Mock-infected Group Research Articles

Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication.

As one of the most important causative agents of severe gastroenteritis in children, piglets, and other young animals, species A rotaviruses have adversely impacted both human health and the global swine industry. Vaccines against rotaviruses (RVs) are insufficiently effective, and no specific treatment is available. To understand the relationships between porcine RV (PoRV) infection and enterocytes in terms of the cellular lipid metabolism, we performed an untargeted liquid chromatography mass spectrometry (LC-MS) lipidomics analysis of PoRV-infected IPEC-J2 cells. Herein, a total of 451 lipids (263 upregulated lipids and 188 downregulated lipids), spanning sphingolipid, glycerolipid, and glycerophospholipids, were significantly altered compared with the mock-infected group. Interestingly, almost all the ceramides among these lipids were upregulated during PoRV infection. LC-MS analysis was used to validated the lipidomics data and demonstrated that PoRV replication increased the levels of long-chain ceramides (C16-ceramide, C18-ceramide, and C24-ceramide) in cells. Furthermore, we found that these long-chain ceramides markedly inhibited PoRV infection and that their antiviral actions were exerted in the replication stage of PoRV infection. Moreover, downregulation of endogenous ceramides with the ceramide metabolic inhibitors enhanced PoRV propagation. Increasing the levels of ceramides by the addition of C6-ceramide strikingly suppressed the replication of diverse RV strains. We further found that the treatment with an apoptotic inhibitor could reverse the antiviral activity of ceramide against PoRV replication, demonstrating that ceramide restricted RV infection by inducing apoptosis. Altogether, this study revealed that ceramides played an antiviral role against RV infection, providing potential approaches for the development of antiviral therapies.IMPORTANCERotaviruses (RVs) are among the most important zoonosis viruses, which mainly infected enterocytes of the intestinal epithelium causing diarrhea in children and the young of many mammalian and avian species. Lipids play an essential role in viral infection. A comprehensive understanding of the interaction between RV and lipid metabolism in the enterocytes will be helpful to control RV infection. Here, we mapped changes in enterocyte lipids following porcine RV (PoRV) infection using an untargeted lipidomics approach. We found that PoRV infection altered the metabolism of various lipid species, especially ceramides (derivatives of the sphingosine). We further demonstrated that PoRV infection increased the accumulation of ceramides and that ceramides exerted antiviral effects on RV replication by inducing apoptosis. Our findings fill a gap in understanding the alterations of lipid metabolism in RV-infected enterocytes and highlight the antiviral effects of ceramides on RV infection, suggesting potential approaches to control RV infection.

Comparative pathogenicity of infectious bronchitis virus Massachusetts and Delmarva (DMV/1639) genotypes in laying hens.

Infectious bronchitis (IB) is a highly contagious and acute viral disease of chicken caused by the infectious bronchitis virus (IBV) of the family Coronaviridae. Even with extensive vaccination against IB by the poultry industry, the occurrence of new IBV genotypes is a continuous challenge encountered by the global poultry industry. This experiment was designed to compare the pathogenicity of two IBV strains belonging to Massachusetts (Mass) and Delmarva DMV/1639 genotypes. Specific pathogen-free laying hens were challenged during the peak of production (30 weeks), keeping a mock-infected control group. During 21 days of observation following infection, a significant drop in egg production with miss-shaped and soft shells was observed in the DMV/1639 IBV-infected hens only. The DMV/1639 IBV infected group showed prolonged and higher cloacal viral shedding compared with the Mass IBV-infected group. At the end of the study (21 days post-infection), the viral genome loads in the respiratory, urogenital, and immune tissues were significantly higher in the DMV/1639 IBV-infected group compared with the Mass IBV-infected group. Macroscopic lesions such as distorted ova leading to egg peritonitis were observed only in the DMV/1639 IBV-infected group. Moreover, microscopic lesion scores were significantly higher in the lung, kidney, cecal tonsils, and oviduct of the DMV/1639 IBV-infected group compared with the Mass IBV-infected group. Finally, the apoptosis index in the kidney, ovary, magnum, isthmus, and shell gland was significantly higher in the DMV/1639 IBV-infected group compared with the control and Mass-infected groups. This study examined the pathogenicity of two IBV genotypes that are impacting the layer industry in North America.

