Divergent Rhabdovirus Discovered in a Patient with New-Onset Nodding Syndrome.

A divergent rhabdovirus was discovered in the bloodstream of a 15-year-old girl with Nodding syndrome from Mundri West County in South Sudan. Nodding syndrome is a progressive degenerative neuropathy of unknown cause affecting thousands of individuals in Sub-Saharan Africa. The index case was previously healthy until she developed head-nodding seizures four months prior to presentation. Virus discovery by VIDISCA-NGS on the patient's plasma detected multiple sequence reads belonging to a divergent rhabdovirus. The viral load was 3.85 × 103 copies/mL in the patient's plasma and undetectable in her cerebrospinal fluid. Further genome walking allowed for the characterization of full coding sequences of all the viral proteins (N, P, M, U1, U2, G, U3, and L). We tentatively named the virus "Mundri virus" (MUNV) and classified it as a novel virus species based on the high divergence from other known viruses (all proteins had less than 43% amino acid identity). Phylogenetic analysis revealed that MUNV forms a monophyletic clade with several human-infecting tibroviruses prevalent in Central Africa. A bioinformatic machine-learning algorithm predicted MUNV to be an arbovirus (bagged prediction strength (BPS) of 0.9) transmitted by midges (BPS 0.4) with an artiodactyl host reservoir (BPS 0.9). An association between MUNV infection and Nodding syndrome was evaluated in a case-control study of 72 patients with Nodding syndrome (including the index case) matched to 65 healthy households and 48 community controls. No subject, besides the index case, was positive for MUNV RNA in their plasma. A serological assay detecting MUNV anti-nucleocapsid found, respectively, in 28%, 22%, and 16% of cases, household controls and community controls to be seropositive with no significant differences between cases and either control group. This suggests that MUNV commonly infects children in South Sudan yet may not be causally associated with Nodding syndrome.

Seroreactive Profiling of Filoviruses in Chinese Bats Reveals Extensive Infection of Diverse Viruses.

Southern China is a hot spot of emerging infectious diseases, in which diverse species of bats dwell, a large group of flying mammals considered natural reservoirs for zoonotic viruses. Recently, divergent filoviruses (FiVs) have been identified in bats within this region, which pose a potential risk to public health, but the true infection situation in bats remains largely unclear. Here, 689 archived bat serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and neutralization assay to investigate the seroprevalence and cross-reactivity of four divergent FiVs and two other viruses (rabies virus and Tuhoko pararubulavirus 1) of different families within the order Mononegavirales Results showed no cross-antigenicity between FiVs and other mononegaviruses but different cross-reactivity among the FiVs themselves. The total FiV seroreactive rate was 36.3% (250/689), with infection by the indigenous Chinese FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Further viral metagenomic analysis of fruit bat tissues also identified the gene sequence of a novel FiV. These results indicate the likely prevalence of other so far unidentified FiVs within the Chinese bat population, with frugivorous Rousettus leschenaultii and Eonycteris spelaea bats and insectivorous Myotis horsfieldii and Miniopterus schreibersii bats being their major reservoirs.IMPORTANCE Bats are natural hosts of many FiVs, from which diverse FiVs were serologically or virologically detected in Africa, Europe, and East Asia. Recently, very divergent FiVs were identified in the Chinese bat population, but their antigenic relationship with other known FiVs remains unknown. Here, we conducted serological characterization and investigation of Chinese indigenous FiVs and prototypes of other viruses in bats. Results indicated that Chinese indigenous FiVs are antigenically distant to other FiVs, and infection of novel or multiple FiVs occurred in Chinese bats, with FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Additionally, besides Rousettus leschenaultii and Eonycteris spelaea bats, the insectivorous Myotis horsfieldii and M. schreibersii bats are highly preferential hosts of FiVs. Seroreactive and viral metagenomic results indicated that more as yet unknown bat-borne FiVs circulate in Southern China, and to uncover them further, investigation and timely surveillance is needed.

