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.

Risk Factors for Parainfluenza Virus Lower Respiratory Tract Disease after Hematopoietic Cell Transplantation.

Parainfluenza virus (PIV) infection can progress from upper respiratory tract infection (URTI) to lower respiratory tract disease (LRTD) in immunocompromised hosts. Risk factors for progression to LRTD and presentation with LRTD without prior URTI are poorly defined. Hematopoietic cell transplant (HCT) recipients with PIV infection were retrospectively analyzed using standardized definitions of LRTD. PIV was detected in 540 HCT recipients; 343 had URTI alone and 197 (36%) had LRTD (possible, 76; probable, 19; proven, 102). Among 476 patients with positive nasopharyngeal samples, the cumulative incidence of progression to probable/proven LRTD by day 40 was 12%, with a median time to progression of 7 days (range, 2 to 40). In multivariable analysis monocytopenia (hazard ratio, 2.22; P = .011), steroid use ≥1mg/kg prior to diagnosis (hazard ratio, 1.89; P = .018), co-pathogen detection in blood (hazard ratio, 3.21; P = .027), and PIV type 3 (hazard ratio, 3.57; P = .032) were associated with increased progression risk. In the absence of all 4 risk factors no patients progressed to LRTD, whereas progression risk increased to >30% if 3 or more risk factors were present. Viral load or ribavirin use appeared to have no effect on progression. Among 121 patients with probable/proven LRTD, 64 (53%) presented LRTD without prior URTI, and decreased lung function before infection and lower respiratory co-pathogens were risk factors for this presentation. Mortality was unaffected by the absence of prior URTI. We conclude that the risk of progression to probable/proven LRTD exceeded 30% with ≥3 risk factors. To detect all cases of LRTD, virologic testing of lower respiratory samples is required regardless of URTI symptoms.

Low Retinol-Binding Protein and Vitamin D Levels Are Associated with Severe Outcomes in Children Hospitalized with Lower Respiratory Tract Infection and Respiratory Syncytial Virus or Human Metapneumovirus Detection.

Retinol binding protein and vitamin D were measured in children aged <5 years hospitalized with lower respiratory tract infection and respiratory syncytial virus and/or human metapneumovirus detections. Low vitamin levels were observed in 50% of the children and were associated with significantly elevated risk of the need for intensive care unit admission and invasive mechanical ventilation.

DAS181 for Treatment of Parainfluenza Virus Infections in Hematopoietic Stem Cell Transplant Recipients at a Single Center.

Parainfluenza virus (PIV) causes severe respiratory infections in hematopoietic stem cell transplant (HSCT) recipients. Currently, no effective therapies are available. DAS181 is a novel antiviral agent that inhibits attachment of PIV to respiratory cells, but clinical data on the use of DAS181 for PIV infection are limited to case reports. We report the clinical manifestations and outcomes of 16 HSCT recipients who received DAS181 daily for the treatment of PIV infection through a compassionate-use protocol or a single-arm clinical trial. Of the 16 patients (clinical trial: 9; compassionate use: 7), 13 were allogeneic HSCT recipients and 8 had graft-versus-host disease. PIV types were 3 (n = 7), 4 (n = 5), 1 (n = 3), and type 3 and 4 coinfection (n = 1). Fourteen patients had pneumonia. All patients presented with cough, 14 had dyspnea, 11 had hypoxia, and 8 had a fever. Patients received 5 to 10 days of treatment. Nine patients (56%) had a complete clinical response after DAS181 therapy and 4 (25%) had a partial response. The 3 patients without a clinical response had coinfections with other pathogens. Of the 7 patients with virologic and spirometric data, 5 had >1-log reduction in nasopharyngeal swab PIV viral load and 4 had improved forced expiratory volumes by the end of treatment. Three patients (19%) died within 30 days and 2 of these deaths were related to PIV infection. Our data suggest that DAS181 may be an effective therapy for PIV pneumonia in HSCT recipients. Randomized placebo-controlled trials are needed to better evaluate its efficacy.

Development of A recombinant nucleocapsid based indirect ELISA for the detection of antibodies to avian metapneumovirus subtypes, A, B, and C.

