Characterization of proinflammatory cytokines profile during acute SARS-CoV-2 infection in people with human immunodeficiency virus.

Persistent inflammation in chronic HIV infection may affect immune responses against SARS-CoV-2 infection. Plasma levels of multiple proinflammatory cytokines during acute SARS-CoV-2 infection were assessed in people with HIV (PWH) with effective cART. There were no significant differences in any of the tested cytokines between COVID-19 severity in PWH, while most of them were significantly higher in individuals with severe disease in HIV-uninfected individuals, suggesting that excess cytokines release by hyper-inflammatory responses does not occur in severe COVID-19 with HIV infection. The strong associations between the cytokines observed in HIV-uninfected individuals, especially between IFN-α/TNF-α and other cytokines, were lost in PWH. The steady state plasma levels of IP-10, ICAM-1, and CD62E were significantly higher in PWH, indicating that PWH are in an enhanced inflammatory state. Loss of the several inter-cytokine correlations were observed in in vitro LPS stimuli-driven cytokines production in PWH. These data suggest that inflammatory responses during SARS-CoV-2 infection in PWH are distinct from those in HIV-uninfected individuals, partially due to the underlying inflammatory state and/or impairment of innate immune cells.

Longitudinal analysis of microbiome composition in Ghanaians living with HIV-1.

Human immunodeficiency virus (HIV) 1 infection is known to cause gut microbiota dysbiosis. Among the causes is the direct infection of HIV-1 in gut-resident CD4+ T cells, causing a cascade of phenomena resulting in the instability of the gut mucosa. The effect of HIV infection on gut microbiome dysbiosis remains unresolved despite antiretroviral therapy. Here, we show the results of a longitudinal study of microbiome analysis of people living with HIV (PLWH). We contrasted the diversity and composition of the microbiome of patients with HIV at the first and second time points (baseline_case and six months later follow-up_case, respectively) with those of healthy individuals (baseline_control). We found that despite low diversity indices in the follow-up_case, the abundance of some genera was recovered but not completely, similar to baseline_control. Some genera were consistently in high abundance in PLWH. Furthermore, we found that the CD4+ T-cell count and soluble CD14 level were significantly related to high and low diversity indices, respectively. We also found that the abundance of some genera was highly correlated with clinical features, especially with antiretroviral duration. This includes genera known to be correlated with worse HIV-1 progression (Achromobacter and Stenotrophomonas) and a genus associated with gut protection (Akkermansia). The fact that a protector of the gut and genera linked to a worse progression of HIV-1 are both enriched may signify that despite the improvement of clinical features, the gut mucosa remains compromised.

High-level resistance to non-nucleos(t)ide reverse transcriptase inhibitor based first-line antiretroviral therapy in Ghana; A 2017 study.

Expanding access to effective antiretroviral therapy (ART) is a major tool for management of Human Immunodeficiency Virus (HIV) infection. However, rising levels of HIV drug-resistance have significantly hampered the anticipated success of ART in persons living with HIV (PLWH), particularly those from Africa. Though great strides have been made in Ghana toward achieving the UNAIDS "95-95-95" target, a substantial number of PLWH receiving ART have not attained viral suppression. This study investigated patterns of drug resistance mutations in ART naïve as well as ART-experienced PLWH receiving first-line regimen drugs from Ghana. In a cross-sectional study, blood samples were collected from HIV-1 infected adults (≥18 years) attending HIV/AIDS clinic at the Eastern Regional Hospital, Koforidua, Ghana from September to October 2017. Viral RNA isolated from plasma were subjected to genotypic drug resistance testing for Protease Inhibitors (PI), Reverse Transcriptase Inhibitors (RTI), and Integrase Strand Transfer Inhibitors (INSTI). A total of 95 (84 ART experienced, 11 ART naïve) HIV-1 infected participants were sampled in this study. Sixty percent (50/84) of the ART-experienced participants were controlling viremia (viral load < 1,000 copies/ml). Of the 95 patient samples, 32, 34, and 33 were successfully sequenced for protease, reverse-transcriptase, and integrase regions, respectively. The dominant HIV-1 subtypes detected were CRF02_AG (70%), and A3 (10%). Major drug resistance associated mutations were only detected for reverse transcriptase inhibitors. The predominant drug resistance mutations were against nucleos(t)ide reverse transcriptase inhibitors (NRTI)-M184V/I and non-nucleos(t)ide reverse transcriptase inhibitors (NNRTI)-K103N. In the ART-experienced group, M184V/I and K103N were detected in 54% (15/28) and 46% (13/28) of individuals, respectively. Both mutations were each detected in 33% (2/6) of ART naïve individuals. Multiclass resistance to NRTI and NNRTI was detected in 57% of ART-experienced individuals and two ART naïve individuals. This study reports high-level resistance to NNRTI-based antiretroviral therapy in PLWH in Ghana. However, the absence of major PI and INSTI associated-mutations is a good signal that the current WHO recommendation of Dolutegravir in combination with an NRTI backbone will yield maximum benefits as first-line regimen for PLWH in Ghana.

