Deborah Middleton

Hendra and Nipah viruses: different and dangerous.

Hendra virus and Nipah virus are highly pathogenic paramyxoviruses that have recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These features led to their classification into the new genus Henipavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. This review provides an overview of henipaviruses and the types of infection they cause, and describes how studies on the structure and function of henipavirus proteins expressed from cloned genes have provided insights into the unique biological properties of these emerging human pathogens.

Comparative pathology of the diseases caused by Hendra and Nipah viruses

Information on the pathogenesis and transmissibility of Hendra and Nipah viruses was obtained by comparing their histopathology. Both viruses induced syncytial cells in vascular tissues and they were primarily vasotropic and/or neurotropic, generating interstitial pneumonia or encephalitis. Nipah virus in pigs was also epitheliotropic in respiratory epithelium and thus contagious.

Pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission.

Bats of the genus Pteropus have been identified as the reservoir hosts for the henipaviruses Hendra virus (HeV) and Nipah virus (NiV). The aim of these studies was to assess likely mechanisms for henipaviruses transmission from bats. In a series of experiments, Pteropus bats from Malaysia and Australia were inoculated with NiV and HeV, respectively, by natural routes of infection. Despite an intensive sampling strategy, no NiV was recovered from the Malaysian bats and HeV was reisolated from only one Australian bat; no disease was seen. These experiments suggest that opportunities for henipavirus transmission may be limited; therefore, the probability of a spillover event is low. For spillover to occur, a range of conditions and events must coincide. An alternate assessment framework is required if we are to fully understand how this reservoir host maintains and transmits not only these but all viruses with which it has been associated.

A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID50 within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

Ecological dynamics of emerging bat virus spillover

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.

Feline Model of Acute Nipah Virus Infection and Protection with a Soluble Glycoprotein-Based Subunit Vaccine

ABSTRACT Nipah virus (NiV) and Hendra virus (HeV) are paramyxoviruses capable of causing considerable morbidity and mortality in a number of mammalian species, including humans. Case reports from outbreaks and previous challenge experiments have suggested that cats were highly susceptible to NiV infection, responding with a severe respiratory disease and systemic infection. Here we have assessed the cat as a model of experimental NiV infection and use it in the evaluation of a subunit vaccine comprised of soluble G glycoprotein (sG). Two groups of two adult cats each were inoculated subcutaneously with either 500 or 5,000 50% tissue culture infective dose(s) (TCID50) of NiV. Animals were monitored closely for disease onset, and extensive analysis was conducted on samples and tissues taken during infection and at necropsy to determine viral load and tissue tropism. All animals developed clinical disease 6 to 9 days postinfection, a finding consistent with previous observations. In a subsequent experiment, two cats were immunized with HeV sG and two were immunized with NiV sG. Homologous serum neutralizing titers were greater than 1:20,000, and heterologous titers were greater than 1:20,000 to 16-fold lower. Immunized animals and two additional naive controls were then challenged subcutaneously with 500 TCID50 of NiV. Naive animals developed clinical disease 6 to 13 days postinfection, whereas none of the immunized animals showed any sign of disease. TaqMan PCR analysis of samples from naive animals revealed considerable levels of NiV genome in a wide range of tissues, whereas the genome was evident in only two immunized cats in only four samples and well below the limit of accurate detection. These results indicate that the cat provides a consistent model for acute NiV infection and associated pathogenesis and an effective subunit vaccine strategy appears achievable.