Nucleocapsid (N) protein is the most highly expressed of all avian metapneumovirus (aMPV) viral proteins and stimulates a substantial immune response in infected animals. Codon optimized recombinant N (rec-N) protein from aMPV subtypes A, B, and C were expressed using the baculoviral expression system in Trichoplusia ni (Tni) insect cells. A mixture of purified rec-N antigens from each subtype was used as a coating antigen and was evaluated in indirect ELISA (iELISA) to assess antibody response in serum samples collected from experimentally infected chickens and turkeys with different aMPV subtypes. Also, archived field serum samples that were collected from different poultry submissions were used. Receiver operating characteristic (ROC) analysis was performed using chicken and turkey serum samples that were confirmed by indirect fluorescent antibody (IFA) test for serostatus (positive n = 270, negative n = 610). The ROC analysis showed sensitivity and specificity of 97 % at a cut-off value of 0.25. The rec-N iELISA was compared with a commercial whole virus-based APV kit. The rec-N iELISA showed comparable results in detecting antibody response in aMPV infected chicken sera but was more sensitive in detecting early antibody response in aMPV infected turkey serum samples. Our results further confirm the presence of aMPV antibodies in Canadian domestic poultry populations. The developed aMPV-rec N iELISA offers a safe and valuable alternative to whole virus-based iELISA for serodiagnosis and seroepidemiological surveillance of the disease in domestic poultry.

Generation of recombinant nucleocapsid protein of human metapneumovirus in baculovirus for detecting antibodies in the Beijing population.

Human metapneumovirus (hMPV) is associated with respiratory infections in both adults and children. Recombinant nucleocapsid (N) proteins of hMPV from two major groups of hMPV in Beijing with a 6x His-tag at the C terminus were constructed in a baculovirus and expressed in transfected Sf21 insect cells. The antigenicity and specificity of the expressed recombinant proteins were examined by immunofluorescence assay and western blotting. Preliminary use of the expressed proteins for antibody detection in 187 serum specimens collected from different age groups in Beijing, China, indicated that the purified recombinant N-His proteins were of good antigenicity and specificity and could be a potential antigen for further seroprevalence study of hMPV, especially in the Chinese population. The positive rate for antibody detection suggested that hMPV from two clusters was co-circulating in Beijing and that most of the people in Beijing have been exposed to the virus by age 60.

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens.

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.

Field estimation of the flock-level diagnostic specificity of an enzyme-linked immunosorbent assay for Avian metapneumovirus antibodies in turkeys.

Routine serologic testing for Avian metapneumovirus (AMPV) infection of turkey flocks at slaughter is currently being used to monitor changes in the occurrence of AMPV infection in endemic areas and can also be used to detect the emergence of infection in currently unaffected areas. Because of the costs associated with false-positive results, particularly in areas that are free of AMPV infection, there is a need to obtain improved estimates of flock-level specificity (SP). The objective of this study was to estimate flock-level SP of a program to monitor AMPV infection in turkey flocks at processing using a standard enzyme-linked immunosorbent assay (ELISA). A study was carried out in which 37 AMPV-free flocks from 7 Midwest operations were followed serologically. Six percent, 3%, and 0.2% of total samples tested AMPV positive at 8 weeks, 12 weeks, and at processing, respectively. Overall, flock-level SP increased as the cutoff increased and as age increased. Flock-level SP at processing was 97%, if a cutoff of 1 was used (the flock was classified as positive if at least 1 sample tested positive), and 100%, if any other cutoff was used. Administration of antibiotics (P = 0.02) and vaccination for Bordetella avium (P = 0.08) were positively associated with the probability of (false) positive test results. These findings suggest possible cross-reactions with other infections and highlight the need to consider variable diagnostic performance depending on farm conditions.

Micronutrient concentrations in respiratory syncytial virus and human metapneumovirus in Yemeni children.

BACKGROUND: Acute respiratory infections (ARI) cause significant childhood mortality. Nutritional homeostasis, particularly micronutrient levels, is important in modulating response to infection. More information is required regarding micronutrient levels in ARI viral infections, especially newly identified viruses such as human metapneumovirus (HMPV). AIM: To describe zinc, copper, selenium and vitamins A and E concentrations in children with respiratory syncytial virus (RSV) and/or HMPV in relation to levels of C-reactive protein (CRP). METHODS: The presence of RSV/HMPV in nasopharyngeal aspirates (NPA) was identified in 246 children using RTPCR. Zinc, copper, selenium and vitamins A and E concentrations were measured using inductive coupled plasma mass spectrometry and high performance liquid chromatography. RESULTS: 183 children had RSV, 39 had HMPV and 24 were co-infected. Zinc concentrations were lower in children with HMPV than in children with RSV or RSV/HMPV co-infection. Copper concentrations were lower in children with RSV than in children with RSV/HMPV or HMPV and zinc/copper ratios were lower in children with HMPV/RSV or RSV than in children with HMPV alone. Retinol and a alpha-tocopherol were lower in children with RSV than in children with HMPV. Most children had low selenium concentrations. Children with RSV and raised CRP (>5 mg/L) had higher copper and lower zinc/copper ratios than those with low CRP (< or =5 mg/L). Children with HMPV and raised CRP had higher copper and lower zinc concentrations than children with low CRP. Children with RSV/HMPV and raised CRP had higher copper concentrations. Children with RSV/HMPV and raised CRP had higher a alpha-tocopherol concentrations. CONCLUSION: The profiles of micronutrients differ in children with RSV and HMPV and are confounded by CRP. These results may guide strategies for micronutrient supplementation in ARI.

