Equine Infectious Anemia Virus Gag Assembly and Export Are Directed by Matrix Protein through trans-Golgi Networks and Cellular Vesicles.

UNLABELLED: Gag intracellular assembly and export are very important processes for lentiviruses replication. Previous studies have demonstrated that equine infectious anemia virus (EIAV) matrix (MA) possesses distinct phosphoinositide affinity compared with HIV-1 MA and that phosphoinositide-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release. In this study, we compared the cellular assembly sites of EIAV and HIV-1. We observed that the assembly of EIAV particles occurred on interior cellular membranes, while HIV-1 was targeted to the plasma membrane (PM) for assembly. Then, we determined that W7 and K9 in the EIAV MA N terminus were essential for Gag assembly and release but did not affect the cellular distribution of Gag. The replacement of EIAV MA with HIV-1 MA directed chimeric Gag to the PM but severely impaired Gag release. MA structural analysis indicated that the EIAV and HIV-1 MAs had similar spatial structures but that helix 1 of the EIAV MA was closer to loop 2. Further investigation indicated that EIAV Gag accumulated in the trans-Golgi network (TGN) but not the early and late endosomes. The 9 N-terminal amino acids of EIAV MA harbored the signal that directed Gag to the TGN membrane system. Additionally, we demonstrated that EIAV particles were transported to the extracellular space by the cellular vesicle system. This type of EIAV export was not associated with multivesicular bodies or microtubule depolymerization but could be inhibited by the actin-depolymerizing drug cytochalasin D, suggesting that dynamic actin depolymerization may be associated with EIAV production. IMPORTANCE: In previous studies, EIAV Gag was reported to localize to both the cell interior and the plasma membrane. Here, we demonstrate that EIAV likely uses the TGN as the assembly site in contrast to HIV-1, which is targeted to the PM for assembly. These distinct assembly features are determined by the MA domain. We also identified two sites in the N terminus of EIAV MA that were important for Gag assembly and release. Furthermore, the observation of EIAV transport by cellular vesicles but not by multivesicular bodies sheds light on the mechanisms underlying EIAV cellular replication.

A pilot study on interaction between donkey tetherin and EIAV stains with different virulent and replication characteristics.

Tetherin (BST-2) is an important host restriction factor that can inhibit the release of a diverse array of enveloped viruses from infected cells. Conversely, to facilitate their release and spread, many viruses have evolved various strategies to overcome the antiviral effect of tetherin in a species-specific manner. During the development of an attenuated equine infectious anemia virus (EIAV) vaccine in our laboratory, we found that serial passage of a field-isolated virulent EIAV strains in horse and donkey as well as the cultivated donkey cells, produces several typical EIAV strains, including EIAVDV, EIAVDLV, and EIAVFDDV, which exhibit distinct virulence and replication features in vivo and in vitro. However, the role of host restriction factors in EIAV evolution during the serial passage is not well understood. This study aimed to evaluate whether these newly generated strains adapt differently to donkey tetherin (do-tetherin) based on their virulence. We found that do-tetherin exerts an inhibition on the release of the viral particles produced by all three strains, albeit with varying intensity: EIAVDV < EIAVDLV < EIAVFDDV. Additionally, all three EIAV strains could counteract the restriction mediated by do-tetherin via their envelope proteins (Env) with varying strength: EIAVDV > EIAVDLV > EIAVFDDV. These results indicate that donkey tetherin is involved in shaping of EIAV evolution during serial passage.

New H6 influenza virus reassortment strains isolated from Anser fabalis in Anhui Province, China.

BACKGROUND: H6 subtype avian influenza viruses are globally distributed and, in recent years, have been isolated with increasing frequency from both domestic and wild bird species as well as infected humans. Many reports have examined the viruses in the context of poultry or several wild bird species, but there is less information regarding their presence in migratory birds. METHODS: Hemagglutination and hemagglutination inhibition tests were used to measure HA activity for different HA subtypes. Whole viral genomes were sequenced and analysed using DNAstar and MEGA 6 to understand their genetic evolution. Pathogenicity was evaluated using a mouse infection model. RESULTS: We isolated 13 strains of H6 virus from faecal samples of migratory waterfowl in Anhui Province of China in 2014. Phylogenetic analysis showed gene reassortment between Eurasian and North American lineages. Five of the identified H6 strains had the ability to infect mice without adaptation. CONCLUSION: Our findings suggest that regular surveillance of wild birds, especially migratory birds, is important for providing early warning and control of avian influenza outbreaks.

