An evolutionarily conserved ubiquitin ligase drives infection and transmission of flaviviruses.

Mosquito-borne flaviviruses such as dengue (DENV) and Zika (ZIKV) cause hundreds of millions of infections annually. The single-stranded RNA genome of flaviviruses is translated into a polyprotein, which is cleaved equally into individual functional proteins. While structural proteins are packaged into progeny virions and released, most of the nonstructural proteins remain intracellular and could become cytotoxic if accumulated over time. However, the mechanism by which nonstructural proteins are maintained at the levels optimal for cellular fitness and viral replication remains unknown. Here, we identified that the ubiquitin E3 ligase HRD1 is essential for flaviviruses infections in both mammalian hosts and mosquitoes. HRD1 directly interacts with flavivirus NS4A and ubiquitylates a conserved lysine residue for ER-associated degradation. This mechanism avoids excessive accumulation of NS4A, which otherwise interrupts the expression of processed flavivirus proteins in the ER. Furthermore, a small-molecule inhibitor of HRD1 named LS-102 effectively interrupts DENV2 infection in both mice and Aedes aegypti mosquitoes, and significantly disturbs DENV transmission from the infected hosts to mosquitoes owing to reduced viremia. Taken together, this study demonstrates that flaviviruses have evolved a sophisticated mechanism to exploit the ubiquitination system to balance the homeostasis of viral proteins for their own advantage and provides a potential therapeutic target to interrupt flavivirus infection and transmission.

Lethal infection caused by Tetratrichomonas gallinarum in black swans (Cygnus atratus).

BACKGROUND: Tetratrichomonas gallinarum is parasitic protozoa with a wide host range. However, its lethal infection is rare reported. CASE PRESENTATION: Here, we described the first lethal cases of T. gallinarum infection in black swans in China. Five black swans died within a week in succession without obvious symptoms except mild diarrhea. At necropsy, severe lesions were observed in caeca with thickened caecal walls and hemorrhages in the mucosa. A large number of moving trophozoites were found in the contents of the cecum by microscopic examination. The livers were enlarged with multiple bleeding spots on the surface. Histopathology of the livers showed mononuclear cell infiltration and moderate hyperplasia of fibrous tissue. The histopathology of the cecum showed that the villi of the cecum were edematous. Finally, the presence of T. gallinarum was determined by specific PCR andin-situ hybridization assay. Additionally, common pathogens that can cause similar symptoms were excluded. CONCLUSIONS: The death of the black swan was caused by T. gallinarum, suggesting that the parasite might be a new threat to the Cygnus birds.

Seroprevalence and Risk Factors of Fascioliasis in Yaks, Bos grunniens, from Three Counties of Gansu Province, China.

The aim of this study was to determine the seroprevalence and risk factors of fascioliasis in yaks, Bos grunniens, from 3 counties of Gansu Province in China. A total of 1,584 serum samples, including 974 samples from white yaks from Tianzhu, 464 from black yaks from Maqu, and 146 from black yaks from Luqu County, were collected and analyzed using ELISA to detect IgG antibodies against Fasciola hepatica. The overall F. hepatica seroprevalence was 28.7% (454/1,584), with 29.2% in white yaks (284/974) and 27.9% in black yaks (170/610). The seroprevalence of F. hepatica in yaks from Tianzhu, Luqu, and Maqu was 29.2%, 22.6%, and 29.5%, respectively. Female yaks (30.9%) had higher F. hepatica seroprevalence than male yaks (23.4%). Also, F. hepatica seroprevalence varied by different age group from 24.1% to 33.8%. Further, the seroprevalence ranged from 21.8% to 39.1% over different seasons. Interestingly, the season and age of yaks were associated with F. hepatica infection in yaks in the investigated areas. These findings provided a basis for further studies on this disease in yaks from 3 counties of Gansu Province in northwestern China, which may ultimately support the development of effective control strategies of fascioliasis in these areas.