Exposure to novel parainfluenza virus and clinical relevance in 2 bottlenose dolphin (Tursiops truncatus) populations.

Parainfluenza virus (PIV) is a leading cause of respiratory infections in humans. A novel virus closely related to human and bovine parainfluenza viruses types 3 (HPIV-3 and BPIV-3), named Tursiops truncatus parainfluenza virus type 1 (TtPIV-1), was isolated from a dolphin with respiratory disease. We developed a dolphin-specific ELISA to measure acute- and convalescent-phase PIV antibodies in dolphins during 1999-2006 with hemograms similar to that of the positive control. PIV seroconversion occurred concurrently with an abnormal hemogram in 22 animals, of which 7 (31.8%) had respiratory signs. Seroprevalence surveys were conducted on 114 healthy bottlenose dolphins in Florida and California. When the most conservative interpretation of positive was used, 11.4% of healthy dolphins were antibody positive, 29.8% were negative, and 58.8% were inconclusive. PIV appears to be a common marine mammal virus that may be of human health interest because of the similarity of TtPIV-1 to BPIV-3 and HPIV-3.

Animal infection studies of two recently discovered African bat paramyxoviruses, Achimota 1 and Achimota 2.

Bats are implicated as the natural reservoirs for several highly pathogenic viruses that can infect other animal species, including man. Here, we investigate the potential for two recently discovered bat rubulaviruses, Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2), isolated from urine collected under urban bat (Eidolon helvum) roosts in Ghana, West Africa, to infect small laboratory animals. AchPV1 and AchPV2 are classified in the family Paramyxoviridae and cluster with other bat derived zoonotic rubulaviruses (i.e. Sosuga, Menangle and Tioman viruses). To assess the susceptibility of AchPV1 and AchPV2 in animals, infection studies were conducted in ferrets, guinea pigs and mice. Seroconversion, immunohistological evidence of infection, and viral shedding were identified in ferrets and guinea pigs, but not in mice. Infection was associated with respiratory disease in ferrets. Viral genome was detected in a range of tissues from ferrets and guinea pigs, however virus isolation was only achieved from ferret tissues. The results from this study indicate Achimota viruses (AchPVs) are able to cross the species barrier. Consequently, vigilance for infection with and disease caused by these viruses in people and domesticated animals is warranted in sub-Saharan Africa and the Arabian Peninsula where the reservoir hosts are present.

Human Metapneumovirus Infection: Pneumonia Risk Factors in Patients With Solid Organ Transplantation and Computed Tomography Findings.

BACKGROUND: Human metapneumovirus (HMPV) is a newly detected pathogen that can cause lower respiratory tract disease. Clinical characteristics, computed tomography (CT) findings, and outcomes of HMPV pneumonia in patients with solid organ transplantation (SOT) have not been well demonstrated. METHODS: Between January 2010 and February 2016, clinical and imaging findings of 59 patients with SOT (types of organ: kidney, 37; liver, 16; heart, 4; and pancreas and kidney, 2) who had HMPV infection detected in nasopharyngeal or bronchoalveolar lavage by reverse transcription polymerase chain reaction were retrospectively evaluated. RESULTS: Most (90%) of the patients were detected between March and June. In the 59 patients with SOT with upper respiratory tract infection (URI), 29 (49%) progressed to lower respiratory tract disease after a median of 7 days (range, 2-31 days). Coinfection was noted in 39% of the patients. In Cox proportional hazards analysis, low lymphocyte count (≤0.7 × 10/µL; hazard ratio, 2.24; 95% confidence interval, 1.04-4.85; P = 0.04) and high C-reactive protein (>10 mg/dL; hazard ratio, 2.93; 95% confidence interval, 1.19-7.21; P = 0.02) at URI diagnosis were associated with HMPV pneumonia. On CT, HMPV pneumonia presented as bilateral ill-defined centrilobular nodules, consolidation and ground-glass opacities, whereas lymphadenopathy or effusion is not common. There were no significantly different imaging CT findings between patients with HMPV infection alone and those with coinfection. CONCLUSIONS: Human metapneumovirus pneumonias were detected in nearly half of patients with SOT showing URI symptoms with positive HMPV, and low lymphocyte count and high C-reactive protein at URI diagnosis were significant factors associated with HMPV pneumonia.