SARS‐CoV‐2 infection worsens Vascular Dementia (VaD)‐associated neuropathology in mice

AbstractBackgroundVascular Dementia (VaD) is the second leading cause of dementia behind Alzheimer’s disease (AD) and has a profound association with the morbidity and mortality of COVID‐19. Recent evidence demonstrates that preexisting vascular dementia is associated with increased COVID‐19 incidence and worsened outcomes after COVID‐19 infection. Collectively, these observations suggest that vascular pathology in VaD such as blood‐brain barrier (BBB) disruption, directly contributes to this association. Previously we and others have demonstrated that SARS‐CoV‐2 binds to the vascular integrin α5β1 receptor and that inhibition of α5β1 integrin with the clinically‐validated peptide ATN‐161 can improve acute SARS‐CoV‐2 infection and reduce SARS‐CoV‐2‐mediated vascular disruption. Furthermore, we have previously demonstrated that ATN‐161 reduces BBB disruption in models of brain ischemia (stroke) and VaD‐modeled chronic bilateral carotid artery stenosis (BCAS), which models reduced blood flow, BBB disruption, neuroinflammation, white matter damage and cognitive decline in VaD. In the present study, we tested the hypothesis that experimental VaD worsens COVID‐19‐associated neuroinflammation and exacerbates subsequent neurovascular damage, which may be targeted therapeutically by ATN‐161.Method10‐week‐old wild‐type (WT) BALB/c male mice were infected with 2×105 PFU mouse‐adapted (MA10) SARS‐CoV‐2 or mock infection 1‐week after BCAS surgery to model the effects of preexisting VaD in mice and subsequent SARS‐CoV‐2 infection. Body weight was monitored daily. The brain was isolated 3‐days post‐infection (dpi) and analyzed for immunohistochemical and mRNA expression of selected markers of neuroinflammation and immune response.ResultBCAS+SARS‐CoV‐2 infected mice exhibited a significant reduction in body weight at 3 dpi compared with the BCAS+mock infected group. Immunohistochemical analysis demonstrated a significant increase in α5 integrin signal in the cortex. SARS‐CoV‐2 infection after BCAS worsened brain and vascular inflammation and glial activation, indicated by increased Iba‐1 (microglia) and GFAP (astrocyte) immunofluorescence and brain (proinflammatory) IL‐1β cytokine transcript levels. Lastly, SARS‐CoV‐2 infection worsens white matter (myelin) integrity as measured by myelin basic protein (MBP) immunofluorescence.ConclusionSARS‐CoV‐2 aggravated VaD‐associated morbidity indicated by elevated white matter damage, glial activation, and neuroinflammation along with increased integrin α5 expression. Integrin α5β1 could, therefore, be a therapeutic target for improving both acute SARS‐CoV‐2 infection, and VaD post‐COVID morbidity and cognitive decline.

Viral infection causes long term changes of the immunological milieu within the heart

Abstract Background The last two decades have brought plentiful insights and new advances in understanding virus-host interactions, and the importance of acute and latent viral infections in the modulation of the structure and function of the mammalian immune system became apparent. Despite abundant evidence demonstrating substential changes after viral infections in the immune cell composition and the local immune response within various organs such as the lungs and the gut, the same phenomena within the heart tissue has remained poorly investigated. Methods C57BL6/J 6–12-week-old female mice were infected intravenously with murine cytomegalovirus (MCMV) or left uninfected. Kinetics of viral replication within the heart were determined using a standard plaque assay. Furthermore, immune cell composition within the heart was determined by flow cytometry during acute and latent viral infection. Results Active MCMV replication within the heart peaked at day 5 post-infection (p.i.) and the replicating viral particles became undetectable by day 12 in all of the infected animals. The initial immune cell infiltration was led by the increase in the NK cell compartment, followed by a substantial increase in the T cell compartment which peaked around day 7 p.i. Furthermore, there were substantial phenotypic changes in the population of cardiac macrophages which became predominantly MHCIIhigh and Ly6Chigh. Most notable was an enlargement within the CD8 T cell population which increased around 60-fold compared to the steady state conditions. Crucially, the changes in the immune cell milieu were maintained long after cessation of active viral replication as the hearts of latently infected mice contained around 2.5 times more leukocytes (CD45+) than the hearts of non-infected animals, around 18% percent of which were CD8 T cells, compared to 5% observed in the mock-infected group. Conclusion Systemic viral infection with a ubiquitous herpesvirus causes long term alterations in the composition and phenotype of the heart immune cells which could have major consequences for myocardial coping with different pathological injuries such as a subsequent microbial infection, ischemic conditions or stress induced by hypertension or volume overload.

