Shawn Todd

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.

Metagenomic study of the viruses of African straw-coloured fruit bats: Detection of a chiropteran poxvirus and isolation of a novel adenovirus

Abstract Viral emergence as a result of zoonotic transmission constitutes a continuous public health threat. Emerging viruses such as SARS coronavirus, hantaviruses and henipaviruses have wildlife reservoirs. Characterising the viruses of candidate reservoir species in geographical hot spots for viral emergence is a sensible approach to develop tools to predict, prevent, or contain emergence events. Here, we explore the viruses of Eidolon helvum, an Old World fruit bat species widely distributed in Africa that lives in close proximity to humans. We identified a great abundance and diversity of novel herpes and papillomaviruses, described the isolation of a novel adenovirus, and detected, for the first time, sequences of a chiropteran poxvirus closely related with Molluscum contagiosum. In sum, E. helvum display a wide variety of mammalian viruses, some of them genetically similar to known human pathogens, highlighting the possibility of zoonotic transmission.

Type III IFNs in Pteropid Bats: Differential Expression Patterns Provide Evidence for Distinct Roles in Antiviral Immunity

Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.

Novel, Potentially Zoonotic Paramyxoviruses from the African Straw-Colored Fruit Bat Eidolon helvum

ABSTRACT Bats carry a variety of paramyxoviruses that impact human and domestic animal health when spillover occurs. Recent studies have shown a great diversity of paramyxoviruses in an urban-roosting population of straw-colored fruit bats in Ghana. Here, we investigate this further through virus isolation and describe two novel rubulaviruses: Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2). The viruses form a phylogenetic cluster with each other and other bat-derived rubulaviruses, such as Tuhoko viruses, Menangle virus, and Tioman virus. We developed AchPV1- and AchPV2-specific serological assays and found evidence of infection with both viruses in Eidolon helvum across sub-Saharan Africa and on islands in the Gulf of Guinea. Longitudinal sampling of E. helvum indicates virus persistence within fruit bat populations and suggests spread of AchPVs via horizontal transmission. We also detected possible serological evidence of human infection with AchPV2 in Ghana and Tanzania. It is likely that clinically significant zoonotic spillover of chiropteran paramyxoviruses could be missed throughout much of Africa where health surveillance and diagnostics are poor and comorbidities, such as infection with HIV or Plasmodium sp., are common.

Co-circulation of diverse paramyxoviruses in an urban African fruit bat population.

Bats constitute a reservoir of zoonotic infections and some bat paramyxoviruses are capable of cross-species transmission, often with fatal consequences. Determining the level of viral diversity in reservoir populations is fundamental to understanding and predicting viral emergence. This is particularly relevant for RNA viruses where the adaptive mutations required for cross-species transmission can be present in the reservoir host. We report the use of non-invasively collected, pooled, neat urine samples as a robust sample type for investigating paramyxoviruses in bat populations. Using consensus PCR assays we have detected a high incidence and genetic diversity of novel paramyxoviruses in an urban fruit bat population over a short period of time. This may suggest a similarly unique relationship between bats and the members of the family Paramyxoviridae as proposed for some other viral families. Additionally, the high rate of bat–human contact at the study site calls for the zoonotic potential of the detected viruses to be investigated further.

Genome Sequence Conservation of Hendra Virus Isolates during Spillover to Horses, Australia

Bat-to-horse transmission of Hendra virus has occurred at least 14 times. Although clinical signs in horses have differed, genome sequencing has demonstrated little variation among the isolates. Our sequencing of 5 isolates from recent Hendra virus outbreaks in horses found no correlation between sequences and time or geographic location of outbreaks.

A Novel Bat Herpesvirus Encodes Homologues of Major Histocompatibility Complex Classes I and II, C-Type Lectin, and a Unique Family of Immune-Related Genes

