Titers Of Infectious Virus Research Articles

Safety and immunogenicity of a live attenuated Rift Valley Fever recombinant arMP-12ΔNSm21/384 vaccine candidate for sheep, goats and calves

Rift Valley fever (RVF) causes serious health and economic losses to the livestock industry as well as a significant cause of human disease. The prevention of RVF in Africa is a global priority, however, available vaccines have only been partially effective. Therefore, the objective of this study was to evaluate the safety and immunogenicity of a live, attenuated recombinant RVFV arMP-12ΔNSm21/384 nucleotide deletion vaccine candidate in domestic ruminants. Evaluation involved testing to determine the infectivity titer of the vaccine virus in Vero cells for industrial scale up vaccine production. Safety experiments were conducted to determine the potential of the vaccine virus to revert to virulence by serial passages in sheep, the possibility of virus spread from vaccinated sheep and calves to unvaccinated animals, and the potential health effects of administering overdoses of the vaccine to sheep, goats and calves. The immunogenicity of 3 doses of 104, 105 and 106 Tissue Culture Infectious Doses50% (TCID50) of the vaccine was assessed in 3 groups of 10 sheep and 3 groups of 10 goats, and doses of 105, 106 and 107 TCID50 was evaluated in 3 groups of 10 calves subcutaenous vaccintation. The results showed that the infectivity titer of the vaccine virus was 108.4 TCID50/ml, that the vaccine did not spread from vaccinated to un-vaccinated animals, there was no evidence of reversion to virulence in sheep and the vaccine overdoses did not cause any adverse effects. The immunogenicity among sheep, goats and calves indicated that doses of 104–106 TCID50 elicited detectable antibody by day 7 post-vaccination (PV) with antibody titers ranging from 0.6 log to 2.1 log on day 14 PV with sustained titers through day 28 PV. Overall, these findings indicated that the RVFV arMP-12ΔNSm21/384 vaccine is a promising candidate for the prevention of RVF among domestic ruminants.

The recruitment of peripheral blood leukocytes to the brain is delayed in susceptible BALB/c compared to resistant C57BL/6 mice during herpes simplex virus encephalitis.

The cerebral immune response induced by herpes simplex virus (HSV) encephalitis (HSE) was evaluated in susceptible BALB/c and resistant C57BL/6 mice. BALB/c and C57BL/6 (named C57BL/6-high) mice were respectively infected intranasally with 1 × 103 and 5 × 105 plaque-forming units (PFUs) of HSV-1. C57BL/6 mice (named C57BL/6-low) infected with a low inoculum (1 × 103 PFUs) of HSV-1 were tested in parallel. Mice were monitored for weight loss, sickness signs, and survival for 21days. The viral load, infectious titers, cytokine/chemokine levels, and peripheral leukocyte infiltration were determined in brain homogenates on days 0 (non-infected), 4, 6, and 8 post-infection (p.i.) by qPCR, plaque assay, ELISA/Luminex™, and flow cytometry, respectively. Our results showed that the mortality of BALB/c mice (67%) was higher compared to those of C57BL/6-low (0%; P ≤ 0.01) and C57BL/6-high (20%; P ≤ 0.05) animals. This higher mortality was associated with increased infectious titers and cytokine/chemokine levels in the brains of BALB/c compared to C57BL/6 mice. Recruitment of inflammatory monocytes, dendritic cells, natural killer, and natural killer T cells to the brain was higher in C57BL/6-high compared to BALB/c animals on day 4 p.i. Infiltration of inflammatory monocytes and T cells in the brain of BALB/c mice was seen on day 6 p.i. Our data suggest that a rapid, sustained, and coordinated recruitment of peripheral leukocytes to the brain of C57BL/6-high mice results in an effective control of viral replication and inflammation whereas the delayed infiltration of immune cells in the brain of BALB/c mice was associated with an exacerbated inflammatory response during HSE.

Mutation of Influenza A Virus PA-X Decreases Pathogenicity in Chicken Embryos and Can Increase the Yield of Reassortant Candidate Vaccine Viruses.

The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

A virus precipitation method for concentration & detection of avian influenza viruses from environmental water resources & its possible application in outbreak investigations.