Transcriptomic analysis reveals upregulated host metabolisms and downregulated immune responses or cell death induced by acute African swine fever virus infection.

The African swine fever virus is a virulent and communicable viral disease that can be transmitted by infected swine, contaminated pork products, or soft tick vectors. Nonstructural proteins encoded by ASFV regulate viral replication, transcription, and evasion. However, the mechanisms underlying the host response to ASFV infection remain incompletely understood. In order to enhance comprehension of the biology and molecular mechanisms at distinct time intervals (6, 12, 24 h) post infection, transcriptome analyses were executed to discern differentially expressed genes (DEGs) between ASFV and mock-infected PAMs. The transcriptomic analysis unveiled a total of 1,677, 2,122, and 2,945 upregulated DEGs and 933, 1,148, and 1,422 downregulated DEGs in ASFV- and mock-infected groups at 6, 12, and 24 h.p.i.. The results of the transcriptomic analysis demonstrated that the infection of ASFV significantly stimulated host metabolism pathways while concurrently inhibiting the expression of various immune responses and cell death pathways. Our study offers crucial mechanistic insights into the comprehension of ASFV viral pathogenesis and the multifaceted host immune responses. The genes that were dysregulated may serve as potential candidates for further exploration of anti-ASFV strategies.

Global quantitative proteomic analysis profiles of host protein expression in response to enterovirus A71 infection in bronchial epithelial cells based on tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS uncovers the key role of proteasome in virus replication

Enterovirus A71 (EV-A71) is a neurotropic human pathogen which mainly caused hand, foot and mouth disease (HFMD) mostly in children under 5 years-old. Generally, EV-A71-associated HFMD is a relatively self-limiting febrile disease, but there will still be a small percentage of patients with rapid disease progression and severe neurological complications. To date, the underlying mechanism of EV-A71 inducing pathological injury of central nervous system (CNS) remains largely unclear. It has been investigated and discussed the changes of mRNA, miRNA and circRNA expression profile during infection by EV-A71 in our previous studies. However, these studies were only analyzed at the RNA level, not at the protein level. It's the protein levels that ultimately do the work in the body. Here, to address this, we performed a tandem mass tag (TMT) peptide labeling coupled with LC-MS/MS approach to quantitatively identify cellular proteome changes at 24 h post-infection (hpi) in EV-A71-infected 16HBE cells. In total, 6615 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the EV-A71- and mock-infected groups, 210 differentially expressed proteins were found, including 86 upregulated and 124 downregulated proteins, at 24 hpi. To ensure the validity and reliability of the proteomics data, 3 randomly selected proteins were verified by Western blot and Immunofluorescence analysis, and the results were consistent with the TMT results. Subsequently, functional enrichment analysis indicated that the up-regulated and down-regulated proteins were individually involved in various biological processes and signaling pathways, including metabolic process, AMPK signaling pathway, Neurotrophin signaling pathway, Viral myocarditis, GABAergic synapse, and so on. Moreover, among these enriched functional analysis, the “Proteasome” pathway was up-regulated, which has caught our attention. Inhibition of proteasome was found to obviously suppress the EV-A71 replication. Finally, further in-depth analysis revealed that these differentially expressed proteins contained distinct domains and localized in different subcellular components. Taken together, our data provided a comprehensive view of host cell response to EV-A71 and identified host proteins may lead to better understanding of the pathogenic mechanisms and host responses to EV-A71 infection, and also to the identification of new therapeutic targets for EV-A71 infection.