Seroprevalence and risk factors of bovine viral diarrhoea virus (BVDV) infection in yaks (Bos grunniens) in northwest China.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is an important pathogen of cattle worldwide, causing reproductive disorders in adult cattle and mucosal disease in calves. However, limited information about BVDV infection in yaks (Bos grunniens) in China is available, especially in white yaks which is a unique yak breed that only lives in Tianzhu Tibetan Autonomous County (TTAC), Gansu Province, northwest China. Therefore, we conducted a cross-sectional study to estimate the seroprevalence and risk factors associated with BVDV infection in 1584 yaks in Gansu province, northwest China, between April 2013 and March 2014 using an indirect ELISA test. The overall seroprevalence of BVDV in yaks was 37.56 % (595/1584), with 45.08 % (275/610) in black yaks and 32.85 % (320/974) in white yaks. Moreover, positive yaks were found in all four regions, varied from 33.22 to 40.31 %. Male yaks had a similar seroprevalence (37.84 %) with that of the female yaks (37.11 %). Season, species and geographical origins of yaks were considered as risk factors analyzed by logistic regression model. To our knowledge, this is the first report of seroprevalence and risk factors associated with BVDV infection in white yaks in China.

A long-lasting protective immunity against chronic toxoplasmosis in mice induced by recombinant rhoptry proteins encapsulated in poly (lactide-co-glycolide) microparticles.

Toxoplasma gondii infection in humans and animals is a worldwide zoonosis. Prevention and control of toxoplasmosis based on vaccination is one of the promising strategies. In the present study, recombinant T. gondii rhoptry proteins 38 and 18 (TgROP38 and TgROP18) were encapsulated into poly (lactide-co-glycolide) (PLG) (1:1), respectively, to obtain the stable water-in-oil-in-water double emulsion. Female Kunming mice were then immunized with the protein vaccines twice at a 2-week interval. Eight weeks after the second immunization, 10 mice from each group were challenged with T. gondii PRU strain (genotype II). The entrapment rates of PLG-rROP38 and PLG-rROP18 ranged from 65.5 to 77.7% and 58.1 to 72.3%, respectively. Immunization of mice with rROP38 and rROP18 proteins encapsulated into PLG microparticles elicited strongly humoral and cell-mediated responses against T. gondii, associated with relatively high levels of total IgG, IgG2a isotype, and IFN-γ, as well as the mixed Th1/Th2 immunity responses. Immunization with various protein vaccines induced significant reduction of the brain cysts after chronic infection with the T. gondii PRU strain, and the most effective protection was achieved in the PLG-rROP38-rROP18-immunized mice, with a cyst reduction of 81.3%. The findings of the present study indicated that recombinant rhoptry antigens encapsulated in PLG could maintain the protein immunogenicity in an extended period and elicit effective protection against chronic T. gondii infection, which has implications for the development of long-lasting vaccines against chronic toxoplasmosis in animals.

[Research Progress and Application Future of MicroRNA-36].

MicroRNA-36 (miR-36) is a recently discovered miRNA family which including at least eight members, and specifically existed in helminths compared with other miRNAs that widely exists in almost all kinds of lives. This paper reviews recent research advances about miR-36 to provide further fundamental information for helminth and miRNA study.

An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants.

Herd immunity achieved through mass vaccination is an effective approach to prevent contagious diseases. Nonetheless, emerging SARS-CoV-2 variants with frequent mutations largely evaded humoral immunity induced by Spike-based COVID-19 vaccines. Herein, we develop a lipid nanoparticle (LNP)-formulated mRNA-based T-cell-inducing antigen, which targeted three SARS-CoV-2 proteome regions that enriched human HLA-I epitopes (HLA-EPs). Immunization of HLA-EPs induces potent cellular responses to prevent SARS-CoV-2 infection in humanized HLA-A*02:01/DR1 and HLA-A*11:01/DR1 transgenic mice. Of note, the sequences of HLA-EPs are highly conserved among SARS-CoV-2 variants of concern. In humanized HLA-transgenic mice and female rhesus macaques, dual immunization with the LNP-formulated mRNAs encoding HLA-EPs and the receptor-binding domain of the SARS-CoV-2 B.1.351 variant (RBDbeta) is more efficacious in preventing infection of SARS-CoV-2 Beta and Omicron BA.1 variants than single immunization of LNP-RBDbeta. This study demonstrates the necessity to strengthen the vaccine effectiveness by comprehensively stimulating both humoral and cellular responses, thereby offering insight for optimizing the design of COVID-19 vaccines.