Induction of local and systemic immune reactions following infection of turkeys with avian Metapneumovirus (aMPV) subtypes A and B.

Most of the studies regarding the immunopathogenesis of avian Metapneumovirus (aMPV) have been done with subtype C of aMPV. Not much is known about the immunopathogenesis of aMPV subtypes A and B in turkeys. Specifically, local immune reactions have not been investigated yet. We conducted two experiments in commercial turkeys. We investigated local and systemic humoral and cell mediated immune reactions following infection with an attenuated vaccine strain of aMPV subtype B (Experiment I) and virulent strains of aMPV subtypes A and B (Experiment II). Turkeys infected with virulent aMPV strains developed mild respiratory signs while birds inoculated with the attenuated aMPV did not show any clinical signs. Virus neutralizing antibodies were detected locally in tracheal washes and systemically in serum as soon as 5-7 days post aMPV infection (PI) independent of the strain used. Virus neutralizing antibody titres peaked at 7 days PI and then antibody levels declined. The peak of serum ELISA antibody production varied between infected groups and ranged from 14 and 28 days PI. All aMPV strains induced an increase in the percentage of CD4+ T cell populations in spleen and Harderian gland at days 7 or 14 PI. Furthermore, as shown in Experiment I, infection with the attenuated aMPV-B strain stimulated spleen leukocytes to release significantly higher levels of interferons (IFNs), interleukin-6 and nitric oxide in ex vivo culture in comparison to virus-free controls up to 7 days PI (P<0.05). As detected by quantitative real time RT-PCR in Experiment II, infection with virulent aMPV induced an increased IFNgamma expression in the Harderian gland in comparison to virus-free controls. IFNgamma expression in the spleen varied between aMPV strains and days PI. Overall, our study demonstrates that aMPV subtypes A and B infection induced humoral and cell mediated immune reactions comparable to subtype C infections. We observed only temporary stimulation of serum virus neutralizing antibodies and of most of the local immune reactions independent of the aMPV strain used. The temporary character of immune reactions may explain the short duration of protection against challenge following aMPV vaccination in the field.

[Human metapneumovirus as a common cause of respiratory tract disease].

Human metapneumovirus (HMPV), the newly identified paramyxovirus, causes respiratory infections in children, immunosuppressed patients, and the elderly in different countries of the world. The epidemiology and clinical manifestations of HMPV infection are similar to those in human respiratory syncytial virus infection. The diagnosis of HMPV infection is based on the polymerase chain reaction detection of viral RNA or the recording of rising serum antibody titers. There are at least two genotypes and several subtypes of HMPV in the human population. The use of cell cultures and laboratory animals have provided new evidence for the pathogenesis of HMPV infection, the specific features of antiviral immunity and enabled recombinant HMPV vaccine candidates to be designed.

Cytokine Profiles in Human Metapneumovirus Infected Children: Identification of Genes Involved in the Antiviral Response and Pathogenesis.

Human metapneumovirus (hMPV) causes severe airway infection in children that may be caused by an unfavorable immune response. The nature of the innate immune response to hMPV in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. We examined nasopharynx aspirates and blood samples from hMPV-infected children without detectable co-infections. The expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mRNA quantification while blood plasma proteins were determined by a multiplex immunoassay. Several genes were significantly up-regulated at mRNA and protein level in the hMPV infected children. Most apparent was the expression of the chemokine IP-10, the pro-inflammatory cytokine IL-18 in addition to the interferon inducible gene ISG54. Interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes IL-1ß and NLRP3. Overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hMPV mediated lung disease and the antiviral response in children with severe infection. Our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hMPV-infected children.

Isolation of Nipah virus from Malaysian Island flying-foxes.