A novel mouse model of SARS‐CoV‐2 induced neuroinflammation

AbstractBackgroundSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection may impact neurological function acutely or chronically, even in the absence of severe respiratory illness. Developing clinically relevant laboratory models to understand the neuropathogenesis of SARS‐CoV‐2 infection is an important step towards unravelling this neurologic consequence. We hypothesize that mouse adapted SARS‐CoV‐2 viral infection will induce neuroinflammation in immuno‐competent C57BL/6J (10 weeks old male) as well as immunodeficient RAG2−/‐ (10 weeks old male) and BALB/c (1 year old female) mice.MethodAll three mouse strains were inoculated intranasally with a dose of 1×103 PFU/mouse (50 µL) of either mock or the mouse‐adapted (MA)10 strain of SARS‐CoV‐2 (BEI resource, NR‐55329). Mice were euthanized on day 2, 3, 7, 15 or 30 post infection and brain samples processed for qRT‐PCR, immunofluorescence, and H&E analysis.ResultSARS‐CoV‐2 MA10 resulted in a significantly higher (p < 0.05) mRNA expression for chemokine ligand 2 (CCL2) and lower (p < 0.05) mRNA expression for the blood‐brain barrier component Claudin‐5 in RAG2−/‐ and WT mice when compared to mock infection. Also, SARS‐CoV‐2 MA10 infection increased microglial expression in 1 year old female BALB/c mice after 2 days of infection, compared to mock infected group. At 30 days post infection, MA10 infected BALB/c mice had a higher perivascular lymphocyte cuffing in the brain.ConclusionThis study demonstrates that the mouse‐adapted MA10 strain of SARS‐CoV‐2 can induce a neuroinflammatory state in the brain and more so in immunodeficient and aging mouse models. These mouse models will enable the investigation of the long‐term neurological effects of SARS‐CoV‐2 infection.

Quantitative proteomic analysis shows involvement of the p38 MAPK pathway in bovine parainfluenza virus type 3 replication

BackgroundBovine parainfluenza virus type 3 (BPIV3) infection often causes respiratory tissue damage and immunosuppression and further results in bovine respiratory disease complex (BRDC), one of the major diseases in dairy cattle, caused huge economical losses every year. However, the pathogenetic and immunoregulatory mechanisms involved in the process of BPIV3 infection remain unknown. However, the pathogenetic and immunoregulatory mechanisms involved in the process of BPIV3 infection remain unknown. Proteomics is a powerful tool for high-throughput identification of proteins, which has been widely used to understand how viruses interact with host cells.MethodsIn the present study, we report a proteomic analysis to investigate the whole cellular protein alterations of MDBK cells infected with BPIV3. To investigate the infection process of BPIV3 and the immune response mechanism of MDBK cells, isobaric tags for relative and absolute quantitation analysis (iTRAQ) and Q-Exactive mass spectrometry-based proteomics were performed. The differentially expressed proteins (DEPs) involved in the BPIV3 invasion process in MDBK cells were identified, annotated, and quantitated.ResultsA total of 116 proteins, which included 74 upregulated proteins and 42 downregulated proteins, were identified as DEPs between the BPIV3-infected and the mock-infected groups. These DEPs included corresponding proteins related to inflammatory response, immune response, and lipid metabolism. These results might provide some insights for understanding the pathogenesis of BPIV3. Fluorescent quantitative PCR and western blotting analysis showed results consistent with those of iTRAQ identification. Interestingly, the upregulated protein MKK3 was associated with the p38 MAPK signaling pathway.ConclusionsThe results of proteomics analysis indicated BPIV3 infection could activate the p38 MAPK pathway to promote virus replication.

NLRP3 Inflammasome-Mediated Pyroptosis Pathway Contributes to the Pathogenesis of Candida albicans Keratitis.