Background & objectives:Avian influenza (AI) viruses have been a major cause of public health concern. Wild migratory birds and contaminated environmental sources such as waterbodies soiled with bird droppings play a significant role in the transmission of AI viruses. The objective of the present study was to develop a sensitive and user-friendly method for the concentration and detection of AI viruses from environmental water sources.Methods:Municipal potable water, surface water from reservoirs and sea were spiked with low pathogenic AI viruses. To concentrate the viruses by precipitation, a combination of potassium aluminium sulphate with milk powder was used. Real-time reverse transcription-polymerase chain reaction was performed for virus detection, and the results were compared with a virus concentration method using erythrocytes. Drinking water specimens from poultry markets were also tested for the presence of AI viruses.Results:A minimum of 101.0 EID50 (50% egg infectious dose)/ml spiked H5N1 and 101.7 EID50/ml spiked H9N2 viruses were detected from spiked potable water; 101.0 and 102.0 EID50/ml spiked H5N1 virus was detected from surface water and seawater samples, respectively. The present method was more sensitive than the erythrocyte-binding method as approximately 10-fold higher infectious virus titres were obtained. AI H9N2 viruses were detected and isolated from water from local poultry markets, using this method.Interpretation & conclusions:Viability and recovery of the spiked viruses were not affected by precipitation. The present method may be suitable for the detection of AI viruses from different environmental water sources and can also be applied during outbreak investigations.

Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID50), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.

Influenza A Virus M2 Protein Apical Targeting Is Required for Efficient Virus Replication.

The influenza A virus (IAV) M2 protein is a multifunctional protein with critical roles in virion entry, assembly, and budding. M2 is targeted to the apical plasma membrane of polarized epithelial cells, and the interaction of the viral proteins M2, M1, HA, and NA near glycolipid rafts in the apical plasma membrane is hypothesized to coordinate the assembly of infectious virus particles. To determine the role of M2 protein apical targeting in IAV replication, a panel of M2 proteins with basolateral plasma membrane (M2-Baso) or endoplasmic reticulum (M2-ER) targeting sequences was generated. MDCK II cells stably expressing M2-Baso, but not M2-ER, complemented the replication of M2-stop viruses. However, in primary human nasal epithelial cell (hNEC) cultures, viruses encoding M2-Baso and M2-ER replicated to negligible titers compared to those of wild-type virus. M2-Baso replication was negatively correlated with cell polarization. These results demonstrate that M2 apical targeting is essential for IAV replication: targeting M2 to the ER results in a strong, cell type-independent inhibition of virus replication, and targeting M2 to the basolateral membrane has greater effects in hNECs than in MDCK cells.IMPORTANCE Influenza A virus assembly and particle release occur at the apical membrane of polarized epithelial cells. The integral membrane proteins encoded by the virus, HA, NA, and M2, are all targeted to the apical membrane and believed to recruit the other structural proteins to sites of virus assembly. By targeting M2 to the basolateral or endoplasmic reticulum membranes, influenza A virus replication was significantly reduced. Basolateral targeting of M2 reduced the infectious virus titers with minimal effects on virus particle release, while targeting to the endoplasmic reticulum resulted in reduced infectious and total virus particle release. Therefore, altering the expression and the intracellular targeting of M2 has major effects on virus replication.

TEST-SYSTEM BASED ON INDIRECT “SANDWICH” ELISA TO DETECT THE LUMPY SKIN DISEASE VIRUS ANTIGEN

Lumpy skin disease is an economically significant disease as it results in decrease in weight gain and milk yield, abortions, mastitis, reproduction disorders, animal emaciation, lesions of respiratory organs and in some cases – death. Today the disease is included in the OIE list and is subject to obligatory notification. The emergence and spread of the disease in the Russian Federation necessitated performance of tests in the framework of laboratory diagnosis method improvement. The test-system based on the indirect “Sandwich” ELISA for diagnosis of lumpy skin disease allowing performance of biomaterial tests within 24 hours was for the first time developed in Russia. The test-system development included antibody preparation in laboratory animals (rabbits and guinea pigs), selection of optimal dilution of capture and detection antibodies, composition of a buffer solution and conditions of the reaction procedure. 50 samples of initial culture antigens of the lumpy skin disease virus as well virus-containing suspensions collected on different stages of purification and concentration were tested using this technique. To confirm the ELISA results all analyzed samples were tested using RT-PCR. Besides, the virus infectivity titer was determined by titration in YaDK-04 (Goat gonad cells). The test specificity was 100%, and analytical sensitivity – 3.5 lg TCD50. The developed “Sandwich” ELISA allows performing tests of 24 antigen samples at 1:2–1:16 dilution simultaneously using 96-well plate and it can be used for lumpy skin disease diagnosis.