A lung-selective delivery of mRNA encoding broadly neutralizing antibody against SARS-CoV-2 infection.

The respiratory system, especially the lung, is the key site of pathological injury induced by SARS-CoV-2 infection. Given the low feasibility of targeted delivery of antibodies into the lungs by intravenous administration and the short half-life period of antibodies in the lungs by intranasal or aerosolized immunization, mRNA encoding broadly neutralizing antibodies with lung-targeting capability can perfectly provide high-titer antibodies in lungs to prevent the SARS-CoV-2 infection. Here, we firstly identify a human monoclonal antibody, 8-9D, with broad neutralizing potency against SARS-CoV-2 variants. The neutralization mechanism of this antibody is explained by the structural characteristics of 8-9D Fabs in complex with the Omicron BA.5 spike. In addition, we evaluate the efficacy of 8-9D using a safe and robust mRNA delivery platform and compare the performance of 8-9D when its mRNA is and is not selectively delivered to the lungs. The lung-selective delivery of the 8-9D mRNA enables the expression of neutralizing antibodies in the lungs which blocks the invasion of the virus, thus effectively protecting female K18-hACE2 transgenic mice from challenge with the Beta or Omicron BA.1 variant. Our work underscores the potential application of lung-selective mRNA antibodies in the prevention and treatment of infections caused by circulating SARS-CoV-2 variants.

Prevalence and genetic diversity of Enterocytozoon bieneusi in nonhuman primates in Northern and Central China.

Background: Enterocytozoon bieneusi is a common enteric pathogen reported in human and many animals. But there are few reports of E. bieneusi in nonhuman primates (NHPs). The aim of this study was to examine the prevalence and molecular characterization of E. bieneusi in NHPs from Northern and Central China. Results: A total of 299 specimens of NHPs were collected. The overall prevalence rate of E. bieneusi was 9.4% (28/299) in NHPs by ribosomal internal transcribed spacer (ITS) amplification, including 10.0% (16/160) in captive NHPs and 8.6% (12/139) in wild NHPs. In captive NHPs, the infection rate was 9.1% in male, 11.5% in female. Infection rate in juvenile ITS (6.7%) was higher than in the adult ITS (5.6%). In different regions, infection rate in Hubei (14.7%) was higher than in Henan (7.6%) and Beijing (7.9%). Five genotypes were found, including 4 known genotypes (D, HND-Ⅰ, EbpC, SHW2) and a novel genotype named NHP1. Genotype D (8/28) and NHP1 (8/28) were the most prevalent, followed by EbpC (6/28), SHW2 (4/28), and HND-Ⅰ (2/28). All the 5 genotypes belonged to zoonotic Group1. Conclusion: These findings could deepen our understanding of E. bieneusi prevalence and genotype distribution in NHPs in China. Our study shows that NHPs may be the reservoir of zoonotic E. bieneusi and might present a potential serious threat.

Infection and chronic disease activate a brain-muscle signaling axis that regulates muscle performance.

Infections and neurodegenerative diseases induce neuroinflammation, but affected individuals often show a number of non-neural symptoms including muscle pain and muscle fatigue. The molecular pathways by which neuroinflammation causes pathologies outside the central nervous system (CNS) are poorly understood, so we developed three models to investigate the impact of neuroinflammation on muscle performance. We found that bacterial infection, COVID-like viral infection, and expression of a neurotoxic protein associated with Alzheimer' s disease promoted the accumulation of reactive oxygen species (ROS) in the brain. Excessive ROS induces the expression of the cytokine Unpaired 3 (Upd3) in insects, or its orthologue IL-6 in mammals, and CNS-derived Upd3/IL-6 activates the JAK/Stat pathway in skeletal muscle. In response to JAK/Stat signaling, mitochondrial function is impaired and muscle performance is reduced. Our work uncovers a brain-muscle signaling axis in which infections and chronic diseases induce cytokine-dependent changes in muscle performance, suggesting IL-6 could be a therapeutic target to treat muscle weakness caused by neuroinflammation.