Evidence of Australian bat lyssavirus infection in diverse Australian bat taxa.

Historically, Australia was considered free of rabies and rabieslike viruses. Thus, the identification of Australian bat lyssavirus (ABLV) in 1996 in a debilitated bat found by a member of the public precipitated both public health consternation and a revision of lyssavirus taxonomy. Subsequent observational studies sought to elaborate the occurrence and frequency of ABLV infection in Australian bats. This paper describes the taxonomic diversity of bat species showing evidence of ABLV infection to better inform public health considerations. Blood and/or brain samples were collected from two cohorts of bats (wild-caught and diagnostic submissions) from four Australian states or territories between April 1996 and October 2002. Fresh brain impression smears were tested for ABLV antigen using fluorescein-labelled anti-rabies monoclonal globulin (CENTOCOR) in a direct fluorescent antibody test; sera were tested for the presence of neutralising antibodies using a rapid fluorescent focus inhibition test. A total of 3,217 samples from 2,633 bats were collected and screened: brain samples from 1,461 wild-caught bats and 1,086 submitted bats from at least 16 genera and seven families, and blood samples from 656 wild-caught bats and 14 submitted bats from 14 genera and seven families. Evidence of ABLV infection was found in five of the six families of bats occurring in Australia, and in three of the four Australian states/territories surveyed, supporting the historic presence of the virus in Australia. While the infection prevalence in the wild-caught cohort is evidently low, the significantly higher infection prevalence in rescued bats in urban settings represents a clear and present public health significance because of the higher risk of human exposure.

Antibodies against Duvenhage virus in insectivorous bats in Swaziland.

Several rabies-related lyssaviruses have been associated with bat species in southern Africa, the rarest of these being Duvenhage virus (DUVV), for which only five isolations have been made over five decades. Three of these were from human fatalities, and it is not known which bat species acts as reservoir. In studying a population of Nycteris thebaica in the kingdom of Swaziland, a landlocked country bordering Mozambique and South Africa, we found evidence of the circulation of a lyssavirus. Virus-neutralization assays indicated DUVV-neutralizing antibodies in 30% of the sera collected from this population of N. thebaica, providing the first indication of a Duvenhage virus circulating in this particular species and the first evidence of a bat lyssavirus circulating in Swaziland bats.

Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a.

The serotine bat (Eptesicus serotinus) accounts for the vast majority of bat rabies cases in Europe and is considered the main reservoir for European bat lyssavirus type 1 (EBLV-1, genotype 5). However, so far the disease has not been investigated in its native host under experimental conditions. To assess viral virulence, dissemination and probable means of transmission, captive bats were infected experimentally with an EBLV-1a virus isolated from a naturally infected conspecific from Germany. Twenty-nine wild caught bats were divided into five groups and inoculated by intracranial (i.c.), intramuscular (i.m.) or subcutaneous (s.c.) injection or by intranasal (i.n.) inoculation to mimic the various potential routes of infection. One group of bats was maintained as uninfected controls. Mortality was highest in the i.c.-infected animals, followed by the s.c. and i.m. groups. Incubation periods varied from 7 to 26 days depending on the route of infection. Rabies did not develop in the i.n. group or in the negative-control group. None of the infected bats seroconverted. Viral antigen was detected in more than 50% of the taste buds of an i.c.-infected animal. Shedding of viable virus was measured by virus isolation in cell culture for one bat from the s.c. group at 13 and 14 days post-inoculation, i.e. 7 days before death. In conclusion, it is postulated that s.c. inoculation, in nature caused by bites, may be an efficient way of transmitting EBLV-1 among free-living serotine bats.

Up-regulation of chemokine gene transcripts and T-cell infiltration into the central nervous system and dorsal root ganglia are characteristics of experimental European bat lyssavirus type 2 infection of mice.