Intensive single-cell analysis reveals immune-cell diversity among healthy individuals.

Immune responses are different between individuals and personal health histories and unique environmental conditions should collectively determine the present state of immune cells. However, the molecular systems underlying such heterogeneity remain elusive. Here, we conducted a systematic time-lapse single-cell analysis, using 171 single-cell libraries and 30 mass cytometry datasets intensively for seven healthy individuals. We found substantial diversity in immune-cell profiles between different individuals. These patterns showed daily fluctuations even within the same individual. Similar diversities were also observed for the T-cell and B-cell receptor repertoires. Detailed immune-cell profiles at healthy statuses should give essential background information to understand their immune responses, when the individual is exposed to various environmental conditions. To demonstrate this idea, we conducted the similar analysis for the same individuals on the vaccination of influenza and SARS-CoV-2. In fact, we detected distinct responses to vaccines between individuals, although key responses are common. Single-cell immune-cell profile data should make fundamental data resource to understand variable immune responses, which are unique to each individual.

Global research alliance in infectious disease: a collaborative effort to combat infectious diseases through dissemination of portable sequencing.

OBJECTIVE: To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION's operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. RESULTS: One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

Dysbiotic Fecal Microbiome in HIV-1 Infected Individuals in Ghana.

HIV-1 infected individuals under antiretroviral therapy can control viremia but often develop non-AIDS diseases such as cardiovascular and metabolic disorders. Gut microbiome dysbiosis has been indicated to be associated with progression of these diseases. Analyses of gut/fecal microbiome in individual regions are important for our understanding of pathogenesis in HIV-1 infections. However, data on gut/fecal microbiome has not yet been accumulated in West Africa. In the present study, we examined fecal microbiome compositions in HIV-1 infected adults in Ghana, where approximately two-thirds of infected adults are females. In a cross-sectional case-control study, age- and gender-matched HIV-1 infected adults (HIV+; n = 55) and seronegative controls (HIV-; n = 55) were enrolled. Alpha diversity of fecal microbiome in HIV+ was significantly reduced compared to HIV- and associated with CD4 counts. HIV+ showed reduction in varieties of bacteria including Faecalibacterium, the most abundant in seronegative controls, but enrichment of Proteobacteria. Ghanaian HIV+ exhibited enrichment of Dorea and Blautia; bacteria groups whose depletion has been reported in HIV-1 infected individuals in several other cohorts. Furthermore, HIV+ in our cohort exhibited a depletion of Prevotella, a genus whose enrichment has recently been shown in men having sex with men (MSM) regardless of HIV-1 status. The present study revealed the characteristics of dysbiotic fecal microbiome in HIV-1 infected adults in Ghana, a representative of West African populations.

Fecal Microbiome Composition in Healthy Adults in Ghana.

Recent studies have indicated an association between gut microbiome composition and various disorders, including infectious diseases. The composition of the microbiome differs among ethnicities and countries, possibly resulting in diversified interactions between host immunity and the gut microbiome. Characterization of baseline microbiome composition in healthy people is an essential step for better understanding of the biological interactions associated with individual populations. However, data on the gut/fecal microbiome have not been accumulated for individuals in West Africa. In the present study, we examined the fecal microbiome composition in healthy adults in Ghana. Toward this, 16S rRNA gene libraries were prepared using bacterial fractions derived from 55 Ghanaian adults, which were then subjected to next-generation sequencing. The fecal microbiome of the Ghanaian adults was dominated by Firmicutes (Faecalibacterium, Subdoligranulum, and Ruminococcaceae UCG-014), Proteobacteria (Escherichia-Shigella and Klebsiella), and Bacteroidetes (Prevotella 9 and Bacteroides), consistent with previous observations in African cohorts. Further, our analysis revealed differences in microbiome composition and a lower diversity of the fecal microbiome in the Ghanaian cohort compared with those reported in non-African countries. This is the first study to describe substantial fecal microbiome data obtained using high-throughput metagenomic tools on samples derived from a cohort in Ghana. The data may provide a valuable basis for determining the association between the fecal microbiome and progression of various diseases in West African populations.