A glucose-like metabolite deficient in diabetes inhibits cellular entry of SARS-CoV-2.

The severity and mortality of COVID-19 are associated with pre-existing medical comorbidities such as diabetes mellitus. However, the underlying causes for increased susceptibility to viral infection in patients with diabetes is not fully understood. Here we identify several small-molecule metabolites from human blood with effective antiviral activity against SARS-CoV-2, one of which, 1,5-anhydro-D-glucitol (1,5-AG), is associated with diabetes mellitus. The serum 1,5-AG level is significantly lower in patients with diabetes. In vitro, the level of SARS-CoV-2 replication is higher in the presence of serum from patients with diabetes than from healthy individuals and this is counteracted by supplementation of 1,5-AG to the serum from patients. Diabetic (db/db) mice undergo SARS-CoV-2 infection accompanied by much higher viral loads and more severe respiratory tissue damage when compared to wild-type mice. Sustained supplementation of 1,5-AG in diabetic mice reduces SARS-CoV-2 loads and disease severity to similar levels in nondiabetic mice. Mechanistically, 1,5-AG directly binds the S2 subunit of the SARS-CoV-2 spike protein, thereby interrupting spike-mediated virus-host membrane fusion. Our results reveal a mechanism that contributes to COVID-19 pathogenesis in the diabetic population and suggest that 1,5-AG supplementation may be beneficial to diabetic patients against severe COVID-19.

Identification and genetic characterization of Contracaecum sp. (Nematoda: Anisakidae) from China.

Contracaecum species are economically important fish-borne larval nematodes with zoonotic significance. In June 2019, more than 100 piscivorous birds died in their habitats close to the Wild Duck Lake, located in Yanqing, northwest of Beijing, China. Post-mortem examination of Black Night Herons (Nycticorax) revealed the presence of numerous anisakid nematodes in the proventriculus. Recovered nematodes were identified as Contracaecum sp. based on morphological description. Phylogenetic analysis of the mitochondrial (mt) genome and the ITS gene showed that sequences of Contracaecum sp. Beijing isolates were grouped into a new individual cluster. Furthermore, the parasite was successfully isolated from fresh dead birds, feces of piscivorous birds, and fish and prevalence ranged from 8.0% to 81.8%. Consequently, our study demonstrated Contracaecum sp. infections in different sources from China, which might constitute a threat to wildlife, aquaculture, and public health.

Molecular Characterization of Cryptosporidium spp. in Brandt's Vole in China.

Cryptosporidium spp. are important intestinal parasites that infect humans and various animals, including wildlife. Currently, few epidemiological data in wild rodents, especially in voles, are available. In the present study, a total of 678 Brandt's vole feces samples were collected from Maodeng Livestock Farm and East Ujimqin, Inner Mongolia. The overall prevalence of Cryptosporidium spp. was 18.7%. Significant differences were not found between genders but between locations and weight groups. Moreover, three known species/genotypes, C. suis, Cryptosporidium environmental sequence and muskrat genotype II, and a novel Cryptosporidium species/genotypes of Brandt's vole was identified. To the best of our knowledge, this is the first report of Cryptosporidium spp. infection in Brandt's vole worldwide. These findings imply Brandt's voles might be a potential source of human cryptosporidiosis.

Isolation and Pathogenic Characterization of Pigeon Paramyxovirus Type 1 via Different Inoculation Routes in Pigeons.