Regulation of Rev expression by the equine infectious anaemia virus tat-rev mRNA Kozak sequence and its potential influence on viral replication.

Rev, an important accessory protein of equine infectious anaemia virus (EIAV), induces the nuclear export of incompletely spliced viral mRNAs. Rev is translated from the tat-rev mRNA through leaky scanning of the tat CUG. In this study, the function of the Kozak sequence at the beginning of the rev ORF was investigated. Deletion or attenuation of the Kozak sequence resulted in expression of an N-terminal 11 aa-truncated Rev in addition to WT Rev. Truncated Rev displayed weaker promotion of Gag expression and processing than WT Rev. Furthermore, EIAV rescued from an infectious molecular clone (pEIAVUK3) with Kozak attenuation exhibited decreased viral replication in host cells in vitro. These results provide a new understanding of the relationship between EIAV Rev expression and viral replication.

The first emergence of paramyxovirus type 12 in wild birds in mainland, China.

Avian paramyxoviruses (APMV) belong to the subfamily Avulavirinae of the family Paramyxoviridae and include 22 distinct subtypes or serotypes (1-22). Avian paramyxovirus serotype 12 (APMV-12) is found sporadically in wild birds worldwide, and reports from only Italy and Taiwan have been published to date; information on its genetic variation and biological characteristics is still limited. In this study, 3 APMV-12 strains, designated WB19, LY9, and LY11, were isolated from 8643 wild bird faecal samples during the annual influenza virus surveillance of wild birds in Guangdong, China between 2018 and 2024, which is first reported in mainland China. The complete genomes of the 3 viruses with 6 gene segments, 3'-N-P-M-F-HN-L-5', were 15,231 nt in length. Phylogenetic analysis based on the whole genome showed that the 3 APMV-12 strains had the highest homology with an APMV-12 strain isolated from Taiwan in 2015, followed by the prototype APMV-12 strains isolated from mallard ducks in Italy in 2005. Genetic analysis of the whole gene of each of them indicated that they were derived from a Eurasian lineage. This study provides additional evidence that wild birds transmit viruses between countries, and this should be monitored to understand APMV transmission, evolution and epidemiology.

Concern regarding H3-subtype avian influenza virus.

The H3-subtype of avian influenza virus (AIV) is one of the most frequently detected low pathogenic avian influenza virus (LPAIV) subtypes in birds and fowls, causing substantial economic loss to the poultry industry. Most importantly, besides poultry, mammals could also be infected with it, such as swines, canines, equines, felines, and humans, posing a serious public health threat. This allows the virus to persist widely in poultry and wild birds for a long time, where it may mix with other subtypes, providing conditions for viral recombination or reassortment. Currently, the monitoring of H3-subtype AIV is inadequate, and there is a lack of effective prevention and control measures for H3-subtype AIV. Here, the epidemiology, phylogeny, and genetic variation of H3-subtype AIV were analyzed, and nonsynonymous and synonymous substitution rates (dN/dS) were calculated. Through these steps, we aimed to clarify the current epidemiological feature and evolutionary characteristics of H3-subtype AIV, and provide an operative reference for future scientific control of H3-subtype AIV.

Evolution and Reassortment of H6 Subtype Avian Influenza Viruses.

The H6 subtype of avian influenza virus (H6 AIV) is the most detected AIV subtype in poultry and wild birds. It causes economic losses to the poultry industry, and the most important, H6 AIV may have the ability to infect mammals, which is a great threat to public health security. In addition, the H6 subtype can serve as a precursor to providing internal genes for other highly pathogenic AIVs, posing a potential threat. H6 AIV currently face to the high positive detection rate and harmless nature of H6 AIV and because not highly effective H6 subtype vaccine available on the market. In this study, we focused on the prevalence of H6 AIV in poultry and wild birds, phylogenetic analysis, genetic variation characteristics, selection analysis, and prevention and control to provide relevant references for the scientific prevention and control of H6 AIV in future.

Emergence and Evolution of Novel Canine-Avian Reassortant H3N2 Influenza A Viruses in Duck in Leizhou Peninsula, China.