PurposeFungal keratitis is a sight-threatening corneal infection caused by fungal pathogens, and the pathogenic mechanisms have not been fully elucidated. The aim of this study was to determine whether NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis contributes to Candida albicans (C. albicans) keratitis and explore the underlying mechanism.MethodsAn in vivo mouse model of C. albicans keratitis and an in vitro culture model of human corneal epithelial cells (HCECs) challenged with heat-killed C. albicans (HKCA) were established in this study. The degree of corneal infection was evaluated by clinical scoring. Gene expression was assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis or immunofluorescence staining was performed to evaluate protein expression. TdT-mediated dUTP nick end labeling (TUNEL) staining was performed to examine the pyroptotic cell death. A lactate dehydrogenase (LDH) release assay was performed to assess cytotoxicity.ResultsCompared with the mock-infected group, we observed that the mRNA levels of NLRP3, caspase-1 (CASP1), interleukin (IL)−1β and gasdermin-D (GSDMD) in C. albicans-infected mice cornea was significantly increased. Our data also demonstrated that the protein expression of NLRP3 and the pyroptosis-related markers apoptosis-associated speck-like protein containing a CARD (ASC), cleaved CASP1, N-GSDMD, cleaved IL-1β and cleaved IL-18 as well as pyroptotic cell death were dramatically elevated in the mouse model of C. albicans keratitis. More importantly, NLRP3 knockdown markedly alleviated pyroptosis and consequently reduced corneal inflammatory reaction in C. albicans keratitis. In vitro, the presence of activated NLRP3 inflammasome and pyroptotic cell death were validated in HCECs exposed to HKCA. Furthermore, the potassium (K+) channel inhibitor glyburide decreased LDH release and suppressed NLRP3 inflammasome activation and pyroptosis in HCECs exposed to HKCA.ConclusionIn conclusion, the current study revealed for the first time that NLRP3 inflammasome activation and pyroptosis occur in C. albicans-infected mouse corneas and HCECs. Moreover, NLRP3 inflammasome-mediated pyroptosis signaling is involved in the disease severity of C. albicans keratitis. Therefore, This NLRP3 inflammasome-dependent pathway may be an attractive target for the treatment of fungal keratitis.

Tandem Mass Tag-Based Quantitative Proteomic Analysis of ISG15 Knockout PK15 Cells in Pseudorabies Virus Infection.

Pseudorabies virus (PRV) is recognized as one of the most important pathogens of swine and poses a serious threat to the swine industry worldwide. Available commercial vaccines fail to protect against the emergence of new PRV strains. Therefore, the new protein targets against PRV highlight the urgent need for uncovering the molecular determinants of host cellular proteins following PRV infection. Interferon-stimulated gene 15 (ISG15) demonstrates an outstanding antiviral response. However, the molecular mechanism of ISG15 that affects PRV replication is incompletely known. Here, we performed a tandem mass tag (TMT)-based approach to quantitatively identify protein expression changes in PRV-infected ISG15 knockout PK15 (ISG15−/−-PK15) cells. In total, 4958 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the PRV- and mock-infected groups, 241 differentially expressed proteins (DEPs) were identified, 162 upregulated and 79 downregulated proteins at 24 h post-infection (hpi), among which AFP, Vtn, Hsp40, Herc5, and Mccc1 may play important roles in PRV propagation. To ensure the validity and reliability of the proteomics data, the randomly selected DEPs were verified by RT-qPCR and Western blot analysis, and the results were consistent with the TMT results. Bioinformatics analyses further demonstrated that the DEPs are mainly involved in various biological processes and signaling pathways, such as signal transduction, the digestive system, and the PI3K-AKT pathway. These findings may provide new insight into molecular mechanisms for PRV infection, which is helpful for identifying potential protein targets for antiviral agents.

Exocellular polysaccharides extracted from mangrove fungus Paecilomyces Lilacinuson present anti-HSV-1 activity in mice

This study examined the anti-HSV-1 activity of EPS extracts isolated from mangrove fungus Paecilomyces Lilacinuson after intraperitoneal administration in mice. Mice were experimentally infected with HSV-1 intracranially and treated intraperitoneally with three different doses of EPS extract (6 g/Kg, 8 g/Kg, and 10 g/Kg) for 7 days. One group of 15 mice was infected with HSV-1 but did not receive any treatment, while another group of 15 mice was mock-infected to remain a control group. Animals were observed twice a day for 14 days after virus infection, searching for clinical signs of weight loss, piloerection, isolation, or retardation movement. Compared with the mock-infected group, mortality was significantly increased (p < 0.05) in the virus-infected group and the groups that received 6 g/Kg and 8 g/Kg EPS extract. Interestingly, no significant differences in mortality were found between the 10 g/Kg EPS extract and the mock-infected group. Mortality in the 10 g/Kg EPS extract group was substantially improved compared with virus-infected(p < 0.05). Additionally, EPS extracts inhibited HSV-1 replication in the mice brain in a dose-dependent manner. Furthermore, the extracts decreased NF-κB protein and mRNA expression and the production of TNF-α in HSV-1-infected mice brain tissue. These effects were also dose-dependent. Our findings suggest that the EPS extract may be a potential candidate for developing an antiviral drug against HSV-1.

Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves.Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Calves Infected with Virulent and Attenuated Mycoplasma bovis Strains Have Upregulated Th17 Inflammatory and Th1 Protective Responses, Respectively.

Mycoplasma bovis is a critical bovine pathogen, but its pathogenesis remains poorly understood. Here, the virulent HB0801 (P1) and attenuated HB0801-P150 (P150) strains of M. bovis were used to explore the potential pathogenesis and effect of induced immunity from calves’ differential transcriptomes post infection. Nine one-month-old male calves were infected with P1, P150, or mock-infected with medium and euthanized at 60 days post-infection. Calves in P1 group exhibited other clinical signs and pathological changes compared to the other two groups. Transcriptome profiles of peripheral blood mononuclear cells revealed seven and 10 hub differentially expressed genes (DEGs) in P1 and P150 groups compared with mock-infected group, respectively. Then, P1-induced pathogenesis was predicted to be associated with enhanced Th17, and P150-induced immunity with Th1 response and expression of ubiquitination-associated enzymes. Association analysis showed that 14 and 11 DEGs were positively and negatively correlated with pathological changes, respectively. Furthermore, up-regulated expression in molecules critical to differentiation of pathogenic Th17 cells in lung and peripheral blood mononuclear cells in P1 group was validated at RNA and protein levels. The results confirmed virulent and attenuated strains might be associated with biased differentiation of pro-inflammatory pathogenic Th17 and Th1 subsets respectively.

Proteomics analysis reveals heat shock proteins involved in caprine parainfluenza virus type 3 infection

BackgroundCaprine parainfluenza virus type 3 (CPIV3) is major pathogen of goat herds causing serious respiratory tract disease and economic losses to the goat industry in China. We analyzed the differential proteomics of CPIV3-infected Madin-Darby bovine kidney (MDBK) cells using quantitative iTRAQ coupled LC-MS/MS. In addition, four DEPs were validated by qRT-PCR and western blot analysis.ResultsQuantitative proteomics analysis revealed 163 differentially expressed proteins (DEPs) between CPIV3-infected and mock-infected groups (p-value < 0.05 and fold change > 1.2), among which 91 were down-regulated and 72 were up-regulated. Gene ontology (GO) analysis showed that these DEPs were involved in molecular functions, cellular components and biological processes. Biological functions in which the DEPs were involved in included diseases, genetic information processing, metabolism, environmental information processing, cellular processes, and organismal systems. STRING analysis revealed that four heat shock proteins (HSPs) included HSPA5, HSPA1B, HSP90B1 and HSPA6 may be associated with proliferation of CPIV3 in MDBK cells. qRT-PCR and western blot analysis showed that the selected HSPs were identical to the quantitative proteomics data.ConclusionTo our knowledge, this is the first report of the proteomic changes in MDBK cells after CPIV3 infection.

Isolation and identification of Singapore grouper iridovirus Hainan strain (SGIV-HN) in China.