Pigeon paramyxovirus type I (PPMV-1) causes regular outbreaks in pigeons and even poses a pandemic threat among chickens and other birds. The birds infected with PPMV-1 mainly show a pathological damage in the respiratory system, digestive system, and nervous system. However, there were few reports on the efficiency of the virus entering the host via routes of different systems. In the present study, a PPMV-1 strain was obtained from a dead wild pigeon in 2016 in Beijing, China. The mean death time (MDT) and the intracerebral pathogenicity (ICPI) of our isolate showed medium virulence. Phylogenetic analysis based on F gene sequence showed that the isolate belonged to subgenotype VIb, class II, which dominated in China in recent years. Then, we evaluated the infection efficiency of different routes. Pigeons were randomly divided into five groups of six as follows: intracephalic (IC), intranasal (IN), and intraoral (IO) infection routes, cohabitation infection (CO), and negative control (N negative). All pigeons were inoculated with 100 µl·106 EID50 PPMV-1 virus. After infection, pathological lesions, virus shedding, body weight change, survival rate, and tissue tropism were tested to compare the efficiency of the different infected routes. The mortality of groups IC, IN, IO, and CO were 100, 66.7, 50, and 33.3%, respectively. Weight loss in group IC was higher than the other groups, followed by groups IN and IO. The lesions observed in PPMV-1-infected pigeons were severe, especially in the lung and intestine in group IC. Viral shedding was observed from 2 dpi in groups IC and IN, but the shedding rate was higher in group IN than group IC. The longest period was in group CO. Tissue tropism experiment showed that our isolate has a wide range of tissue distribution, and the virus titer in the heart and intestine of group IC and in the brain of group IN was higher. Our data may help us to evaluate the risk of transmission of PPMV-1.

Identification of Enterocytozoon bieneusi and Cryptosporidium spp. in farmed wild boars (Sus scrofa) in Beijing, China.

Enterocytozoon bieneusi and Cryptosporidium spp. are opportunistic pathogen that can infected humans and other animals. However, the data on the prevalence and genotypes of the parasites in captive wild boars is not available in Beijing, China. In this study, a total of 257 fecal specimens of wild boars were collected. The overall prevalence of E. bieneusi and Cryptosporidium spp. was 42.0% (108/257) and 5.8%, respectively. Higher infection rate of E. bieneusi was discovered in the wild boar ≤2 months old (58.3%). The differences between the feeding pattern and gender were not significant. Furthermore, eight genotypes of E. bieneusi were determined by analyzing the internal transcribed spacer (ITS) of the rRNA gene, including seven known genotypes and one novel genotype. Phylogenetic analysis revealed that all the eight genotypes belonged to the zoonotic potential Group 1. For Cryptosporidium spp., no significant differences were found between groups of gender, age and feeding pattern. Only C. scrofarum was identified in the investigated samples. The findings suggest that wild boar could be reservoirs of E. bieneusi and C. scrofarum which could be potentially transmitted to humans and other animals.

Highly pathogenic H5N6 avian influenza virus outbreak in Pavo cristatus in Jiangxi Province, China.

Highly pathogenic avian influenza (HPAI) is a persistent threat to poultry, wild birds, humans, and other mammals. The continually evolving HPAI H5N6 virus has induced great losses in breeding industries in growing regions around the world. In this study, we confirmed an outbreak of the HPAI H5N6 virus in captive Pavo cristatus in Jiangxi Province, China. The causative agents H5N6 viruses were isolated and designated JS01, JS02, and K10. Animal experiments showed that all three isolates exhibited high pathogenicity to chickens, but they need adaption to effectively infect mice. A phylogenetic analysis showed that all three isolates were clustered in H5 clade 2.3.4.4c. No novel genetic reassortant was found in JS01, JS02, and K10 viruses. It was estimated that JS01, JS02, and K10 H5N6 viruses were direct descendants of the H5N6 virus circulating in South of China. The estimated divergence time from tMRCAs was anywhere between May 2014 to June 2016. Although the number of outbreaks of avian influenza decreased significantly in 2018, the threat from avian influenza to public health remains serious. Enhanced active surveillance is required to monitor the transmission and evolution of H5 influenza viruses.

First report of Enterocytozoon bieneusi and Cryptosporidium spp. in peafowl (Pavo cristatus) in China.