Avian-to-mammal transmission and mammalian adaptation of avian influenza virus (AIV) are threats to public health and of great concern. The H3 subtype of influenza virus has low pathogenicity and is widely distributed in humans, canines, equines and avians. In 2018-2019, we isolated six H3N2 subtype influenza viruses from 329 samples acquired from ducks on the Leizhou Peninsula, China, as part of an ongoing virus surveillance program. All viruses were analyzed by whole-genome sequencing with subsequent genetic comparison and phylogenetic analysis. Phylogenetic analysis demonstrated that reassortment of these viruses has occurred among different hosts and subtypes. Some of the H3 AIV isolates have similar genes as subtypes H5 and H7 of highly pathogenic avian influenza viruses (HPAIVs). Most importantly, one strain of H3N2 virus is a novel reassortant influenza virus containing HA and PB2 segments from canine H3N2 virus. The time of most recent common ancestor (tMRCA) data indicated that this reassortant H3N2 virus might have emerged in 2011-2018. The findings suggest that the viruses studied here have undergone multiple reassortment events. Our results provide a framework for understanding the molecular basis of host-range shifts of influenza viruses and we should pay more attention to canine which lived with avian together.

Effects of Rhodotorula mucilaginosa on the Immune Function and Gut Microbiota of Mice.

As a dominant species among marine yeasts, Rhodotorula benthica accounts for ~50% of all marine yeasts. Rhodotorula is rich in a variety of bioactive substances and commonly used in the production of carotenoids by microbial fermentation and is worth developing. Therefore, the present study used a strain of Rhodotorula mucilaginosa isolated from the coastal waters of the South China Sea as the target yeast to investigate its impact on the immune function and gut microbiota of mice. A total of 200 mice were randomly divided into gavage groups and control group and garaged for 30 consecutive days at different concentration. Samples were collected on day 15 and day 30 of gavage administration. The results showed that R. mucilaginosa ZTHY2 could increase the thymus and spleen indices of mice, and its effect on the thymus index was more significant after long-term gavage administration. Short-term (15 days) gavage administration of R. mucilaginosa suspension enhanced delayed hypersensitivity in mice, increased serum IgG, IgA, and IL-2. Long-term (30 days) gavage administration of R. mucilaginosa suspension significantly enhanced the phagocytosis of macrophages in mice and significantly increased serum TNF-α and INF-γ. R. mucilaginosa ZTHY2 altered the structure of the gut microbiota of mice at the phylum and genus levels, leading to an increased relative abundance of Firmicutes and Lactobacillus and a decreased relative abundance of Bacteroidetes. This strain increased the beneficial intestinal bacteria and reduced the harmful intestinal bacteria in mice. This study provides experimental evidence and lays the foundation for the future development and application of this strain as a microecological source of carotenoids.

Integrated mRNA and microRNA Transcriptome Sequencing Characterizes Sequence Variants and mRNA-microRNA Regulatory Networks in Grass Carp Fibroblasts Infected with Virulent and Attenuated GCRV.

Grass carp hemorrhagic disease is a fatal disease caused by the grass carp reovirus (GCRV). The aberrant regulation of transcripts has been implicated in many types of diseases. In the present study, we characterized mRNA and miRNA transcriptomes of different virulent GCRVs using RNA sequencing (RNA-Seq). One hundred eighteen miRNAs were identified as being differentially expressed between different virulent viruses in grass carp fibroblasts. Eight miRNAs were selected to verify the RNA-Seq results using RT-PCR and mRNA methods. In total, 996 differentially expressed mRNA genes were identified in grass carp fibroblasts, while 901 miRNA-mRNA target pairs were observed to be inversely regulated in grass carp fibroblasts. Integrated mRNA/miRNA expression profiling analysis results showed that the most influenced processes were the immune response and cell death. Three miRNAs were shown to exhibit the same expression patterns when two different methods were used and had important functions during viral infection. These results provide insights into the miRNA-mediated regulation of mRNA and valuable resources on transcript variation and regulation during GCRV infection, which are potentially useful for mechanistic and drug studies.

Mitochondrial damage are involved in Aflatoxin B1-induced testicular damage and spermatogenesis disorder in mice.

