Snakehead vesiculovirus (SHVV) infection alters striped snakehead (Ophicephalus striatus) cells (SSN-1) glutamine metabolism and apoptosis pathways.

Snakehead vesiculovirus (SHVV) causes enormous economic losses in snakehead fish (Ophicephalus striatus) culture. Understanding replication mechanisms of virus is considerable significance in preventing and treating viral disease. In our previous studies, we have reported that glutamine starvation could significant inhibit the replication of SHVV. Furthermore, we also showed that SHVV infection could cause apoptosis of striped snakehead fish cells (SSN-1). However, the underlying mechanisms remain enigmatic. To decipher the relationships among the viral infection, glutamine starvation and apoptosis, SSN-1 cells transcriptomic profilings of SSN-1 cells infected with or without SHVV under glutamine deprived condition were analyzed. RNA-seq was used to identify differentially expressed genes (DEGs). Our data revealed that 1215 up-regulated and 226 down-regulated genes at 24 h post-infection were involved in MAPK, apoptosis, RIG-1-like and toll-like receptors pathways and glutamine metabolism. Subsequently, DEGs of glutamine metabolism and apoptosis pathways were selected to validate the sequencing data by quantitative real-time PCR (qRT-PCR). The expression patterns of both transcriptomic data and qRT-PCR were consistent. We observed that lack of glutamine alone could cause mild cellular apoptosis. However, lack of glutamine together with SHVV infection could synergistically enhance cellular apoptosis. When the cells were cultured in complete medium with glutamine, overexpression of glutaminase (GLS), an essential enzyme for glutamine metabolism, could significantly enhance the SHVV replication. While, SHVV replication was decreased in cells when GLS was knocked down by specific siRNA, indicating that glutamine metabolism was essential for viral replication. Furthermore, the expression level of caspase-3 and Bax was significantly decreased in SHVV infected cells with GLS overexpression. By contrast, they were significantly increased in SHVV infected cells with GLS silence by SiRNA, indicating that SHVV infection activated the Bax and caspase-3 pathways to induce apoptosis independent of glutamine. Our results reveal that SHVV replication and starvation of glutamine could synergistically promote the cellular apoptosis, which will pave a new way for developing strategies against the vial infection.

MiR-214 inhibits snakehead vesiculovirus (SHVV) replication by targeting host GS.

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.

Australian bat lyssavirus

BACKGROUND: Australian bat lyssavirus (ABLV) causes human illness that is indistinguishable from classical rabies. All Australian bats have the potential to carry and transmit ABLV, and potentially risky human exposures to bats are common. ABLV infection has resulted in three human deaths in Australia since 1996. OBJECTIVE: The aim of this article is to equip general practitioners (GPs) to assist in the prevention and management of potential ABLV exposures in Australia, including complex clinical scenarios that are not fully addressed in current national guidelines. DISCUSSION: Potential ABLV exposures are frequently encountered in general practice. GPs play a critical role in risk mitigation for groups such as veterinarians and wildlife carers, and in triggering urgent multidisciplinary responses to potential exposures. Timely notification of the public health unit following a potential exposure is crucial to ensure appropriate assessment and access to correct treatment. Complex exposure scenarios require careful consideration.

Sleep and behavior during vesicular stomatitis virus induced encephalitis in BALB/cJ and C57BL/6J mice.

Intranasal application of vesicular stomatitis virus (VSV) produces a well-characterized model of viral encephalitis in mice. Within one day post-infection (PI), VSV travels to the olfactory bulb and, over the course of 7 days, it infects regions and tracts extending into the brainstem followed by clearance and recovery in most mice by PI day 14 (PI 14). Infectious diseases are commonly accompanied by excessive sleepiness; thus, sleep is considered a component of the acute phase response to infection. In this project, we studied the relationship between sleep and VSV infection using C57BL/6 (B6) and BALB/c mice. Mice were implanted with transmitters for recording EEG, activity and temperature by telemetry. After uninterrupted baseline recordings were collected for 2 days, each animal was infected intranasally with a single low dose of VSV (5×10(4) PFU). Sleep was recorded for 15 consecutive days and analyzed on PI 0, 1, 3, 5, 7, 10, and 14. Compared to baseline, amounts of non-rapid eye movement sleep (NREM) were increased in B6 mice during the dark period of PI 1-5, whereas rapid eye movement sleep (REM) was significantly reduced during the light periods of PI 0-14. In contrast, BALB/c mice showed significantly fewer changes in NREM and REM. These data demonstrate sleep architecture is differentially altered in these mouse strains and suggests that, in B6 mice, VSV can alter sleep before virus progresses into brain regions that control sleep.

