Michael K. Lo

Date palm sap linked to Nipah virus outbreak in Bangladesh, 2008.

INTRODUCTION We investigated a cluster of patients with encephalitis in the Manikgonj and Rajbari Districts of Bangladesh in February 2008 to determine the etiology and risk factors for disease. METHODS We classified persons as confirmed Nipah cases by the presence of immunoglobulin M antibodies against Nipah virus (NiV), or by the presence of NiV RNA or by isolation of NiV from cerebrospinal fluid or throat swabs who had onset of symptoms between February 6 and March 10, 2008. We classified persons as probable cases if they reported fever with convulsions or altered mental status, who resided in the outbreak areas during that period, and who died before serum samples were collected. For the case-control study, we compared both confirmed and probable Nipah case-patients to controls, who were free from illness during the reference period. We used motion-sensor-infrared cameras to observe bats contact of date palm sap. RESULTS We identified four confirmed and six probable case-patients, nine (90%) of whom died. The median age of the cases was 10 years; eight were males. The outbreak occurred simultaneously in two communities that were 44 km apart and separated by a river. Drinking raw date palm sap 2-12 days before illness onset was the only risk factor most strongly associated with the illness (adjusted odds ratio 25, 95% confidence intervals 3.3-∞, p<0.001). Case-patients reported no history of physical contact with bats, though community members often reported seeing bats. Infrared camera photographs showed that Pteropus bats frequently visited date palm trees in those communities where sap was collected for human consumption. CONCLUSION This is the second Nipah outbreak in Bangladesh where date palm sap has been implicated as the vehicle of transmission. Fresh date palm sap should not be drunk, unless effective steps have been taken to prevent bat access to the sap during collection.

The emergence of Nipah virus, a highly pathogenic paramyxovirus.

Nipah virus first emerged in Malaysia and Singapore between 1998 and 1999, causing severe febrile encephalitis in humans with a mortality rate of close to 40%. In addition, a significant portion of those recovering from acute infection had relapse encephalitis and long-term neurological defects. Since its initial outbreak, there have been numerous outbreaks in Bangladesh and India, in which the mortality rate rose to approximately 70%. These subsequent outbreaks were distinct from the initial outbreak, both in their epidemiology and in their clinical presentations. Recent developments in diagnostics may expedite disease diagnosis and outbreak containment, while progress in understanding the molecular biology of Nipah virus could lead to novel therapeutics and vaccines for this deadly pathogen.

Characterization of Nipah Virus from Outbreaks in Bangladesh, 2008-2010

New genotyping scheme facilitates classification of virus sequences.

Nipah virus infection outbreak with nosocomial and corpse-to-human transmission, Bangladesh.

Particularly vulnerable are health care workers who do not use personal protective equipment and persons who do not wash hands after traditional burial practices.

Determination of the henipavirus phosphoprotein gene mRNA editing frequencies and detection of the C, V and W proteins of Nipah virus in virus-infected cells.

The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.

Development of a Neutralization Assay for Nipah Virus Using Pseudotype Particles

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

The C, V and W proteins of Nipah virus inhibit minigenome replication.

Nipah virus (NiV) is a recently emergent, highly pathogenic, zoonotic paramyxovirus of the genus Henipavirus. Like the phosphoprotein (P) gene of other paramyxoviruses, the P gene of NiV is predicted to encode three additional proteins, C, V and W. When the C, V and W proteins of NiV were tested for their ability to inhibit expression of the chloramphenicol acetyltransferase (CAT) reporter gene in plasmid-based, minigenome replication assays, each protein inhibited CAT expression in a dose-dependent manner. The C, V and W proteins of NiV also inhibited expression of CAT from a measles virus (MV) minigenome, but not from a human parainfluenzavirus 3 (hPIV3) minigenome. Interestingly, the C and V proteins of MV, which have previously been shown to inhibit MV minigenome replication, also inhibited NiV minigenome replication; however, they were not able to inhibit hPIV3 minigenome replication. In contrast, the C protein of hPIV3 inhibited minigenome replication of hPIV3, NiV and MV. Although there is very limited amino acid sequence similarity between the C, V and W proteins within the paramyxoviruses, the heterotypic inhibition of replication suggests that these proteins may share functional properties.

Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses ☆

Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention.

Distinct and Overlapping Roles of Nipah Virus P Gene Products in Modulating the Human Endothelial Cell Antiviral Response

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis in up to 75% of infected humans. Like other paramyxoviruses, NiV employs co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternative reading frame. There is evidence from both in vitro and in vivo studies to show that the P gene products play a role in NiV pathogenesis. We have developed a reverse genetic system to dissect the individual roles of the NiV P gene products in limiting the antiviral response in primary human microvascular lung endothelial cells, which represent important targets in human NiV infection. By characterizing growth curves and early antiviral responses against a number of recombinant NiVs with genetic modifications altering expression of the proteins encoded by the P gene, we observed that multiple elements encoded by the P gene have both distinct and overlapping roles in modulating virus replication as well as in limiting expression of antiviral mediators such as IFN-β, CXCL10, and CCL5. Our findings corroborate observations from in vivo hamster infection studies, and provide molecular insights into the attenuation and the histopathology observed in hamsters infected with C, V, and W-deficient NiVs. The results of this study also provide an opportunity to verify the results of earlier artificial plasmid expression studies in the context of authentic viral infection.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.