Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake virus.

BACKGROUND: Mosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur Lake virus (EBIV), belonging to the genus Orthobunyavirus within the family Peribunyaviridae, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. METHODS: To investigate the range of mosquito vectors for OYAV (strain SZC50) and EBIV (strain Cu20-XJ), the susceptibility of four mosquito species (Culex pipiens pallens, Cx. quinquefasciatus, Aedes albopictus, and Ae. aegypti) was measured through artificial oral infection. Then, mosquito species with a high infection rate (IR) were chosen to further evaluate the dissemination rate (DR), transmission rate (TR), and transmission efficiency. The viral RNA in each mosquito sample was determined by RT-qPCR. RESULTS: The results revealed that for OYAV, Cx. pipiens pallens had the highest IR (up to 40.0%) among the four species, but the DR and TR were 4.8% and 0.0%, respectively. For EBIV, Cx. pipiens pallens and Cx. quinquefasciatus had higher IR compared to Ae. albopictus (1.7%). However, the EBIV RNA and infectious virus were detected in Cx. pipiens pallens, with a TR of up to 15.4% and a transmission efficiency of 3.3%. CONCLUSIONS: The findings indicate that Cx. pipiens pallens was susceptible to OYAV but had an extremely low risk of transmitting the virus. Culex pipiens pallens and Cx. quinquefasciatus were susceptible to EBIV, and Cx. pipiens pallens had a higher transmission risk to EBIV than Cx. quinquefasciatus.

Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus.

Mosquito-borne viruses cause various infectious diseases in humans and animals. Tibet orbivirus (TIBOV), a newly identified arbovirus, efficiently replicates in different types of vertebrate and mosquito cells, with its neutralizing antibodies detected in cattle and goats. However, despite being isolated from Culicoides midges, Anopheles, and Culex mosquitoes, there has been a notable absence of systematic studies on its vector competence. Thus, in this study, Aedes aegypti and Culex pipiens pallens were reared in the laboratory to measure vector susceptibility through blood-feeding infection. Furthermore, RNA sequencing was used to examine the overall alterations in the Ae. aegypti transcriptome following TIBOV infection. The results revealed that Ae. aegypti exhibited a high susceptibility to TIBOV compared to Cx. p. pallens. Effective replication of the virus in Ae. aegypti midguts occurred when the blood-feeding titer of TIBOV exceeded 105 plaque-forming units mL-1. Nevertheless, only a few TIBOV RNA-positive samples were detected in the saliva of Ae. aegypti and Cx. p. pallens, suggesting that these mosquito species may not be the primary vectors for TIBOV. Moreover, at 2 dpi of TIBOV, numerous antimicrobial peptides downstream of the Toll and Imd signaling pathways were significantly downregulated in Ae. aegypti, indicating that TIBOV suppressed mosquitos' defense to survive in the vector at an early stage. Subsequently, the stress-activated protein kinase JNK, a crucial component of the MAPK signaling pathway, exhibited significant upregulation. Certain genes were also enriched in the MAPK signaling pathway in TIBOV-infected Ae. aegypti at 7 dpi.IMPORTANCETibet orbivirus (TIBOV) is an understudied arbovirus of the genus Orbivirus. Our study is the first-ever attempt to assess the vector susceptibility of this virus in two important mosquito vectors, Aedes aegypti and Culex pipiens pallens. Additionally, we present transcriptome data detailing the interaction between TIBOV and the immune system of Ae. aegypti, which expands the knowledge about orbivirus infection and its interaction with mosquitoes.

Development and evaluation of RT-qPCR assays for two neglected orthobunyaviruses: Oya virus and Ebinur Lake virus.

OBJECTIVES: Oya virus (OYAV) and Ebinur lake virus (EBIV) belong to the genus Orthobunyavirus within the Peribunyaviridae family, and both are recognized as the novel virus with potential threat to the animal or public health. Given their potential to cause outbreaks and their detection in diverse samples across different regions, the need for a reliable and efficient molecular detection method for OYAV and EBIV becomes imperative. METHODS: The S-segment of OYAV and EBIV was used for designing specific primer and probe sets, which were employed in a real-time reverse transcription quantitative PCR (RT-qPCR) assay. The analytical performance of these assays, encompassing specificity, sensitivity, reproducibility, and fitness for purpose, was thoroughly evaluated across various sample matrices. RESULTS: The developed RT-qPCR assays were very specific to their respective targets. Both assays were highly reproducible (%CV<3) and sensitive with the 95% limit of detection (LOD) of 0.80 PFU/mL for OYAV primer probe set and 0.37 PFU/mL for EBIV primer probe set. Furthermore, the assays fitness for purpose was good as it could detect the specific viruses in virus-spiked serum samples, virus-inoculated mosquito samples, field caught mosquitoes and biting midge samples. CONCLUSIONS: Our study has successfully developed specific, sensitive, and reliable RT-qPCR assays for the detection of OYAV and EBIV. These assays hold great promise for their potential application in clinical and field samples in the future.