Bioluminescence imaging of live infected salmonids reveals that the fin bases are the major portal of entry for Novirhabdovirus.

Although Novirhabdovirus viruses, like the Infectious hematopietic necrosis virus (IHNV), have been extensively studied, limited knowledge exists on the route of IHNV entry during natural infection. A recombinant IHNV (rIHNV) expressing the Renilla luciferase gene was generated and used to infect trout. A noninvasive bioluminescence assay was developed so that virus replication in live fish could be followed hours after infection. We provide here evidence that the fin bases are the portal of entry into the fish. Confirmation was brought by the use of a nonpathogenic rIHNV, which was shown to persist in fins for 3 weeks postinfection.

G, N, and P gene-based analysis of Chandipura viruses, India.

An encephalitis outbreak in 2003 in children from India was attributed to Chandipura virus. Sequence analyses of G, N, and P genes showed 95.6%-97.6% nucleotide identity with the 1965 isolate (G gene, 7-11 amino acid changes); N and P genes were highly conserved.

Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio).

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10(6) 50% tissue culture infective doses (TCID50) of SHRV/ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10(5) TCID50 of SHRV/ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host.

There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-G(I)); (ii) two copies of the G gene, one from each serotype (VSIV-G(NJ)G(I)); or (iii) a single copy of the G(NJ) gene instead of the G(I) gene (VSIV-G(NJ)). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-G(I)), VSNJV (VSIV-G(NJ)), or both (VSIV-G(NJ)G(I)), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-G(NJ) and VSIV-G(NJ)G(I) were attenuated. However, in swine, a natural host for VSV, the G(NJ) glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-G(I). These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.

Effect of mosquito salivary gland treatment on vesicular stomatitis New Jersey virus replication and interferon alpha/beta expression in vitro.

The sensitivity of vesicular stomatitis (VS) viruses to interferon (IFN)-mediated antiviral effects has been well documented. Previous studies in our laboratory have shown the ability of mosquito saliva to enhance vesicular stomatitis New Jersey (VSNJ) virus infection in mice. To investigate the effect of mosquito saliva on virus replication and IFN alpha/beta expression, virus titers were analyzed at various time points after infection in cells that were treated with mosquito salivary gland homogenate (SGH). Salivary gland treatment of mouse fibroblast cells (L929) resulted in a significant increase in virus growth kinetics compared with untreated controls. In contrast, Vero cells, which are deficient in the IFN alpha/beta response, did not yield increased viral titers in the time points examined. Treatment of L929 cells with an IFN alpha/beta neutralizing antibody also slightly increased virus yield. Ribonuclease protection assays revealed that induction of IFN alpha2 expression was reduced in L929 cells treated with SGH. Modulation of IFN alpha/beta by mosquito saliva may be a critical determinant of the transmission and pathogenesis of VSNJ virus.

Propagation of viruses on micropatterned host cells.

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.

Effects of promyelocytic leukemia protein on virus-host balance.

The cellular promyelocytic leukemia protein (PML) associates with the proteins of several viruses and in some cases reduces viral propagation in cell culture. To examine the role of PML in vivo, we compared immune responses and virus loads of PML-deficient and control mice infected with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV). PML(-/-) mice exhibited accelerated primary footpad swelling reactions to very-low-dose LCMV, higher swelling peaks upon high-dose inoculation, and higher viral loads in the early phase of systemic LCMV infection. T-cell-mediated hepatitis and consequent mortality upon infection with a hepatotropic LCMV strain required 10- to 100-times-lower inocula despite normal cytotoxic T-lymphocyte reactivity in PML(-/-) mice. Furthermore, PML deficiency rendered mice 10 times more susceptible to lethal immunopathology upon intracerebral LCMV inoculation. Accordingly, 10-times-lower VSV inocula elicited specific neutralizing-antibody responses, a replication-based effect not observed with inactivated virus or after immunization with recombinant VSV glycoprotein. These in vivo observations corroborated our results showing more virus production in PML(-/-) fibroblasts. Thus, PML is a contributor to innate immunity, defining host susceptibility to viral infections and to immunopathology.