In late 1998, Nipah virus emerged in peninsular Malaysia and caused fatal disease in domestic pigs and humans and substantial economic loss to the local pig industry. Surveillance of wildlife species during the outbreak showed neutralizing antibodies to Nipah virus mainly in Island flying-foxes (Pteropus hypomelanus) and Malayan flying-foxes (Pteropus vampyrus) but no virus reactive with anti-Nipah virus antibodies was isolated. We adopted a novel approach of collecting urine from these Island flying-foxes and swabs of their partially eaten fruits. Three viral isolates (two from urine and one from a partially eaten fruit swab) that caused Nipah virus-like syncytial cytopathic effect in Vero cells and stained strongly with Nipah- and Hendra-specific antibodies were isolated. Molecular sequencing and analysis of the 11,200-nucleotide fragment representing the beginning of the nucleocapsid gene to the end of the glycoprotein gene of one isolate confirmed the isolate to be Nipah virus with a sequence deviation of five to six nucleotides from Nipah virus isolated from humans. The isolation of Nipah virus from the Island flying-fox corroborates the serological evidence that it is one of the natural hosts of the virus.

Intravenous ribavirin by constant infusion for serious influenza and parainfluenzavirus infection.

Three patients with severe lower respiratory tract influenza or parainfluenzavirus infections were treated with continuous ribavirin infusion, given as a 5 mg/kg/hour (h) loading infusion for 8 h followed by 1.5 mg/kg/h for 2 to 6 days. This regimen was generally well tolerated. Plasma ribavirin concentrations were 40 to 60 microM in two patients during the continuous infusion phase and lower concentrations were detectable in tracheobronchial secretions. In temporal association with ribavirin administration, viral shedding diminished in one patient and ceased in two patients, one of whom had developed virus resistant to amantadine. The strategy of continuous ribavirin infusion warrants controlled testing for its antiviral and possible clinical effectiveness.

A novel covalent enzyme-linked immunoassay (CELIA) for simultaneously measuring free and immune complex bound antibodies with a defined specificity. II. Application to immune complexes containing viral antigens in human sera.

The coupling of viral antigens from parainfluenza virus (PIV-1), cytomegalovirus (CMV) and human immunodeficiency virus (HIV-1) to chemically functionalized polystyrene plates has permitted us to develop a covalent enzyme-linked immunoassay (CELIA) for measuring the titers of free antibody (Ab) and immune complex (IC) bound Ab directed against each of these viruses. The method was first validated for experimentally produced IC (PIV-anti-PIV) and then applied to the analysis of IC in human sera. In the case of a renal transplant patient with CMV viremia whose free Ab titers were less than 100, the method unambiguously permitted the IC bound anti-CMV titers to be determined. In the case of a survey for HIV-1 Ab, it also allowed us to identify a sub-group of seropositives with IC anti-HIV. In view of the ease and rapidity with which CELIA can be performed, this technology should enable determinations of IC bound Ab of defined specificity to be undertaken routinely in seroepidemiological surveys.

Diagnosis of nipah virus encephalitis by electron microscopy of cerebrospinal fluid.

BACKGROUND: between 1998 and 1999, an outbreak of potentially fatal viral encephalitis erupted among pig farm workers in West Malaysia, and later spread to Singapore where abattoir workers were afflicted. Although Japanese encephalitis virus was initially suspected, the predominant aetiologic agent was subsequently confirmed to be Nipah virus, a novel paramyxovirus related to but distinct from Hendra virus. OBJECTIVE: to describe a case of Nipah virus encephalitis in a pig farm worker from Malaysia. STUDY DESIGN: the clinical, laboratory and radiological findings of this patient were scrutinized. Special emphasis was placed on the electron microscopic analysis of the cerebrospinal fluid (CSF) specimen from this patient. RESULTS: the neurological deficits indicative of cerebellar involvement were supported by the magnetic resonance imaging that showed prominent cerebellar and brainstem lesions. CSF examination provided further evidence of viral encephalitis. Complement fixation and/or RT-PCR assays were negative for Japanese encephalitis, herpes simplex, measles and mumps viruses. ELISA for detecting IgM and IgG antibodies against Hendra viral antigens were equivocal for the CSF specimen, and tested initially negative for the first serum sample but subsequently positive for the repeat serum sample. Transmission electron microscopy of negatively-stained preparations of CSF revealed enveloped virus-like structures fringed with surface projections as well as nucleocapsids with distinctive helical and herringbone patterns, features consistent with those of other paramyxoviruses, including Hendra virus. CONCLUSION: this case report reiterates the relevant and feasible role of diagnostic electron microscopy for identifying and/or classifying novel or emerging viral pathogens for which sufficiently specific and sensitive tests are lacking.