifestations and insufficient notification by clinical physicians. Supported by accumulating basic research, the discovery of diagnostic and therapeutic molecular targets is the key to overcome these problems.

[Diagnosis of an Imported Case of Plasmodium ovale Infection in Nanping City, Fujian].

An imported case previously misdiagnosed as vivax malaria was reviewed. The epidemiological data and blood sample were collected. The detection was conducted by microscopy, rapid diagnostic test (RDT) and nested PCR. The case was finally comfirmed as the first imported case of Plasmodium ovale infection in Nanping.

[Identification of three common sandfies in southern Xinjiang with multiplex PCR].

Based on the variable part of mtDNA CO I gene sequence, a multiplex PCR method was developed for the identification of the three common sandflies (Phlebotomus longiductus, Ph. wui, and Ph. alexandri) in southern Xinjiang. The results demonstrated that this multiplex PCR method was reliable, and could be used to identify the three Phlebotomus species. The PCR product of CO I gene from Ph. longiductus, Ph. wui and Ph. alexandri was 248, 632, and 395 bp, respectively.

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.

Mosquito defensin facilitates Japanese encephalitis virus infection by downregulating the C6/36 cell-surface antiviral protein HSC70B.

Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death and disability whose primary vector is the Culex mosquito. Viral infection induces a series of antimicrobial peptide responses in mosquitoes, and the effector defensin enhances JEV replication in mosquitoes. However, the underlying mechanisms by which defensin enhances JEV are not fully understood. Here, we found that mosquito defensin could downregulate the antiviral protein HSC70B and enhance virus infection in mosquitoes. The cell-surface protein HSC70B was significantly downregulated by JEV infection and defensin treatment. Low levels of HSC70B were beneficial to JEV infection in mosquitoes. Taken together, these findings show that defensin and HSC70B axis facilitates JEV infection in the mosquito.

[Cultivation of pathogenic free-living amoebae].

The isolation and culture of pathogenic free-living amoebae are useful in the diagnosis and research. This review focuses on the methods of isolation and cultivation of pathogenic free-living amoebae, including sample treatment, culture conditions, passage culture, pathogen detection, and maintenance.

[On revision of the tribe aedini mosquitoes taxa record in China, with a proposed new classification system (Diptera: Culicidae)].

This paper reports the rectification results of the tribe aedini mosquitoes formerly recorded in China, using the classification system proposed by Reinert during the recent years. Among all the 171 species of Chinese aedini mosquitoes examined, 160 species could be included in the new classification system. The other 11 species were listed in traditional taxonomic status for further study. The proposed new classification system of the Chinese aedini mosquitoes contained 29 genera, i.e. Aedes, Armigeres, Ayurakitia, Bothaella*, Bruceharrisonius*, Christophersiomyia*, Collessius*, Danielsia*, Downsiomyia*, Edwardsaedes*, Finlaya*, Fredwardsius*, Gilesius*, Heizmannia, Himalaius*, Hopkinsius*, Hulecoeteomyia*, Jihlienius*, Kenknightia*, Luius*, Mucidus*, Neomelaniconion*, Ochlerotatus, Phagomyia*, Scutomyia*, Stegomyia*, Tanakaius*, Udaya, and Verrallina. Among them, 22 genera (*) were new records in China. Besides, the authors made a significant revision to the following 4 species recorded formerly in "Fauna Sinica, Insecta Vol. 8, Diptera: Culicidae": Ae. (Edw.) antuensis as the synonym of Ed. pingpaensis, while Ae. (Sin.) occidentayunnanus, Ae. (Och.) flavidorsalis, and Ae. (Fin.) subsimilis should be rectified as Hz. (Mat.) occidentayunnana, Oc. albineus, and Ud. subsimilis, respectively.

The emerged genotype I of Japanese encephalitis virus shows an infectivity similar to genotype III in Culex pipiens mosquitoes from China.

Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that represents the most significant etiology of childhood viral neurological infections throughout the Asia. During the last 20 years, JEV genotype dominance has shifted from genotype III (GIII) to genotype I (GI). To date, the exact mechanism of this displacement is still not known. Culex (Cx.) mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle. In this study, we used Cx. pipiens mosquitoes from China as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift. We exposed female Cx. pipiens mosquitoes orally to either GI or GIII JEV strains. Midgut, whole mosquitoes, secondary organs, and salivary glands of JEV-infected mosquitoes were collected at 7 and 14 days of post infection (dpi) and subjected to measure the infection rate, replication kinetics, dissemination rate and transmission potential of the infected JEV strains in Cx. pipiens mosquitoes by 50% tissue culture infective dose assay. We found that Cx. pipiens mosquito was competent vector for both GI and GIII JEV infection, with similar infection rates and growth kinetics. After the establishment of infection, Cx. pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar rates of dissemination. A few GI-infected mosquito salivary glands (16.2%) were positive for GI virus, whereas GIII virus was undetectable in GIII-infected mosquito salivary glands at 7 dpi. However, 29.4% (5/17) and 36.3% (8/22) were positive for GI- and GIII-infected mosquito salivary glands at 14 dpi, respectively, showing an increase in JEV positive rate. No statistical difference in the transmission rate between GI- and GIII-infected mosquitoes was detected. Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx. pipiens mosquitoes, suggesting that Cx. pipiens mosquitoes from China may not play a critical role in JEV genotype shift. Although the current data were obtained solely from Cx. pipiens mosquitoes, it is likely that the conclusion drawn could be extrapolated to the role of mosquitoes in JEV genotype shift.

[On a new checklist of the anopheline mosquitoes in China with rectification for some specific names].

This paper presents a new revised "Checklist of the Anopheline Mosquitoes in China" based on the development of the mosquito-taxonomic researches during the years of 1988-2007. The new checklist contained 61 species (subspecies) of anopheline mosquitoes all in China. Twelve species among the past records were omitted because of their invalid specific names which were allocated into following categories: (1) A doubtful record in China, with no typical specimen up to date since last century, e.g. Anopheles campestris reported in Yunnan; (2) Misidentification: An. atroparvus and An. indiensis; (3) Confirmed as synonyms by hybridizing experiments or molecular identification, including 9 species as follows: An. changes, An. dazhaius, An. kiangsuensis, An. anthropophagus, An. kunmingensis, An. xiaokuanus, An. junlianensis, An. yutsushiroensis (part) and An. fluviatilis. Meanwhile, the following rectified 4 anopheline mosquito species should be added to the new checklist: An. belenrae, An. lester, An. pulls, and An. baimaii.

[Maturation of dendritic cells and activation of B-lymphocytes in spleens of ICR mice infected with chloroquine-resistant Plasmodium berghei].

OBJECTIVE: To investigate the relation between activation of B-cells and maturation of dendritic cells (DC) in the spleens of ICR mice infected with chloroquine-resistant (RC) or chloroquine-sensitive (N) strain of Plasmodium berghei. METHODS: Spleens were taken after the mice were infected with N or RC strains of P. berghei and attained certain degree of parasitemia. Changes of B-cells and DCs were examined by pathological method, immunohistochemistry and immunofluorescence methods, transmission electron microscopy (TEM) and flow cytometry technology. RESULTS: Proliferation of white pulps in the spleen of mice infected with RC strain was found as compared to that with N strain. The percentage of cluster of differentiation (CD) 45R/B220, CD19 cells increased in the spleen cells, number of medium and small lymphocytes increased in the germinal centers, the immature and mature plasma cells also increased in the red pulps of spleen in RC strain-infected mice. On the contrary, in the N strain-infected mice spleen, the white pulps were reduced and the red pulps were filled with parasite-infected red blood cells; less small lymphocytes, immature and mature plasma cells were observed in red pulps. The number of CD11c DCs increased, especially in the periarteriolar lymphoid sheath, T cell area; the expression of major histocompatibility complex II (MHC II) on DC was up-regulated in RC strain-infected mice as compared to that in N strain-infected mice. TEM showed that the DCs in RC strain-infected mice spleens were more active than that in N strain-infected mice. CONCLUSION: Infection of RC strain P. berghei increases mature DCs in the spleen, which induces the proliferation of B cells and immune response.

Postoperative infection caused by Acinetobacter baumannii misdiagnosed as a free-living amoeba species in a humeral head hemiarthroplasty patient: a case report.