European bat lyssaviruses (EBLV) types 1 and 2 are closely related to classical rabies virus (RABV), and are capable of causing rabies in terrestrial mammals, including humans. The authors have investigated the murine host innate immune response in the brain, salivary gland, spinal cord, and blood, following peripheral inoculation with EBLV-2. In the brain, increases in Toll-like receptor-3 (TLR-3) transcript preceded overt disease, with a range of inflammatory gene transcripts increasing during the clinical stage of infection. This included transcripts for interleukin-6 (IL-6), interferon-gamma (IFN-gamma), and CXC chemokine ligand 10 (CXCL10). In the salivary gland, there was a small but significant increase of CXCL10 gene transcript and a limited increase in 2'-5' oligoadenylate synthetase (2'-5' OAS1) transcript. In the blood, there was an increase in levels of IFN-gamma and virus-neutralizing antibodies (VNAs) were detected prior to the appearance of clinical signs. These changes were associated with severe lymphocyte infiltration observed within the spinal cord and dorsal root ganglia (DRG), which was dominated by T lymphocytes and associated with widespread inflammatory changes. The authors speculate that the increase of inflammatory cytokines and chemokines in response to EBLV-2 infection leads to a dramatic increase in T-cell infiltration and provides evidence for a robust immune response to lyssavirus infection that may not commonly occur in RABV infection.

Cytokine production by human leukocytes with different expressions of natural antiviral immunity and the effect of antibodies against interferons and TNF-alpha.

INTRODUCTION: Two activities of innate antiviral immunity were studied: the resistance of human peripheral blood mononuclear cells (PMBCs) ex vivo to viral infection and the production of cytokines. MATERIALS AND METHODS: Samples of blood were taken from healthy blood donors and from persons with frequent infections of the upper respiratory system. PMBCs were isolated by gradient centrifugation. Vesicular stomatitis virus (VSV) was used as the indicatory virus to infect PMBCs. The cytokines: IFN, TNF, and IL-6 were titrated by biological methods and IL-10 by ELISA. RESULTS: Blood donors were divided for two groups: those with VSV-resistant and those with VSV-sensitive PMBCs and secretion of cytokines by them was compared. The resistant PMBCs produced more cytokines than the sensitive ones. A statistically significant difference, was found only in the case of the IFNs. To examine the contribution of IFNs and TNF in maintaining resistance, leukocytes from both groups were treated with specific anti-cytokine antibodies. The authors' previous study showed that the elimination of spontaneous IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha from resistant leukocytes resulted in increased VSV replication This indicates the important role of cytokines. In VSV-sensitive PMBCs, anti-IFN-alpha showed the opposite effect (decreased virus replication). In the absence of spontaneous IFN-alpha, disturbances in cytokine production were observed. CONCLUSIONS: Complete resistance of PMBC to VSV infection is accompanied by higher cytokine release, The paradoxical effect of anti-IFN-alpha on virus replication in leukocytes sensitive to viral infection may be attributed to changes in the cytokine profile balance, i.e. high TNF production by VSV-infected leukocytes and a complete reduction of IL-6 production.

Pre-exposure rabies booster vaccinations: a literature review.

In Europe, more attention is turning towards human infection with European bat lyssaviruses (EBLVs). Following the death of a bat conservationist from EBLV in Scotland, in 2002, the Department of Health in the United Kingdom (UK) recommended that all bat workers receive prophylactic rabies vaccination. This systematic literature review aims to review the evidence base for current UK policy on rabies booster vaccination. Ten papers met the inclusion criteria and were reviewed. Most of the papers were prospective cohort studies with follow-up ending after the first booster vaccination. One year after a three dose intramuscular primary rabies vaccination course, 87.9-100 % of participants had a rabies antibody level > or = 0.5 IU/ml, before the first booster. It may, therefore, be prudent for the UK to reduce its current recommended interval, primary course to first booster, from two years to one year. More research, with longer follow-up, is required to enable recommendations on subsequent boosters to be made.