Respiratory Syncytial Virus Matrix (M) Protein Interacts with Actin In Vitro and in Cell Culture.

The virus–host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1–256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for M–actin interaction in transporting virion components in the infected cell.

Rapid quantification of vesicular stomatitis virus in Vero cells using Laser Force Cytology

The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells. LFC uses a combination of optical and fluidic forces to interrogate single cells without the use of labels or antibodies. Using a combination of variables measured by the Radiance™ LFC instrument (LumaCyte), an infection metric was developed that correlates well with the viral titer as measured by TCID50 and shortens the timeframe from infection to titer determination from 3 days to 16 h (a 4.5 fold reduction). A correlation was also developed between in-process cellular measurements and the viral titer of collected supernatant, demonstrating the potential for real-time infectivity measurements. Overall, these results demonstrate the utility of LFC as a tool for rapid infectivity measurements throughout the vaccine development process.

TM6SF2 Promotes Lipidation and Secretion of Hepatitis C Virus in Infected Hepatocytes

Hepatitis C virus (HCV) co-opts the very-low-density lipoprotein pathway for morphogenesis, maturation, and secretion, and circulates as lipoviroparticles (LVPs). We investigated the functions and underlying mechanisms of the lipid-associated TM6SF2 protein in modulating LVP formation and the HCV life cycle. We knocked down or overexpressed TM6SF2 in hepatic cellsand examined HCV infection, measuring viral RNA andprotein levels and infectious LVP titers. The density of secreted LVPswas evaluated by iodixanol gradient assay. We measured levels and patterns of TM6SF2 in liver biopsies from 73 patients with chronic hepatitis C, livers of HCV-infected humanized Alb-uPA/SCID/beige mice, and HCV-infected Huh7.5.1 cells. TM6SF2 knockdown in hepatocytes reduced viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation, or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatants. In HCV-infected cells, TM6SF2 overexpression resulted in production of more infectious LVPs in the lower-density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice, and liver tissues of HCV patients. TM6SF2 messenger RNA levels correlated positively with HCV RNA levels in liver biopsies from patients. SREBF2 appears to mediate the ability of HCVto increase the expression of TM6SF2 in hepatic cells. In studies of cells, mice and human liver tissues, we found TM6SF2 is required for maturation, lipidation, and secretion of infectious LVPs. HCV, in turn, up-regulates expression of TM6SF2 to facilitate productive infection.

Adaptive mutation F772S-enhanced p7-NS4A cooperation facilitates the assembly and release of hepatitis C virus and is associated with lipid droplet enlargement

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and liver cancer worldwide. Adaptive mutations play important roles in the development of the HCV replicon and its infectious clones. We and others have previously identified the p7 mutation F772S and the co-presence of NS4A mutations in infectious HCV full-length clones and chimeric recombinants. However, the underlying mechanism of F772S function remains incompletely understood. Here, we investigated the functional role of F772S using an efficient JFH1-based reporter virus with Core-NS2 from genotype 2a strain J6, and we designated J6-p7/JFH1-4A according to the strain origin of the p7 and NS4A sequences. We found that replacing JFH1-4A with J6-4A (wild-type or mutated NS4A) or genotype 2b J8-4A severely attenuated the viability of J6-p7/JFH1-4A. However, passage-recovered viruses that contained J6-p7 all acquired F772S. Introduction of F772S efficiently rescued the viral spread and infectivity titers of J6-p7/J6-4A, which reached the levels of the original J6-p7/JFH1-4A and led to a concomitant increase in RNA replication, assembly and release of viruses with J6-specific p7 and NS4A. These data suggest that an isolate-specific cooperation existed between p7 and NS4A. NS4A exchange- or substitution-mediated viral attenuation was attributed to the RNA sequence, and no p7-NS4A protein interaction was detected. Moreover, we found that F772S-enhanced p7-NS4A cooperation was associated with the enlargement of intracellular lipid droplets. This study therefore provides new insights into the mechanisms of adaptive mutations and facilitates studies on the HCV life cycle and virus–host interaction.