In recent years, with the rapid development of marine farming activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing heavy economic losses. Singapore grouper iridovirus (SGIV) is one of the most important viruses causing disease in fish. In the present study, we isolated and identified a virus from diseased groupers by coculturing the affected tissue cells with grouper spleen cells. The genome of the isolated virus shared 99.83% nucleotide sequence homology with those of SGIV reference strains in the GenBank database. The virus clustered with SGIV on an evolutionary tree constructed based on "major capsid protein" (MCP) amino acid sequences, so it was designated 'Singapore grouper iridovirus Hainan' (SGIV-HN). To evaluate the pathogenic potential of SGIV-HN in fish, orange-spotted groupers were infected by intraperitoneal injection with the virus. Infected groupers began to die from the fourth day after infection, and survivors tended to be stable by the eighth day. The death rate was 83.33%. In a mock-infected control group, only two fish died, and the mortality rate was 6.67%. Dissection showed that the fish had enlarged spleenswith hemorrhage, and enlarged cells were visible with Giemsa staining. This is the first report of isolation of SGIV from naturally infected fish in China, and we show that SGIV-HN is highly infectious, causing massive deaths in groupers.

Transcriptome analysis of Aeromonas hydrophila infected hybrid sturgeon (Huso dauricus\xd7Acipenser schrenckii)

The hybrid sturgeon (Huso dauricus × Acipenser schrenckii) is an economically important species in China. With the increasing aquaculture of hybrid sturgeon, the bacterial diseases are a great concern of the industry. In this study, de novo sequencing was used to compare the difference in transcriptome in spleen of the infected and mock infected sturgeon with Aeromonas hydrophila. Among 187,244 unigenes obtained, 87,887 unigenes were annotated and 1,147 unigenes were associated with immune responses genes. Comparative expression analysis indicated that 2,723 differently expressed genes between the infected and mock-infected group were identified, including 1,420 up-regulated and 1,303 down-regulated genes. 283 differently expressed anti-bacterial immune related genes were scrutinized, including 168 up-regulated and 115 down-regulated genes. Ten of the differently expressed genes were further validated by qRT-PCR. In this study, toll like receptors (TLRs) pathway, NF-kappa B pathway, class A scavenger receptor pathway, phagocytosis pathway, mannose receptor pathway and complement pathway were shown to be up-regulated in Aeromonas hydrophila infected hybrid sturgeon. Additionally, 65,040 potential SSRs and 2,133,505 candidate SNPs were identified from the hybrid sturgeon spleen transcriptome. This study could provide an insight of host immune genes associated with bacterial infection in hybrid sturgeon.

Transcriptional Profile and Integrative Analyses of Long Noncoding RNAs in Primary Human Corneal Epithelial Cells in Response to HSV-1 Infection

ABSTRACT Purpose Long noncoding RNAs (lncRNAs) have been demonstrated to have important regulatory functions in diverse cellular processes; however, the role of lncRNAs in the pathogenesis of herpes simplex keratitis (HSK) remains poorly understood. Methods Primary human corneal epithelial cells (HCECs) were infected with herpes simplex virus-1 (HSV-1) and the total RNAs extracted from both the infected group and the mock-infected group subjected to microarray analysis to identify the differential expression of lncRNAs and mRNAs. We also performed bioinformatic analysis including gene ontology (GO) analysis, pathway analysis and co-expression network analysis. Results Compared with mock-infected group, the expression of thousands of lncRNAs and mRNAs were significantly changed, and the microarray results were validated by qRT-PCR. The most enriched GOs targeted by up-regulated transcripts were defense response, intrinsic component of plasma membrane and cytokine activity，and the most enriched GOs targeted by the down-regulated transcripts were cellular metabolic process, intracellular part and poly (A) RNA binding. Pathway analysis indicated that the most correlated pathways for up- and down-regulated transcripts were cytokine-cytokine receptor interaction and RNA transport, respectively. Conclusions Our study identified the genome-wide profile of lncRNAs and mRNAs expression in primary corneal epithelial cells with HSV-1 infection. These transcriptomic data together with subsequent bioinformatic analysis will provide us with novel clue to the insight into molecular mechanism and potential therapeutic targets of HSK. Further studies are expected to verify the potentially functional genes and pathways and explore the critical lncRNAs. Abbreviations Long noncoding RNAs: lncRNAs; herpes simplex virus-1: HSV-1; herpes simplex virus keratitis: HSK; human corneal epithelial cells: HCECs

Protective effects and immunomodulation on piglets infected with rotavirus following resveratrol supplementation.