The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti.

Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.

Portable sequencer in the fight against infectious disease.

Infectious disease is still a major threat in the world today. Five decades ago, it was considered soon to be eradicated, but the adaptation of pathogens to environmental pressure, such as antimicrobials, encouraged the emergence and reemergence of infectious disease. The fight with infectious disease starts with prevention, diagnosis, and treatment. Diagnosis can be upheld by observing the cause of disease under the microscope or detecting the presence of nucleic acid and proteins of the pathogens. The molecular techniques span from classical polymerase chain reaction (PCR) to sequencing the nucleic acid composition. Here, we are reviewing the works have been undertaken to utilize a portable sequencer, MinION, in various aspects of infectious disease management.

Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.

On-Site MinION Sequencing.

DNA sequencing has reached an unprecedented level with the advent of Oxford Nanopore Technologies' MinION. The low equipment investment, ease of library preparation, small size, and powered only by a laptop computer enable the portability for on-site sequencing. MinION has had its role in clinical, biosecurity, and environmental fields. Here, we describe the many facets of on-site sequencing with MinION. First, we will present some field works using MinION. We will discuss the requirements for targeted or whole genome sequencing and the challenges faced by each technique. We will also elaborate the bioinformatics procedures available for data analysis in the field. MinION has greatly changed the way we do sequencing by bringing the sequencer closer to the biodiversity. Although numerous limitations exist for MinION to be truly portable, improvements of procedures and equipment will enhance MinION's role in the field.

Nanopore sequencing of drug-resistance-associated genes in malaria parasites, Plasmodium falciparum.

Here, we report the application of a portable sequencer, MinION, for genotyping the malaria parasite Plasmodium falciparum. In the present study, an amplicon mixture of nine representative genes causing resistance to anti-malaria drugs is diagnosed. First, we developed the procedure for four laboratory strains (3D7, Dd2, 7G8, and K1), and then applied the developed procedure to ten clinical samples. We sequenced and re-sequenced the samples using the obsolete flow cell R7.3 and the most recent flow cell R9.4. Although the average base-call accuracy of the MinION sequencer was 74.3%, performing >50 reads at a given position improves the accuracy of the SNP call, yielding a precision and recall rate of 0.92 and 0.8, respectively, with flow cell R7.3. These numbers increased significantly with flow cell R9.4, in which the precision and recall are 1 and 0.97, respectively. Based on the SNP information, the drug resistance status in ten clinical samples was inferred. We also analyzed K13 gene mutations from 54 additional clinical samples as a proof of concept. We found that a novel amino-acid changing variation is dominant in this area. In addition, we performed a small population-based analysis using 3 and 5 cases (K13) and 10 and 5 cases (PfCRT) from Thailand and Vietnam, respectively. We identified distinct genotypes from the respective regions. This approach will change the standard methodology for the sequencing diagnosis of malaria parasites, especially in developing countries.

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.

BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

Single nucleotide polymorphisms in genes encoding penicillin-binding proteins in ß-lactamase-negative ampicillin-resistant Haemophilus influenzae in Japan.

OBJECTIVE: ß-Lactamase-negative ampicillin-resistant Haemophilus influenzae is a common opportunistic pathogen of hospital- and community-acquired infections, harboring multiple single nucleotide polymorphisms in the ftsI gene, which codes for penicillin-binding protein-3. The objectives of this study were to perform comprehensive genetic analyses of whole regions of the penicillin-binding proteins in H. influenzae and to identify additional single nucleotide polymorphisms related to antibiotic resistance, especially to ampicillin and other cephalosporins. RESULTS: In this genome analysis of the ftsI gene in 27 strains of H. influenzae, 10 of 23 (43.5%) specimens of group III genotype ß-lactamase-negative ampicillin-resistant H. influenzae were paradoxically classified as ampicillin-sensitive phenotypes. Unfortunately, we could not identify any novel mutations that were significantly associated with ampicillin minimum inhibitory concentrations in other regions of the penicillin-binding proteins, and we reconfirmed that susceptibility to ß-lactam antibiotics was mainly defined by previously reported SNPs in the ftsI gene. We should also consider detailed changes in expression that lead to antibiotic resistance in the future because the acquisition of resistance to antimicrobials can be predicted by the expression levels of a small number of genes.

A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer.