Enterocytozoon bieneusi and Cryptosporidium spp. are important pathogens causing diarrhea in humans and animals. However, few studies have been conducted on the infection of E. bieneusi and Cryptosporidium spp. in peafowl up to now. The purpose of the present study was to determine the prevalence and the involved genotypes of Cryptosporidium spp. and E. bieneusi in peafowl in Beijing and Jiangxi Province, China. In total, 258 peafowl fecal samples were collected. Overall, both Cryptosporidium spp. and E. bieneusi had the same prevalence, i.e. 6.59% (17/258). Higher infection rates of E. bieneusi and Cryptosporidium spp. were found in the adolescent peafowl. The prevalence of E. bieneusi in Beijing and Jiangxi Province was 5.23% and 8.57% respectively. For Cryptosporidium spp., the prevalence was 4.58% and 9.52% in Beijing and Jiangxi Province, respectively. Three zoonotic genotypes of E. bieneusi were confirmed, including two known genotypes, genotype Peru 6 and D, and one novel genotype, JXP1. Two avian specific species/genotypes of Cryptosporidium, Avian genotype Ⅲ and Goose genotype Ⅰ, were identified. To our knowledge, this is the first report of E. bieneusi and Cryptosporidium spp. occurrence in peafowl in China. The findings suggest that peafowl could be reservoirs of E. bieneusi and Cryptosporidium spp. which could be potentially transmitted to humans and other animals, and the present survey have implications for controlling E. bieneusi and Cryptosporidium spp. infection in peafowl.

Prevalence and molecular characterization of Trichomonas gallinae from domestic pigeons in Beijing, China.

Trichomonas gallinae is a globally distributed protozoan parasite, mainly affect the upper avian digestive tract and can bring huge economic losses to pigeon industry. The objective of the present study was to determine the prevalence and genotypes of T. gallinae in Beijing, China. A total of 569 samples of throat swabs of pigeon were collected from pigeon farms in Shunyi District, Fangshan District, Daxing District and Miyun District of Beijing. The overall prevalence was 28.30%. The significant difference in infection rates was not observed between regions, but was found between age groups. The highest prevalence was nestling pigeons (33.16%), followed by adolescent pigeons (30.05%) and breeding pigeons (20.59%). Moreover, genotype A and B of T. gallinae were identified by sequencing the ITS1/5.8S/ITS2 regions and phylogenetic analysis. To our knowledge, this is the first report to display the prevalence and genotype of T. gallinae from Beijing, China.

First Report of Coxiella burnetii Seroprevalence in Tibetan Sheep in China.

Query (Q) fever is a vector-borne zoonosis caused by the obligate intracellular pathogen Coxiella burnetii. Animals, including dogs, cats, cattle, and sheep, can be infected by C. burnetii. However, little information is available about C. burnetii infection in Tibetan sheep in China. Antibodies against C. burnetii in Tibetan sheep serums, which were collected from Maqu County, Tianzhu County and Nyingchi Prefecture, China, between 2011 and 2013, were examined by enzyme-linked immunosorbent assay (ELISA). Overall, 14.39% (304/2112, 95% confidence interval [CI] 12.90-15.89) of the examined Tibetan sheep were positive for C. burnetii infection. The C. burnetii seroprevalence in male and female Tibetan sheep was 12.94% (95% CI 10.26-15.61) and 14.98% (95% CI 13.18-16.78), respectively. The seroprevalence varied from 13.19% (95% CI 10.08-16.30) to 14.96% (95% CI 12.31-17.62) in Tibetan sheep in different areas. In addition, the seroprevalence ranged from 13.16% (95% CI 9.83-16.50) to 15.10% (95% CI 13.15-17.04) in different age groups. The results of the present survey indicate that C. burnetii infection is highly prevalent in Tibetan sheep in China. This is the first report of C. burnetii infection in Tibetan sheep in China, extending the host range for C. burnetii. Further studies are warranted to understand better the epidemiological situation of C. burnetii transmission in these areas and elsewhere.