Dysregulation of tryptophan metabolism and distortion of cell signaling after oral exposure to ethanol and Kynurenic acid.

Kynurenic acid (KYNA), an unavoidable tryptophan metabolite during fermentation is naturally blended with alcohol in all alcoholic beverages. Thus, alcohol drinking inevitably results in co-intake of KYNA. Effects of alcohol or KYNA on human health have been widely studied. However, the combined effects of both remain unknown. Here we report that alcohol and KYNA have a synergistic impact of on global gene expression, especially the gene sets related to tryptophan metabolism and cell signaling. Adult mice were exposed to alcohol (ethanol) and/or KYNA daily for a week. Transcriptomes of the brain, kidney and liver were profiled via bulk RNA sequencing. Results indicate that while KYNA alone largely promotes, and alcohol alone mostly inhibits gene expression, alcohol and KYNA co-administration has a stronger inhibition of global gene expression. Tryptophan metabolism is severely skewed towards kynurenine pathway by decreasing tryptophan hydroxylase 2 and increasing tryptophan dioxygenase. Quantification of tryptophan metabolic enzymes corroborates the transcriptional changes of these enzymes. Furthermore, the co-administration greatly enhances the GnRH signaling pathway. This research provides critical data to better understand the effects of alcohol and KYNA in mix on human health.

Biosafety risks and mitigation strategies for mosquito infection in Arthropod Containment Level-2 laboratory / 中国热带医学

@#Arthropods of medical importance such as mosquitoes, ticks and sandflies are one of the key drivers of arthropod-borne diseases outbreak, posing a great threat to global public health security. For further understanding the transmission mechanisms of arthropod-borne diseases and establishing the prevention and control measures, a series of experiments of arthropods infection need to be carried out under laboratory conditions. Besides the regular biosafety requirements, some specific considerations need to be taken into account when performing arthropod infection and the infected arthropod rearing. Except for the physical containment composed of biosafety facilities, a comprehensive assessment of the biosafety risks during operations and corresponding preventive measures are also critical to eliminate or mitigate the biosafety risks. In this paper, we introduce our practice in handling mosquito infection with Risk Group 2 pathogens in Arthropod Containment Level-2 (ACL-2) laboratory, with an aim to provide a reference for researchers in related fields.

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti's following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

Vector competence and immune response of Aedes aegypti for Ebinur Lake virus, a newly classified mosquito-borne orthobunyavirus.

The global impact of mosquito-borne diseases has increased significantly over recent decades. Ebinur Lake virus (EBIV), a newly classified orthobunyavirus, is reported to be highly pathogenic in adult mice. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. Here, Aedes aegypti was applied to evaluate EBIV infection and dissemination in mosquitos. Our experiments indicated that Ae. aegypti had the possibility to spread EBIV (with a transmission rate of up to 11.8% at 14 days post-infection) through biting, with the highest viral dose in a single mosquito's saliva reaching 6.3 plaque-forming units. The highest infection, dissemination and ovary infection rates were 70%, 42.9%, and 29.4%, respectively. The high viral infection rates in Ae. aegypti ovaries imply the possibility of EBIV vertical transmission. Ae. aegypti was highly susceptible to intrathoracic infection and the saliva-positive rate reached 90% at 10 days post-infection. Transcriptomic analysis revealed Toll and Imd signaling pathways were implicated in the mosquito's defensive response to EBIV infection. Defensin C and chitinase 10 were continuously downregulated in mosquitoes infected via intrathoracic inoculation of EBIV. Comprehensive analysis of the vector competence of Ae. aegypti for EBIV in laboratory has indicated the potential risk of EBIV transmission through mosquitoes. Moreover, our findings support a complex interplay between EBIV and the immune system of mosquito, which could affect its vector competence.

Organ-differential responses to ethanol and kynurenic acid, a component of alcoholic beverages in gene transcription.