BACKGROUND: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. METHODS: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. RESULTS: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. CONCLUSIONS: Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

Ribavirin inhibits Zika virus (ZIKV) replication in vitro and suppresses viremia in ZIKV-infected STAT1-deficient mice.

Zika fever, a mosquito-borne infectious disease caused by Zika virus (ZIKV), is an epidemic disease for which no effective therapy has been established. The recent outbreaks of ZIKV in Brazil and French Polynesia have been linked to a considerable increase in the incidence of fetal microcephaly and other diseases such as Guillain-Barre syndrome. Because there is currently no specific therapy or vaccine, the early exploitation of a method to prevent expansion of ZIKV is a high priority. To validate commonly used antiviral drugs, we evaluated the effect of ribavirin, a drug used to treat hepatitis C with interferon-ß (IFN-ß), on ZIKV replication. In mammalian cells, we observed an inhibitory effect of ribavirin on ZIKV replication and ZIKV-induced cell death without cytotoxic effect. Furthermore, we found that STAT1-deficient mice, which lack type I IFN signaling, were highly sensitive to ZIKV infection and exhibited lethal outcome. Ribavirin abrogated viremia in ZIKV-infected STAT-1-deficient mice. These data suggest that the inhibition of viral RNA-dependent RNA polymerases may be effective for treatment of ZIKV infection. Our data provide a new insight into the mechanisms for inhibition of ZIKV replication and prevention of Zika fever.

Serotyping dengue virus with isothermal amplification and a portable sequencer.

The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples. Here, we report the development of a novel method to detect and genotype tropical disease pathogens, using dengue fever as a model. By combining the sequencer with isothermal amplification that only requires a water bath, we were able to amplify and sequence target viral genomes with ease. Starting from a serum sample, the entire procedure could be finished in a single day. The analysis of blood samples collected from 141 Indonesian patients demonstrated that this method enables the clinical identification and serotyping of the dengue virus with high sensitivity and specificity. The overall successful detection rate was 79%, and a total of 58 SNVs were detected. Similar analyses were conducted on 80 Vietnamese and 12 Thai samples with similar performance. Based on the obtained sequence information, we demonstrated that this approach is able to produce indispensable information for etiologically analyzing annual or regional diversifications of the pathogens.

Genetic polymorphisms in Plasmodium falciparum chloroquine resistance genes, pfcrt and pfmdr1, in North Sulawesi, Indonesia.

BACKGROUND: Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. RESULTS: Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). CONCLUSIONS: Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.

Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus

Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan. Methods: Serotype 2 ThNH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai (Ae. f. miyarai), Aedes galloisi (Ae. galloisi) and Aedes albopictus (Ae. albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test. Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai, Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection. All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally admin-istered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes (P>0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar (P=0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species (Japanese and Thai). Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island (near Okinawa Island) and Ae. galloisi from Hokkaido (Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.

Dengue transmission model by means of viremic adult immuno-competent mouse.

BACKGROUND: Dengue virus infection manifests in three distinct forms in humans: dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Infection with the virus is a fatal disease; no vaccine is available and prevention depends on interruption of the chain of transmission. The study of dengue viral transmission by mosquitoes is hindered due to the lack of an affordable animal model. In general, immuno-competent mice are used as a simple and inexpensive animal model, but mice are not susceptible to dengue virus infection and therefore viremia will not occur following the inoculation of the virus in such mice. Here, we report a method for creating artificial viremia in immuno-competent mice, and further demonstrate the use of viremic mice to simultaneously infect a large number of Aedes aegypti. METHODS: We infected K562 cells with DENV-2 in the presence of an antibody against DENV-4. We then incubated the cells for 2 d before injecting the infected cells into C3H mice. After 5 h incubation, we allowed 100-150 female Aedes aegypti to feed on blood from the mice directly. We collected blood samples from the mice and from randomly selected Ae. aegypti at 2, 6, 12, and 24 h post-blood meal and screened the samples for DENV-2 genome as well as for virus concentration. RESULTS: Our procedure provided high virus concentrations in the mice for at least 7 h after viral inoculation. We found that 13 out of 14 randomly picked mosquitoes were infected with DENV-2. High concentrations of virus were detected in the mosquitoes until at least 12 h post-infection. CONCLUSIONS: Using the viremic immuno-competent mouse, we show that mass infection of Ae. aegypti is achievable. Compared to other infection techniques using direct inoculation, membrane-feeding, or immuno-deficient/humanized mice, we are confident that this method will provide a simpler and more efficient infection technique.