In vitro phagocytosis of opsonized latex beads by HD11 cells as a method to assess the general opsonization potential of chicken serum

ABSTRACTOpsonins, an important arm of the innate immune system, are various soluble proteins, which play a critical role in destruction of invading pathogens directly or via engulfment of pathogens through the intermediate of phagocytosis. The diversity of opsonin profiles is under genetic influence and may be associated with variation in disease resistance. The aim of this study was to set up an assay to determine serum opsonophagocytic potential (OPp) for chicken sera by flow cytometry and to evaluate the assay using samples from different chicken lines. Two chicken lines selected for high and low concentrations of mannose-binding lectin, a known opsonin, in serum were used to establish the method. Furthermore, the presumed “robust” Hellevad chickens and two other commercial chicken lines (Hisex and Bovans) were tested to evaluate OPp as a parameter reflecting general immune competence. The results showed that Hellevad and Bovans chickens had higher OPp than Hisex chickens. There were no correlations between concentrations of total IgY or mannose-binding lectin and OPp. However, a strong positive correlation was observed between vaccine-induced infectious bronchitis virus titres and OPp. Moreover, inverse relationships were observed between concentrations of total serum IgM as well as natural antibody levels, and OPp. In conclusion, in vitro opsonophagocytosis assessment and determination of OPp may be of relevance when addressing general innate immunocompetence.RESEARCH HIGHLIGHTSA flow cytometry method was developed to assess poultry serum opsonophagocytosis potential.This method is based on serum-opsonin-coated polystyrene beads and HD11 cell phagocytosis.Serum samples from different commercial chicken lines were compared.Opsonophagocytic potential may be included in assay panels for general immune competence of poultry.

Pregnancy preserves pulmonary function following influenza virus infection in C57BL/6 mice.

Pregnancy is associated with significant anatomic and functional changes to the cardiopulmonary system. Using pregnant C57BL/6 mice, we characterized changes in pulmonary structure and function during pregnancy in healthy animals and following infection with influenza A virus (IAV). We hypothesized that pregnancy-associated alterations in pulmonary physiology would contribute to the more severe outcome of IAV infection. Nonpregnant and pregnant females (at embryonic day 10.5) were either mock-infected or infected with 2009 H1N1 IAV for assessment of pulmonary function, structure, and inflammation at 8 days postinoculation. There were baseline differences in pulmonary function, with pregnant females having greater lung compliance, total lung capacity, and fixed lung volume than nonpregnant females. Following IAV infection, both pregnant and nonpregnant females exhibited reduced circulating progesterone, which in nonpregnant females was associated with increased pulmonary resistance and decreased lung compliance, minute ventilation, and oxygen diffusing capacity compared with uninfected nonpregnant females. In pregnant females, reduced concentrations of progesterone were associated with adverse pregnancy outcomes, but measures of pulmonary function were preserved following IAV infection and were not significantly different from uninfected pregnant mice. Following IAV infection, infectious virus titers and total numbers of pulmonary leukocytes were similar between pregnant and nonpregnant females, but the histological density of pulmonary inflammation was reduced in pregnant animals. These data suggest that pregnancy in mice is associated with significant alterations in pulmonary physiology but that these changes served to preserve lung function during IAV infection. Pregnancy-associated alterations in pulmonary physiology may serve to protect females during severe influenza.

Following Acute Encephalitis, Semliki Forest Virus is Undetectable in the Brain by Infectivity Assays but Functional Virus RNA Capable of Generating Infectious Virus Persists for Life.

Alphaviruses are mosquito-transmitted RNA viruses which generally cause acute disease including mild febrile illness, rash, arthralgia, myalgia and more severely, encephalitis. In the mouse, peripheral infection with Semliki Forest virus (SFV) results in encephalitis. With non-virulent strains, infectious virus is detectable in the brain, by standard infectivity assays, for around ten days. As we have shown previously, in severe combined immunodeficient (SCID) mice, infectious virus is detectable for months in the brain. Here we show that in MHC-II-/- mice, with no functional CD4 T-cells, infectious virus is also detectable in the brain for long periods. In contrast, in the brains of CD8-/- mice, virus RNA persists but infectious virus is not detectable. In SCID mice infected with SFV, repeated intraperitoneal administration of anti-SFV immune serum rapidly reduced the titer of infectious virus in the brain to undetectable, however virus RNA persisted. Repeated intraperitoneal passive transfer of immune serum resulted in maintenance of brain virus RNA, with no detectable infectious virus, for several weeks. When passive antibody transfer was stopped, antibody levels declined and infectious virus was again detectable in the brain. In aged immunocompetent mice, previously infected with SFV, immunosuppression of antibody responses many months after initial infection also resulted in renewed ability to detect infectious virus in the brain. In summary, antiviral antibodies control and determine whether infectious virus is detectable in the brain but immune responses cannot clear this infection from the brain. Functional virus RNA capable of generating infectious virus persists and if antibody levels decline, infectious virus is again detectable.