ABSTRACT Herpesviruses or herpesviral sequences have been identified in various bat species. Here, we report the isolation, cell tropism, and complete genome sequence of a novel betaherpesvirus from the bat Miniopterus schreibersii (MsHV). In primary cell culture, MsHV causes cytopathic effects (CPE) and reaches peak virus production 2 weeks after infection. MsHV was found to infect and replicate less efficiently in a feline kidney cell, CRFK, and failed to replicate in 13 other cell lines tested. Sequencing of the MsHV genome using the 454 system, with a 224-fold coverage, revealed a genome size of 222,870 bp. The genome was extensively analyzed in comparison to those of related viruses. Of the 190 predicted open reading frames (ORFs), 40 were identified as herpesvirus core genes. Among 93 proteins with identifiable homologues in tree shrew herpesvirus (THV), human cytomegalovirus (HCMV), or rat cytomegalovirus (RCMV), most had highest sequence identities with THV counterparts. However, the MsHV genome organization is colinear with that of RCMV rather than that of THV. The following unique features were discovered in the MsHV genome. One predicted protein, B125, is similar to human herpesvirus 6 (HHV-6) U94, a homologue of the parvovirus Rep protein. For the unique ORFs, 7 are predicted to encode major histocompatibility complex (MHC)-related proteins, 2 to encode MHC class I homologues, and 3 to encode MHC class II homologues; 4 encode the homologues of C-type lectin- or natural killer cell lectin-like receptors;, and the products of a unique gene family, the b149 family, of 16 members, have no significant sequence identity with known proteins but exhibit immunoglobulin-like beta-sandwich domains revealed by three-dimensional (3D) structural prediction. To our knowledge, MsHV is the first virus genome known to encode MHC class II homologues.

Recombinant Hendra viruses expressing a reporter gene retain pathogenicity in ferrets

BackgroundHendra virus (HeV) is an Australian bat-borne zoonotic paramyxovirus that repeatedly spills-over to horses causing fatal disease. Human cases have all been associated with close contact with infected horses.MethodsA full-length antigenome clone of HeV was assembled, a reporter gene (GFP or luciferase) inserted between the P and M genes and transfected to 293T cells to generate infectious reporter gene-encoding recombinant viruses. These viruses were then assessed in vitro for expression of the reporter genes. The GFP expressing recombinant HeV was used to challenge ferrets to assess the virulence and tissue distribution by monitoring GFP expression in infected cells.ResultsThree recombinant HeV constructs were successfully cloned and rescued; a wild-type virus, a GFP-expressing virus and a firefly luciferase-expressing virus. In vitro characterisation demonstrated expression of the reporter genes, with levels proportional to the initial inoculum levels. Challenge of ferrets with the GFP virus demonstrated maintenance of the fatal phenotype with disease progressing to death consistent with that observed previously with the parental wild-type isolate of HeV. GFP expression could be observed in infected tissues collected from animals at euthanasia.ConclusionsHere, we report on the first successful rescue of recombinant HeV, including wild-type virus and viruses expressing two different reporter genes encoded as an additional gene cassette inserted between the P and M genes. We further demonstrate that the GFP virus retained the ability to cause fatal disease in a well-characterized ferret model of henipavirus infection despite the genome being an extra 1290 nucleotides in length.

Isolation of multiple novel paramyxoviruses from pteropid bat urine

Bats have been found to harbour a number of new emerging viruses with zoonotic potential, and there has been a great deal of interest in identifying novel bat pathogens to determine the risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid; however, virus isolation is still a challenge, and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports, we have described the isolation of Hendra virus, Menangle virus and Cedar virus in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.

Duration of Maternal Antibodies against Canine Distemper Virus and Hendra Virus in Pteropid Bats

Old World frugivorous bats have been identified as natural hosts for emerging zoonotic viruses of significant public health concern, including henipaviruses (Nipah and Hendra virus), Ebola virus, and Marburg virus. Epidemiological studies of these viruses in bats often utilize serology to describe viral dynamics, with particular attention paid to juveniles, whose birth increases the overall susceptibility of the population to a viral outbreak once maternal immunity wanes. However, little is understood about bat immunology, including the duration of maternal antibodies in neonates. Understanding duration of maternally derived immunity is critical for characterizing viral dynamics in bat populations, which may help assess the risk of spillover to humans. We conducted two separate studies of pregnant Pteropus bat species and their offspring to measure the half-life and duration of antibodies to 1) canine distemper virus antigen in vaccinated captive Pteropus hypomelanus; and 2) Hendra virus in wild-caught, naturally infected Pteropus alecto. Both of these pteropid bat species are known reservoirs for henipaviruses. We found that in both species, antibodies were transferred from dam to pup. In P. hypomelanus pups, titers against CDV waned over a mean period of 228.6 days (95% CI: 185.4–271.8) and had a mean terminal phase half-life of 96.0 days (CI 95%: 30.7–299.7). In P. alecto pups, antibodies waned over 255.13 days (95% CI: 221.0–289.3) and had a mean terminal phase half-life of 52.24 days (CI 95%: 33.76–80.83). Each species showed a duration of transferred maternal immunity of between 7.5 and 8.5 months, which was longer than has been previously estimated. These data will allow for more accurate interpretation of age-related Henipavirus serological data collected from wild pteropid bats.