Rotavirus (RV), belonging to Reoviridae family, is the leading cause of acute severe viral diarrhea in children (under 5 years old) and infant animals worldwide. Although vaccines are commonly used to prevent infection, episodes of diarrhea caused by RV frequently occur. Thus, this study was conducted to determine whether resveratrol had protective effects against RV infection in piglets. Following pretreatment with resveratrol dry suspension through adding into the basal diet for 3 weeks, the piglets were orally challenged with RV. We found that resveratrol could alleviate diarrhea induced by RV infection. Resveratrol-treatment inhibited the TNF-α production, indicating that the anti-RV activity of resveratrol may be achieved by reducing the inflammatory response. The IFN-γ level was elevated in 10mg/kg/d resveratrol-treated group and 30mg/kg/d resveratrol-treated group after RV infection. The ratios of CD4+/CD8+ in resveratrol-treated groups were the same as that in mock infected group, suggesting that resveratrol could maintain the immune function in RV-infected piglets. It was found that resveratrol could alleviate diarrhea induced by RV infection. These results revealed that resveratrol dry suspension could be a new control measure for RV infection.

Restricted Infection and Cytokine Expression in Primary Murine Astrocytes Induced by the H5N1 Influenza Virus

Central nervous system (CNS) dysfunction caused by infection with neurovirulent viruses is of interest, and may play an important role in many neurodegenerative diseases. Since neuronal functions are believed to be partly regulated by cytokines produced by astrocytes, neuroinflammation and neurodegeneration caused by H5N1 influenza virus infection could be due to abnormal cytokine production of those infected astrocytes. In the present study, cytokine responses of murine astrocytes following H5N1 virus infection were investigated. Primary astrocytes from neonatal outbred ICR mice were isolated and used to investigate cytopathology upon infection with the H5N1 virus (A/Chicken/Thailand/CUK2/04). Thereafter, cell lysates at 6, 24, 48, and 72 hours-post infection (hpi) were collected and subjected to quantification of cytokine gene expression, including TNF-α, IL-1β, IL-2, IL-6 and IL-12β, by quantitative RT-PCR. The results revealed that infection with the H5N1 virus in primary murine astrocytes was restricted, and resulted in abortive virus infection. However, this abortive infection in the astrocytes was found to result in significant upregulation of IL-1β mRNA expression at 72 hpi compared with the mock-infected group, while the mRNA expression of IL-2 and IL-12β was observed to have undergone significant down-regulation at 6-72 hpi and 24-48 hpi, respectively. The results of the present study could support the role of astrocytes in neuroinflammation and neurodegeneration.

Glucagon-like peptide-1 receptor signaling attenuates RSV-induced type 2 responses and immunopathology

Abstract Glucagon-like peptide-1 receptor (GLP-1R) agonists are a well-accepted and safe treatment for Type II diabetes. Although GLP-1R agonists mainly act to potentiate insulin and suppress glucagon secretion, recent evidence suggests that GLP-1R signaling also has anti-inflammatory effects. Severe RSV-associated illness is partially caused by type 2-associated immunopathology. We hypothesized that GLP-1R signaling inhibits type 2-mediated immunopathology during infection with RSV 12/12-6, a strain of RSV that was isolated from a hospitalized infant with severe lower respiratory tract infection and bronchiolitis. We show here that GLP-1R agonist treatment decreased airway inflammation, airway reactivity, and airway mucus production in RSV 12/12-6-infected mice. GLP-1R agonist treatment decreased total lung IL-13 and IL-33 levels, with concurrent decreases in lung IL-13-producing group 2 innate lymphoid cell (ILC2), CD4+ Th2 cell, and basophil numbers as well as IL-33-producing epithelial cells. The GLP-1R agonist prevented airway inflammation, and did not impact viral load, anti-viral interferon and antibody production during secondary RSV infection. Relative to the respective mock-infected groups, RSV-infected GLP-1R agonist treated mice did not have increased weight loss compared to vehicle treated mice. A phenome-wide association study (PheWAS) identified a link between GLP-1R signaling and acute bronchitis and bronchiolitis in humans. These studies demonstrate that GLP-1R signaling protects against type 2-mediated immunopathology during RSV infection. GLP-1R agonists are the first known FDA-approved agents to inhibit IL-33 and may represent a novel treatment strategy for RSV bronchiolitis.