Excessive alcohol (ethanol) use has long been known to affect human health negatively. However, the underlying molecular basis is incompletely understood. Moreover, consumption of alcohol is often mixed with kynurenic acid (KYNA), an abundant tryptophan metabolite produced during fermentation. The combined effect of ethanol and KYNA on host gene expression has not been investigated. The current study used mice as the model to interrogate the impact of ethanol and/or KYNA on global gene transcription. Adult male mice were administered with 2 g/kg ethanol and/or 0.1 mg/kg KYNA by gavage once a day for a week. Three organs: brain, kidney, and liver were collected and their total RNAs extracted for transcriptome sequencing and quantitative real-time PCR. Gene ontology, Kyoto encyclopedia of genes, and genomes pathway analyses revealed that alcohol affects the three organs differentially. Furthermore, the gene expression profile from alcohol and KYNA co-administration was significantly different from that of alcohol or KYNA administration alone. Strikingly, Indolamine 2,3-dioxygenase 1, a rate-limiting enzyme in tryptophan metabolism, was significantly increased in the brain after a combined exposure of alcohol and KYNA, suggesting that Trp metabolism was skewed towards the kynurenine pathway in the brain. Our systemic analysis provides new insights into the mechanism whereby alcohol and KYNA affects organ functions.

Metabolism of Oxalate in Humans: A Potential Role Kynurenine Aminotransferase/Glutamine Transaminase/Cysteine Conjugate Betalyase Plays in Hyperoxaluria.

Hyperoxaluria, excessive urinary oxalate excretion, is a significant health problem worldwide. Disrupted oxalate metabolism has been implicated in hyperoxaluria and accordingly, an enzymatic disturbance in oxalate biosynthesis can result in the primary hyperoxaluria. Alanine-glyoxylate aminotransferase-1 and glyoxylate reductase, the enzymes involving glyoxylate (precursor for oxalate) metabolism, have been related to primary hyperoxalurias. Some studies suggest that other enzymes such as glycolate oxidase and alanine-glyoxylate aminotransferase-2 might be associated with primary hyperoxaluria as well, but evidence of a definitive link is not strong between the clinical cases and gene mutations. There are still some idiopathic hyperoxalurias, which require a further study for the etiologies. Some aminotransferases, particularly kynurenine aminotransferases, can convert glyoxylate to glycine. Based on biochemical and structural characteristics, expression level, and subcellular localization of some aminotransferases, a number of them appear able to catalyze the transamination of glyoxylate to glycine more efficiently than alanine glyoxylate aminotransferase-1. The aim of this minireview is to explore other undermining causes of primary hyperoxaluria and stimulate research toward achieving a comprehensive understanding of underlying mechanisms leading to the disease. Herein, we reviewed all aminotransferases in the liver for their functions in glyoxylate metabolism. Particularly, kynurenine aminotransferase-I and III were carefully discussed regarding their biochemical and structural characteristics, cellular localization, and enzyme inhibition. Kynurenine aminotransferase-III is, so far, the most efficient putative mitochondrial enzyme to transaminate glyoxylate to glycine in mammalian livers, which might be an interesting enzyme to look for in hyperoxaluria etiology of primary hyperoxaluria and should be carefully investigated for its involvement in oxalate metabolism.

Kynurenine aminotransferase 3/glutamine transaminase L/cysteine conjugate beta-lyase 2 is a major glutamine transaminase in the mouse kidney.

BACKGROUND: Kynurenine aminotransferase 3 (KAT3) catalyzes the transamination of Kynurenine to kynurenic acid, and is identical to cysteine conjugate beta-lyase 2 (CCBL2) and glutamine transaminase L (GTL). GTL was previously purified from the rat liver and considered as a liver type glutamine transaminase. However, because of the substrate overlap and high sequence similarity of KAT3 and KAT1, it was difficult to assay the specific activity of each KAT and to study the enzyme localization in animals. METHODS: KAT3 transcript and protein levels as well as enzyme activity in the liver and kidney were analyzed by regular reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, biochemical activity assays combined with a specific inhibition assay, and western blotting using a purified and a highly specific antibody, respectively. RESULTS: This study concerns the comparative biochemical characterization and localization of KAT 3 in the mouse. The results showed that KAT3 was present in both liver and kidney of the mouse, but was much more abundant in the kidney than in the liver. The mouse KAT3 is more efficient in transamination of glutamine with indo-3-pyruvate or oxaloacetate as amino group acceptor than the mouse KAT1. CONCLUSIONS: Mouse KAT3 is a major glutamine transaminase in the kidney although it was named a liver type transaminase. GENERAL SIGNIFICANCE: Our data highlights KAT3 as a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney. This suggests tissue localizations of KAT3/GTL/CCBL2 in other animals may be carefully checked.