EBLV-2 prevalence in the United Kingdom as determined by surveillance testing.

Five cases of EBLV-2 have been detected in the UK since 1996, with all wildlife cases in the Daubenton's bat: one on the south coast in Sussex in 1996, one in Lancashire in 2002, another in 2003, one in Surrey in 2004 and a human fatality in Angus, Scotland, in 2002. As a result of the human case, a seroprevalence study, aimed primarily at the Daubenton's bat was conducted in 2003 in Scotland and at some sites in England. In Scotland, 198 Daubenton's, 20 Natterer's and 6 pipistrelles were caught at 19 sites and analysed, while in England 67 Daubenton, 2 Brandts/ Whiskered and 4 pipistrelle bats were analysed from four sites in Lancashire. Analysis of blood was performed by a modified fluorescent antibody virus neutralisation test (mFAVN) to determine antibody titre to EBLV-2. Ignoring those sites where we had a priori reason to expect infected bats, the overall seroprevalence was between 0.7-5.1 % (95 % confidence interval), with a maximum likelihood estimate of 2.2 %. Mouth swabs were taken and tested for virus genome by RT-PCR and live virus by tissue culture isolation. All of the PCR and isolation results were negative suggesting that none of the bats sampled were actively excreting virus. This suggests a low level of active infection in Britain and raises the possibility that bats may recover following exposure to EBLV-2.

Transmission of vesicular stomatitis New Jersey virus to cattle by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae).

Laboratory-reared Culicoides sonorensis Wirth & Jones were infected with vesicular stomatitis virus serotype New Jersey (family Rhabdoviridae, genus Vesiculovirus, VSNJV) through intrathoracic inoculation. After 10-d incubation at 25 degrees C, these insects were allowed to blood feed on four steers. Two other steers were exposed to VSNJV through intralingual inoculation with 10(8) tissue culture infective dose50 VSNJV. All six steers became seropositive for VSNJV. The results demonstrate the ability of C. sonorensis to transmit VSNJV to livestock. Only the animals intralingually inoculated with VSNJV showed clinical signs in the form of vesicles at the site of inoculation. Uninfected C. sonorensis allowed to feed on the exposed animals did not become infected with VSNJV. Animals infected by C. sonorensis showed a slower antibody response compared with intralingually inoculated animals. This is probably because of different amounts of virus received via insect transmission and syringe inoculation. A significant difference was found in the serum acute-phase protein alpha-1-acid glycoprotein in animals that received VSNJV through C. sonorensis transmission. These animals had previously been exposed to insect attack in the field compared with intralingually inoculated animals and C. sonorensis-infected animals that had been protected from insect attack. The failure to observe clinical signs of vesicular stomatitis through transmission of VSNJV by C. sonorensis may explain widespread subclinical infections during vesicular stomatitis epidemics.

Impact of macrophage and dendritic cell subset elimination on antiviral immunity, viral clearance and production of type 1 interferon.

We report herein that vesicular stomatitis virus (VSV) induced a concurrent primary Th1 (T helper 1) and Th2 cytokine response detectable ex vivo. Liposome-encapsulated clodronate-mediated elimination of CD8- marginal dendritic cells (DCs) and splenic macrophages (m Phi), but not CD8+ interdigitating DCs, prior to infection resulted in a markedly diminished chemokine and Th1 (IL-2, interferon-gamma) cytokine response, although the Th2 response (IL-4) remained relatively intact. Repopulation with marginal DCs and marginal metallophilic macrophages (MMM) restored Th1 cytokine profiles but did not restore chemokine responsiveness or reduce VSV-induced morbidity/mortality. Chemokine competency returned approximately 4 weeks post-depletion, which correlated temporally with repopulation of the spleen with marginal zone macrophages (MZM) and red pulp macrophages (RPM). Unexpectedly, virus-induced morbidity persisted for over 1 month post-depletion and was associated with virus dissemination and distinctive histological lesions in the liver. Depletion of interferon-producing plasmacytoid dendritic cells did not account for virus-induced morbidity because serum levels of type I interferon were not diminished in Cl2MBP-liposome-treated mice. Thus, distinct m Phi subsets are critical for chemokine production and viral clearance, and, in their absence, VSV disseminates even in the presence of high titers of interferon.