BACKGROUND: Acinetobacter baumannii is ubiquitous, facultative intracellular, and opportunistic bacterial pathogen. Its unique abilities allow it to survive in a diverse range of environments, including health care settings, leading to nosocomial infections. And its exceptional ability to develop resistance to multiple antibiotics leaves few drug options for treatment. It has been recognized as a leading cause of nosocomial pneumonia and bacteremia over the world. CASE PRESENTATION: In this case, a 73-year-old woman presented with a Neer Group VI proximal humeral fracture. Six hours after a successfully performed hemiarthroplasty, she developed continuous fever. Clinical examination revealed that the vitals were regular. Laboratory and radiographic examinations revealed only elevated procalcitonin levels. Blood culture revealed no bacterial or fungal growth. Cooling treatment and empirical broad-spectrum antibiotic therapy showed no apparent effect. CONCLUSIONS: We report a postoperative infection caused by Acinetobacter baumannii. The infectious pathogen was identified via molecular DNA sequencing and was initially misidentified as a free-living amoeba species upon microscopic examinations. The patient was mistreated with antiamebic combination therapy. Her symptoms persisted for over 4 months and were eventually followed by her death.

[New channels and transporters as anti-malaria drug targets].

In order to get more nutrition from outside the erythrocyte, new channels were induced by malaria parasite. These channels play an important role in physiology of the parasitized cell. They are of interest both as potential targets in their own right and as potential drug targeting routes capable of mediating the entry of cytotoxic drugs into the appropriate compartment of the infected cell. It is hoped that this new anti-malarial strategy will help to create a sustainable anti-malaria-drug-development portfolio for the treatment of malaria.

[Investigation of mosquito abundance and composition around the Rare Birds National Nature Reserve of Yancheng, Jiangsu Province].

OBJECTIVE: To investigate the mosquito abundance and their relative species composition within and outside the Rare Birds National Nature Reserve of Yancheng, Jiangsu Province. METHODS: Sampling was carried out between May and Oct. 2004 at two weeks interval in two foci (the Reserve and nearby residential district) in Sheyang County. Mosquitoes were collected with the modified CDC light trap. Density was calculated, and species were identified. Environmental temperatures, rainfall and relative humidity were monitored during the study. RESULTS: A total of 40,912 mosquitoes were captured in the two foci. The sampled mosquitoes were identified as 4 species belonging to three genera (Anopheles sinensis, Culex pipiens pallens, Cx. tritaeniorhynchus, and Armigeres subalbatus). The most abundant mosquito species was An. sinensis and Cx pipiens pallens, which accounted for 97.7% of the whole number. 92% and 8% of the total amount of mosquitoes were collected from the nature reserve and residential district respectively. The most abundant species in the nature reserve and residential district was An. sinensis (60.6%) and Cx. pipiens pallens (76%), respectively. Within the nature reserve, there were two peaks occurred in adult abundance, in mid- and late July and mid-Sept. The abundance of mosquitoes in the area was positively correlated to the temperature (r = 0.765, P = 0.005). CONCLUSION: The wetland is an ideal breeding place for An. sinensis and Cx pipiens pallens. The peaks of mosquito abundance are in mid- and late July and mid-Sept. It is of importance to carry out surveillance on mosquito vectors with pathogen-transmitting potential.

[Human natural infection of Plasmodium knowlesi].

A blood film slide taken from a patient previously diagnosed as vivax malaria in Mojiang County, Yunnan Province, showing atypical forms. The ring forms had multinuclei, and the late trophozoites trended to form band. The schizonts and gametocytes were somewhat alike to Plasmodium vivax. PCR amplification confirmed that the patient was infected by P. knowlesi.

[Molecular identification of naturally acquired Plasmodium knowlesi infection in a human case].

OBJECTIVE: To confirm the diagnosis of a human case with atypical vivax-malaria from Yunnan Province by molecular technique. METHODS: DNA was extracted from blood films of unidentified sample, and of four known Plasmodium species (P. vivax, P. falciparum, P. knowlesi, and P. cynomolgi). A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of genus- and species-specific (two human malaria species and P. knowlesi) was introduced. RESULTS: The PCR amplification with primer pair specific for P. knowlesi produced a single fragment of 150 bp. Sequence analysis showed that the amplified fragment was identical to the sequence of P. knowlesi. CONCLUSION: The patient was naturally infected with P. knowlesi.