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress.

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target.IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.

P.1.016 - Molecular linking of influenza infection to cellular pathology of protein misassembly: the case of Disrupted-in-Schizophrenia 1 (DISC1)

Chronic mental illnesses such as schizophrenia or recurrent affective disorders are heterogeneous in their biological causes behind the clinical, syndromatic diagnosis. Viral infection has been linked to chronic mental illnesses both by direct mechanisms and indirect mechanisms through immune stimulation. For example, studies showed that intrauterine influenza infection could be a risk factor for chronic mental illness [1]. It is also established that viral infection can lead to a change in proteostasis impairing regulatory protein networks and leading to disease. The most dramatic consequences of disrupted proteostasis are the so-called protein misfolding diseases that show as a phenotypical hallmark, microscopically visible deposits of one or several proteins, and cell degeneration. More subtle forms of proteostatic imbalance exist where protein deposits are not microscopically visible and the neurons do not die but remain functionally impaired, for example in some forms of chronic mental illness such as schizophrenia [2]. We previously reported insoluble Disrupted-in-schizophrenia 1 (DISC1) protein in a subpopulation of patients with chronic mental illness [3]. This gene/protein is involved in vulnerability to chronic mental disorders, and was discovered mutant and segregating in a large Scottish pedigree with a chromosomal translocation leading to a 3′ truncation of the gene [4]. Subsequent genetic association studies in multiple populations of different ethnicities also support the involvement of this protein in mental illness. For the present study, our hypothesis was that influenza infection could cause significant long-term impairments of specific proteostatic pathways leading to aggregated DISC1 protein in chronic mental illness patients [5]. Neuroblastoma cell lines expressing inducible full-length, non-mutant DISC1 protein were infected with WSN33 influenza (H1N1 strain). The effect of viral infection was investigated by immunocytochemistry, biochemical and functional assays. Furthermore, infected mice with WSN33 influenza where investigated for insoluble DISC1 deposits by immunohistochemistry. Antiviral drugs were tested in order to reverse the influenza-induced DISC1 aggregates. Influenza infection of cell lines induced DISC1 misassembly but not misassembly of the protein TDP43 known to be mislocalized in ALS, but not in mental illness or influenza, and therefore serving as a control for specificity of DISC1 mis-assembly upon influenza infection in cellular models. Appearance of DISC1 deposits was dependent on infectious influenza virus titer, and the infection time. Functional consequences of influenza-induced DISC1 misassembly will be reported, as well as our investigations of influenza-infected mice that are still ongoing. We conclude that influenza infection leads to a very specific proteostatic change, including the misassembly of DISC1, which has functional consequences for dopamine homeostasis. Thus a selective reprogramming of cellular homeostasis, as measured by proteostasis, may be a novel molecular mechanism by which influenza infection can lead to brain disease.

Inactivation of chikungunya virus in blood components treated with amotosalen/ultraviolet A light or amustaline/glutathione.

Chikungunya virus, a mosquito-borne arbovirus, often co-circulates with the Zika, dengue, and yellow fever viruses in Aedes mosquito-infested areas where cases of arbovirus transfusion-transmitted infections have been reported. Building on past experience to help maintain the availability of safe components during major outbreaks of chikungunya virus in La Reunion, Italy, and Thailand and of Zika virus in the Pacific, the Caribbean, and the Americas, pathogen inactivation is a mitigation strategy to reduce the risk of transfusion-transmitted infection. Inactivation of chikungunya virus was investigated for platelets in 100% plasma using amotosalen/ultraviolet A light, and in red blood cells using amustaline/glutathione. Platelets in 100% plasma and red blood cells (RBCs) were spiked with chikungunya virus. Infectious chikungunya virus titers were measured in contaminated blood products before and after treatment with amotosalen/ultraviolet A light for platelets in 100% plasma and after treatment with amustaline/glutathione for RBCs. Viral infectivity was quantified by plaque assay. The mean chikungunya virus infectivity titers before inactivation were 6.50 log10 plaque-forming units/mL for platelets in 100% plasma and 7.60 log10 plaque-forming units/mL for RBCs. No infectivity was detected after amotosalen/ultraviolet A light or amustaline/glutathione treatment, corresponding to greater than 6.5 log10 plaque-forming units/mL and greater than 7.1 log10 plaque-forming units/mL of inactivation, respectively. Robust levels of chikungunya virus inactivation were achieved for platelets in 100% plasma and for RBC components. The licensed amotosalen/ultraviolet A light technology and the amustaline/glutathione pathogen-reduction system under development may provide an opportunity for comprehensive mitigation of the risk of chikungunya virus transfusion-transmitted infection by plasma, platelets, and RBCs.

Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production.

Infectious HCV carrying reporter genes have further applications in understanding the HCV life cycle including replication, viral assembly and release. In this study, a full-length 3039bp LacZ gene was inserted into the derivative of JFH1-AM120 to develop an additional reporter virus. The results showed that the recombinant reporter virus JFH1-AM120-LacZ can replicate and produce lower titers of infectious virus. However, insertion of the LacZ gene in the C-terminal region of the NS5A in HCV JFH1-AM120-LacZ decreased viral replication and dramatically impaired the production of infectious viral particles. Moreover, the JFH1-AM120-LacZ reporter virus lost the LacZ gene after serial passage. Nevertheless, the JFH1-AM120-LacZ reporter virus displayed the entire life cycle of HCV, from replication to production of infectious virus, in Huh7.5 cells. This study demonstrates that the NS5A region of HCV JFH1-AM120 has the capacity to accommodate large foreign genes up to 3,039 bp and suggests that other relatively large gene inserts can be accommodated at this site.

A flavonoid compound library screen revealed potent antiviral activity of plant-derived flavonoids on human enterovirus A71 replication

Hand Foot Mouth Disease (HFMD), resulting from human enterovirus A71 (HEVA71) infection can cause severe neurological complications leading to fatality in young children. Currently, there is no approved antiviral for therapeutic treatment against HEVA71 infection. In this study, a 500-compound flavonoid library was screened to identify potential inhibitors of HEVA71 using high-throughput immunofluorescence-based phenotypic screening method. Two lead flavonoid compounds, ST077124 and ST024734 at the non-cytotoxic concentration of 50 μM were found to be effective antivirals that inhibited replication of HEVA71, reducing infectious viral titers by 3.5 log10 PFU/ml and 2.5 log10 PFU/ml respectively. Our study revealed that ST077124 is a specific antiviral compound that inhibits human enteroviruses while ST024734 exhibited antiviral activity against human enteroviruses as well as dengue virus type-2. We also identified that both compounds affected the viral RNA transcription and translation machinery of HEVA71 but did not interfere with the viral internal ribosomal entry site (IRES) activity. Hence, our findings strongly suggest that ST077124 and ST024734 are effective antiviral compounds of minimal cytotoxicity and could serve as promising therapeutic agents against HEVA71 infection.

Lytic viral replication and immunopathology in a cytomegalovirus-induced mouse model of secondary hemophagocytic lymphohistiocytosis

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a rare immunological disorder caused by unbridled activation of T cells and macrophages, culminating in a life-threatening cytokine storm. A genetic and acquired subtype are distinguished, termed primary and secondary HLH, respectively. Clinical manifestations of both forms are frequently preceded by a viral infection, predominantly with herpesviruses. The exact role of the viral infection in the development of the hemophagocytic syndrome remains to be further elucidated.MethodsWe utilized a recently developed murine model of cytomegalovirus-associated secondary HLH and dissected the respective contributions of lytic viral replication and immunopathology in its pathogenesis.ResultsHLH-like disease only developed in cytomegalovirus-susceptible mouse strains unable to clear the virus, but the severity of symptoms was not correlated to the infectious viral titer. Lytic viral replication and sustained viremia played an essential part in the pathogenesis since abortive viral infection was insufficient to induce a full-blown HLH-like syndrome. Nonetheless, a limited set of symptoms, in particular anemia, thrombocytopenia and elevated levels of soluble CD25, appeared less dependent of the viral replication but rather mediated by the host’s immune response, as corroborated by immunosuppressive treatment of infected mice with dexamethasone.ConclusionBoth virus-mediated pathology and immunopathology cooperate in the pathogenesis of full-blown virus-associated secondary HLH and are closely entangled. A certain level of viremia appears necessary to elicit the characteristic HLH-like symptoms in the model.