Potentiation of vesicular stomatitis New Jersey virus infection in mice by mosquito saliva.

Saliva of arthropod vectors can modulate vertebrate host immunological functions in many ways. To investigate if vesicular stomatitis New Jersey virus (VSNJ) infection could be potentiated by arthropod saliva, mice in three different age groups (3 days, 3 weeks, or > 8 months) were exposed to VSNJ-infected mosquitoes or were needle injected with an equivalent dose of VSNJ (titre 1.5-3 logs). Previous studies have demonstrated that VS viruses do not replicate in mice older than 3 weeks of age. Infection was monitored by examining serum for the presence of VSNJ at 2 days postinfection (PI) or for neutralizing antibody on days 7 and 14 PI. All 3-day-old mice succumbed to viral infection by mosquito transmission or delivery by injection. Ninety-four percent of the 3-week-old mice bitten by infected mosquitoes developed antibody, whereas antibody was detected in only 13% of inoculated mice. Adult mice developed neutralizing antibody (73%) when fed upon by infected mosquitoes, but only 11% developed antibody when virus was injected. Day 2 serum samples from 3-week and adult age groups were negative by virus isolation. These data indicate that mosquito mediated delivery of VSNJ exacerbates virus infection in mice older than 3 weeks.

Regulation of the BBB during viral encephalitis: roles of IL-12 and NOS.

Intranasal infection of mice by Vesicular Stomatitis Virus (VSV) often leads to breakdown of the blood-brain barrier (BBB). The role of Interleukin 12 (IL-12) and nitric oxide synthase (NOS) was examined here. Wild-type (WT), NOS-1 knockout (KO), and NOS-3 KO mice were infected with VSV and treated with either IL-12 or medium. IL-12 treatment of uninfected hosts did not result in pathology. In contrast with WT and NOS-1 KO mice, where extensive gross and ultrastructural correlation of BBB breakdown were evident following infection, in NOS-3 KO mice, integrity of the BBB was observed. Thus NOS-3 activity in astrocytes, endothelial cells, or ependymal cells may play an essential role in regulating the BBB.

Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus.

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.

Potentiation by thyroxine of interferon-gamma-induced antiviral state requires PKA and PKC activities.

Added to HeLa cells previously exposed to recombinant human interferon (IFN)-gamma for 20 h, thyroid hormone [L-thyroxine (T4)] in physiological concentrations potentiates the antiviral action of IFN-gamma by more than 100-fold in 4 h. We examined protein kinase activities for their contributions to the mechanism of this posttranslational effect of thyroid hormone. Added concurrently with thyroid hormone, the protein kinase C (PKC) inhibitor CGP-41251 (5 nM) blocked T4 potentiation of IFN-gamma action. Coincubated with CGP-41251, phorbol 12-myristate 13-acetate (PMA) reversed the effect of the inhibitor on thyroid hormone action. U-73122 (10 nM), a phospholipase C inhibitor, also blocked hormone potentiation. KT-5720 (500 nM), a protein kinase A (PKA) inhibitor, completely inhibited the T4 effect, whereas 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) restored hormone action in the presence of KT-5720. In the absence of T4, 8-BrcAMP and PMA, added together to cells in the 4-h paradigm, fully reproduced hormone potentiation of the antiviral effect of IFN-gamma. Incubated individually with IFN-gamma-treated cells, the two agonists had no potentiating action. Thyroid hormone apparently must activate both PKA and PKC in the nongenomic pathway of IFN-gamma action to enhance antiviral activity in HeLa cells.

Vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity.

Vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS) when intranasally applied. We have examined cellular inflammatory changes in the CNS following VSV infection. As early as 1 day postinfection (p.i.), astrocytes were activated in the olfactory bulb (OB). This was followed by activation of microglia, first observed in the OB at day 3 p.i. Expression of inducible nitric oxide synthase was observed in activated microglia in the OB at day 3 p.i., and increased inducible nitric oxide synthase expression coincided with decreased virus titers in tissue homogenates. Expression of major histocompatibility complex (MHC) class I molecules on astrocytes and microglial, endothelial, and ependymal cells was also rapidly induced and followed by induced expression of MHC class II molecules on astrocytes and microglial and endothelial cells. Consistent with the pattern of viral dissemination, MHC molecules were expressed temporally from the rostral-to-caudal direction. Infiltration of CD8+ cells was observed as early as 1 day p.i. in the OB. CD4+ cells were detected in the OB at day 4 p.i. Increasing T-cell infiltration coincided with decreased virus titers. In contrast, B-cell infiltration of the CNS was not detected until day 14 p.i., after the virus was cleared and mice were showing behavioral signs of recovery. Breakdown of the blood-brain barrier was detected beginning at day 6 p.i., was most severe at day 8 p.i., and was followed by full recovery. Collectively, these data show that both innate immunity (production of nitric oxide) and acquired immunity (expression of MHC molecules and T-cell infiltration) are activated following VSV infection in the CNS.

The glycoprotein of viral hemorrhagic septicemia virus (VHSV): antigenicity and role in virulence.

In order to study the antigenic structure of the G protein of VHSV, we produced several anti-G monoclonal antibodies (MAbs) and used 4 neutralizing MAbs (NMAbs) to select resistant (MAR) mutants. Each MAR mutant was confronted with the 4 NMAbs in a neutralization test, and also with our panel of MAbs in surface plasmon resonance (SPR) analysis to determine the extent of their relatedness. Determination of the sequence of the entire G gene of representative MAR mutants allowed us to map the mutations responsible for the resistant phenotypes. We identified several locations on the G protein sequence, which represent, most probably, critical positions within the binding sites of the neutralizing MAbs. In addition, the MAR mutants selected with a cross-reactive MAb exhibited a reduced pathogenicity for fish. This indicated that the regions bearing the point mutations selected with MAb C10 were probably involved in the determination of the virulent phenotype.

Pathogenesis of viral haemorrhagic septicaemia virus: cellular aspects.

Leucocyte populations from rainbow trout subjected to experimental viral haemorrhagic septicaemia virus (VHSV) infections under in vivo and in vitro conditions were analysed by flow cytometry. Quantitative analysis shows that only a low percentage of the leucocytes support viral replication. Lymphocytes, compared with monocytes granulocytes and macrophages, are the least susceptible sub-population. A decrease in the amount of the phagocytic activity was observed after the cells were infected with VHSV. Obvious modifications were observed through dot plot profiles in the composition of the cell sub-populations and in the cell morphology. In addition to a direct effect of VHSV on the macrophages, a systemic effect is produced. This could also be related to the stress induced by the experimental infection process.

Changes in sciatic nerve and spinal cord function induced by a CNS viral infection.

Electrophysiologic response characteristics of mouse sciatic nerve and spinal cord were investigated following CNS infection with the temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutant G31 KS5. Measurements were obtained before clinical symptoms of the virus-induced CNS disease appeared. Sciatic nerve peak conduction velocities were not different between virus and control inoculated groups. For all control groups, sciatic nerve response (SNR) recovery, characterized by the amplitude ratio of double pulse-evoked responses, followed a facilitation-depression time course. By 4 days after VSV inoculation, the time course of SNR recovery changed with the SNR amplitude ratios significantly depressed compared to control. Crossed spinal responses (CSRs) were measured from one sciatic nerve in response to stimulation of the contralateral sciatic nerve. For all control groups, CSR recovery, as characterized by area ratios calculated from single and double pulse evoked responses, followed a facilitation-depression time course. By 5 days after VSV inoculation, the time course of CSR recovery changed with the CSR area ratios significantly depressed compared to control. The results show that simple electrophysiologic techniques can be used to detect virus-induced changes in sciatic nerve and spinal cord previously undetectable by clinical measures.