Aflatoxin B1 (AFB1) is an unavoidable environmental pollutants, which seriously endangers human and animal health. AFB1 has male reproductive toxicity, yet the underlying mechanisms remain inconclusive. Mitochondra are a kind of crucial organelle for maintaining spermatogenesis in testis. Thus, we hypothesized that AFB1 can impair mitochondria to aggravate testicular damage and spermatogenesis disorder. To verify this hypothesis, 48 male mice were intragastrically administered with 0, 0.375, 0.75 or 1.5 mg/kg body weight AFB1 for 30 days, respectively. In this study, we found AFB1 caused testicular histopathological lesions and spermatogenesis abnormalities, with the elevation of oxidative stress (increased H2O2, whereas decreased SOD and GSH). Significant mitochondria structure damage of germ cells and Leydig cells, MMP loss, ATP contents reduction, and inhibited activities of mitochondrial complexes I-IV in mice testis were found in AFB1 treatment groups. Besides, AFB1 inhibited mitochondrial biogenesis and mitochondrial dynamics, presenting as the decreased mRNA and protein expressions of PGC-1α, Nrf1, Tfam, Drp1, Fis1, Mfn1 and Opa1. The results revealed that the mitochondrial damage were involved in AFB1-induced testicular damage and spermatogenesis disorder, providing a considerable direction to clarify potential mechanisms of AFB1 reproductive toxicity.

Molecular evolution and amino acid characteristics of newly isolated H9N2 avian influenza viruses from Liaoning Province, China.

H9N2 is widespread among poultry and humans. Though this subtype is not lethal to either species, it can cause considerable financial losses for farmers and threaten human health. In this study, 10 new H9N2 avian influenza viruses (AIVs) produced by reassortment were isolated from domestic birds in Liaoning Province between March 2012 and October 2014. Nucleotide sequence comparisons indicate that the internal genes of one of these strains are highly similar to those of human H7N9 viruses. Amino acid substitutions and deletions occurred in the HA and NA proteins separately, indicating that all 10 of these isolates may have an enhanced ability to infect mammals. A cross-hemagglutinin inhibition assay conducted with two vaccine strains that are broadly used in China suggests that antisera against vaccine candidates cannot completely inhibit the new isolates. Two of the 10 newly isolated viruses could replicate in respiratory organs of infected BALB/c mice without adaption, suggesting that these isolates can potentially infect mammals. The continued surveillance of poultry is important to provide early warning and control of AIV outbreaks. Our results highlight the high genetic diversity of AIV and the need for more extensive AIV surveillance.

Lycopene alleviates AFB1-induced immunosuppression by inhibiting oxidative stress and apoptosis in the spleen of mice.

Lycopene (LYC) has been reported to exhibit antioxidant and immunoprotective activities, and our previous studies confirmed that LYC can alleviate multiple tissue damage induced by aflatoxin B1 (AFB1). However, it is unclear whether LYC could relieve the AFB1-induced immunosuppression. Thus, forty-eight male mice were randomly allocated and treated with LYC (5 mg kg-1) and/or AFB1 (0.75 mg kg-1) by intragastric administration for 30 days. We found that LYC alleviated AFB1-induced immunosuppression by relieving splenic structure injury and increasing the spleen weight, spleen coefficient, T lymphocyte subsets, the contents of IL-2, IFN-γ and TNF-α in serum, as well as the mRNA expression of IL-2, IFN-γ and TNF-α in spleen. Furthermore, LYC inhibited oxidative stress induced by AFB1via decreasing the levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2) and malondialdehyde (MDA), while enhancing the total antioxidant capacity (T-AOC) and antioxidant enzyme activities. In addition, LYC also restrained splenic apoptosis through blocking mitochondria-mediated apoptosis in AFB1 intoxicated mice, presenting as the increase of mitochondrial membrane potential, and the decrease of cytoplasmic Cyt-c protein expression, cleaved Caspase-3 protein expression, Caspase-3/9 activities and mRNA expressions, as well as balancing the mitochondrial protein and mRNA expressions of Bax and Bcl-2. These results indicate that LYC can alleviate AFB1-induced immunosuppression by inhibiting oxidative stress and mitochondria-mediated apoptosis of mice spleen.

T-2 toxin impairs male fertility by disrupting hypothalamic-pituitary-testis axis and declining testicular function in mice.