Cytokine levels in Chandipura virus associated encephalopathy in children.

An association of Chandipura (CHP) virus with an explosive outbreak of encephalitis in children from India affecting 349 children with 55% mortality was recently reported. To understand the role of cytokines in the pathogenesis of CHP infection, 14 paediatric encephalitis cases admitted to a tertiary care hospital and 5 age-matched apparently healthy control children were studied. At the time of sampling, post onset of disease was < or =2 d (Group A, n = 4) and >2 d (Group B, n = 10). Concentrations of IL-2, IFN-gamma, TNF-alpha and IL-6 in mitogen stimulated PBMC supernatants of patients and controls were assessed by ELISA. IL-2 levels in Group A and B were significantly higher compared with controls (28.4+/-21.9 vs <7.8, p=0.01, 269.4+/-311.0.vs <7.8, p = 0.01). IFN-gamma levels were significantly elevated in both the groups compared with controls (394.4+/-107.7 vs 13.9+/-20.9, p = 0.01, 339.5+/-244.9 vs 13.9+/-20.9, p = 0.01). TNF-alpha and IL-6 levels were significantly higher in Group B compared with controls (573.1+/-472.5 vs 113.4.+/-148.3, p = 0.01, 486.2+/-145.7 vs 113.8+/-82.4, p = 0.003). Cytokine levels were not significantly different in Groups A and B.

CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection.

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell-independent encephalitis, and infection resulted in a dramatic early up-regulation of chemokine gene expression. Similar marked up-regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV-infected mice, only LCMV-induced T cell-dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus-activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+-dependent CNS inflammation.

A survey of expressed genes in the leukocytes of Japanese flounder, Paralichthys olivaceus, infected with Hirame rhabdovirus.

We constructed a cDNA library of Japanese flounder, Paralichthys olivaceus, leukocytes that were infected with Hirame rhabdovirus (HRV) in order to analyze some of the genes that are induced and expressed by virus infection in the immune system. Four hundred and fifty-two partial sequences representing 300 cDNA clones were obtained from the 5' and/or 3' ends of inserts derived from the Japanese flounder leukocyte cDNA library. About three-quarters of the 300 cDNA clones (217 clones, 72.3%) represented known genes in the public databases, whereas the remaining 83 (27.7%) of the clones did not show any significant homology with the sequences in the public databases. Clones matching known genes were classified into 12 categories according to their function or distribution. Only 40 (18.4%) of the 217 known genes showed homology with fish genes deposited in the database. Thirty (10%) of the clones, encoding 21 different sequences, and representing several categories, were identified as putative biodefense genes or genes associated with the immune response. Nineteen of the 21 putative biodefense or immune response-related cDNAs have not been previously reported in fish genes or cDNAs.

Differential diagnosis of fish pathogenic rhabdoviruses by reverse transcriptase-dependent polymerase chain reaction.

Reverse transcriptase-dependent polymerase chain reaction (RT-PCR) was applied to the detection and differentiation of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) using primer pairs designed for the amplification of glycoprotein G-specific gene fragments of the two viruses. The products of 443 bp (VHS) and 548 bp (IHN), respectively, were amplified from the total RNA extracts of RTG-2 cells infected with a total of 9 different strains of either VHS virus or IHN virus. Restriction analysis using FokI, and DNA sequencing of the PCR products demonstrated specificity of the amplification. The RT-PCR amplification of VHSV or IHNV G-genes was found to be a simple, highly specific and sensitive method allowing differential diagnosis of VHS and IHN within 8 h.

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.