A recombinant Hendra virus G glycoprotein-based subunit vaccine protects ferrets from lethal Hendra virus challenge

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are two deadly zoonotic viruses for which no vaccines or therapeutics have yet been approved for human or livestock use. In 14 outbreaks since 1994 HeV has been responsible for multiple fatalities in horses and humans, with all known human infections resulting from close contact with infected horses. A vaccine that prevents virus shedding in infected horses could interrupt the chain of transmission to humans and therefore prevent HeV disease in both. Here we characterise HeV infection in a ferret model and show that it closely mirrors the disease seen in humans and horses with induction of systemic vasculitis, including involvement of the pulmonary and central nervous systems. This model of HeV infection in the ferret was used to assess the immunogenicity and protective efficacy of a subunit vaccine based on a recombinant soluble version of the HeV attachment glycoprotein G (HeVsG), adjuvanted with CpG. We report that ferrets vaccinated with a 100 μg, 20 μg or 4 μg dose of HeVsG remained free of clinical signs of HeV infection following a challenge with 5000 TCID₅₀ of HeV. In addition, and of considerable importance, no evidence of virus or viral genome was detected in any tissues or body fluids in any ferret in the 100 and 20 μg groups, while genome was detected in the nasal washes only of one animal in the 4 μg group. Together, our findings indicate that 100 μg or 20 μg doses of HeVsG vaccine can completely prevent a productive HeV infection in the ferret, suggesting that vaccination to prevent the infection and shedding of HeV is possible.

Invasion of the Central Nervous System in a Porcine Host by Nipah Virus

ABSTRACT Nipah virus, a newly emerged zoonotic paramyxovirus, infects a number of species. Human infections were linked to direct contact with pigs, specifically with their body fluids. Clinical signs in human cases indicated primarily involvement of the central nervous system, while in pigs the respiratory system was considered the primary virus target, with only rare involvement of the central nervous system. Eleven 5-week-old piglets were infected intranasally, orally, and ocularly with 2.5 × 105 PFU of Nipah virus per animal and euthanized between 3 and 8 days postinoculation. Nipah virus caused neurological signs in two out of eleven inoculated pigs. The rest of the pigs remained clinically healthy. Virus was detected in the respiratory system (turbinates, nasopharynx, trachea, bronchus, and lung in titers up to 105.3 PFU/g) and in the lymphoreticular system (endothelial cells of blood and lymphatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen with titers up to 106 PFU/g). Virus presence was confirmed in the nervous system of both sick and apparently healthy animals (cranial nerves, trigeminal ganglion, brain, and cerebrospinal fluid, with titers up to 107.7 PFU/g of tissue). Nipah virus distribution was confirmed by immunohistochemistry. The study presents novel findings indicating that Nipah virus invaded the central nervous system of the porcine host via cranial nerves as well as by crossing the blood-brain barrier after initial virus replication in the upper respiratory tract.

Establishment, immortalisation and characterisation of pteropid bat cell lines

Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Multiple Infections with Seasonal Influenza A Virus Induce Cross-Protective Immunity against A(H1N1) Pandemic Influenza Virus in a Ferret Model

BACKGROUND An age bias toward children and young adults has been reported for infection and hospitalizations with pandemic H1N1 influenza (A[H1N1]pdm) in the 2009 and 2010 influenza seasons in the Southern and Northern Hemispheres. Serological analysis of prepandemic samples has shown a higher incidence of cross-reactive antibodies to A(H1N1)pdm virus in older populations; conserved T cell epitopes between viruses have been identified. The contribution of preexisting immunity to seasonal influenza to protection against A(H1N1)pdm infection was analyzed in a ferret model. METHODS Ferrets were pre-infected with influenza A viruses and/or vaccinated with inactivated influenza viruses with adjuvant. Infection after challenge was assessed by measuring shedding virus, transmission to naive animals, and seroconversion. RESULTS Homologous vaccination reduced the incidence of infection and delayed transmission. Pre-infection with virus induced sterilizing immunity to homologous challenge. One prior infection with seasonal influenza A virus improved clearance of A(H1N1)pdm virus. Prior infection with A(H1N1)pdm virus reduced shedding after seasonal influenza A challenge. Two infections with seasonal influenza A viruses reduced the incidence of infection, the amount and duration of virus shedding, and the frequency of transmission following A(H1N1)pdm challenge. CONCLUSION These data suggest the reduced incidence and severity of infection with A(H1N1)pdm virus in the adult population during the 2009-2010 influenza season may be a result of previous exposure to seasonal influenza A viruses.