T-2 toxin could impair male reproductive function. But, the toxicity mechanism is still unclear. In this study, male Kunming mice were orally administrated with T-2 toxin at the doses of 0, 0.5, 1 or 2 mg/kg body weight for 28 days. The fertility, body weight, reproductive organs volume, daily sperm production (DSP), and sperm malformation rate were detected. The expressions of testosterone (T) biosynthetic enzymes, luteinizing hormone (LH)-receptor, follicle stimulating hormone (FSH)-receptor and androgen binding protein (ABP) in testis were detected. The serum hormone level of gonadotropin-releasing hormone (GnRH), FSH, LH, T and progesterone (P), and the mRNA expression of GnRH, GnRH-receptor, LH and FSH were measured. These results demonstrated that T-2 toxin decreased body weight, reproductive organs volume and DSP, increased sperm malformation rate. T-2 toxin impaired fertility by decreasing the mating index, fertility index, numbers of implantation sites and viable fetuses, and increasing the number of animal with resorptions. Meantime, T-2 suppressed testicular function by inhibiting T biosynthesis and decreasing FSHR, LHR and ABP expression. Furthermore, the serum reproductive hormone contents and key factors expression of hypothalamic-pituitary-testis (HPT) axis were decreased by T-2 toxin. In summary, T-2 toxin impaired the male fertility by disrupting HPT axis and impairing testicular function.

Detection of reassortant avian influenza A (H11N9) virus in wild birds in China.

Human infectious avian influenza virus (AIV) H7N9 emerged in China in 2013. The N9 gene of H7N9, which has the ability to cause death in humans, originated from an H11N9 influenza strain circulating in wild birds. To investigate the frequency and distribution of the N9 gene of the H11N9 and H7N9 influenza virus circulating in wild birds between 2006 and 2015, 35,604 samples were collected and tested. No H7N9 but four strains of the H11N9 subtype AIV were isolated, and phylogenetic analyses showed that the four H11N9 viruses were intra-subtype and inter-subtype reassortant viruses. A sequence analysis revealed that all six internal genes of A/wild bird/Anhui/L306/2014 (H11N9) originated from an H9N2 AIV isolated in Korea. The H9N2 strain, which is an inner gene donor reassorted with other subtypes, is a potential threat to poultry and even humans. It is necessary to increase monitoring of the emergence and spread of H11N9 AIV in wild birds.

A 627K variant in the PB2 protein of H9 subtype influenza virus in wild birds.

BACKGROUND: Wild birds are gaining increasing attention as gene-mixing reservoirs for influenza viruses. To investigate the molecular properties of the viruses isolated and epidemiological analysis of H9N2 subtype AIV in wild birds, we studied samples obtained over two years (2014-2015) from wetlands in Anhui province, China. METHODS: A total of 4534 samples were collected from migratory waterfowl in Anhui in 2014-2015, and 8 strains of H9 subtype AIV were isolated. RESULTS: Phylogenetic analysis showed different degrees of gene segment reassortment in H9 viruses between the Eurasian lineage and the North American lineage. Most importantly, two viruses harbored the E627K mutation in the polymerase PB2 (PB2) protein. This is the first report of the mutation of this virus from low pathogenicity to high pathogenicity in wild birds. CONCLUSIONS: The continued surveillance of wild birds, especially migratory birds, is important to provide early warning and control of AIV outbreaks. Our results highlight the high genetic diversity of AIV along the Eurasian-Australian migration flyway and the need for more extensive AIV surveillance in eastern China.

Epidemic of wild-origin H1NX avian influenza viruses in Anhui, China.

BACKGROUND: As the natural hosts of avian influenza viruses (AIVs), aquatic and migratory birds provide a gene pool for genetic transfer among species and across species, forming transient "genome constellations." This work describes the phylogenetic dynamics of H1NX based on the complete molecular characterization of eight genes of viruses that were collected from 2014 to 2015 in Anhui Province, China. METHODS: Hemagglutination and hemagglutination inhibition tests were used to determine the hemagglutination (HA) activity of the HA subtypes. The entire genomes of the viruses were sequenced on an ABI PRISM 3500xl DNA Analyzer. The sequences were genetically analysed to study their genetic evolution using DNASTAR and MEGA 6. The pathogenic effects of the viruses were evaluated using mouse infection models. RESULTS: Seven strains of the H1 subtype avian influenza virus were isolated. Phylogenetic analysis indicated natural recombination of the H1 influenza viruses between the Eurasian lineage and the North American lineage. Some genes had high sequence identity with A/bean goose/Korea/220/2011(H9N2), which is a typical case involving viral reassortment between the Eurasian lineage and the North American lineage. The results of infection experiments in mice showed that the viruses could acquire the ability to multiply in mouse respiratory organs without adaptation. CONCLUSIONS: These findings suggest that continued surveillance of wild birds, particularly migratory birds, is important to provide early warning of possible H1 influenza epidemics and to understand the ecology of the virus.