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.     

Qualitative release assessment to estimate the likelihood of henipavirus entering the United Kingdom

The genus Henipavirus includes Hendra virus (HeV) and Nipah virus (NiV), for which fruit bats (particularly those of the genus Pteropus) are considered to be the wildlife reservoir. The recognition of henipaviruses occurring across a wider geographic and host range suggests the possibility of the virus entering the United Kingdom (UK). To estimate the likelihood of henipaviruses entering the UK, a qualitative release assessment was undertaken. To facilitate the release assessment, the world was divided into four zones according to location of outbreaks of henipaviruses, isolation of henipaviruses, proximity to other countries where incidents of henipaviruses have occurred and the distribution of Pteropus spp. fruit bats. From this release assessment, the key findings are that the importation of fruit from Zone 1 and 2 and bat bushmeat from Zone 1 each have a Low annual probability of release of henipaviruses into the UK. Similarly, the importation of bat meat from Zone 2, horses and companion animals from Zone 1 and people travelling from Zone 1 and entering the UK was estimated to pose a Very Low probability of release. The annual probability of release for all other release routes was assessed to be Negligible. It is recommended that the release assessment be periodically re-assessed to reflect changes in knowledge and circumstances over time.     

The Distribution of Henipaviruses in Southeast Asia and Australasia: Is Wallace’s Line a Barrier to Nipah Virus?

Nipah virus (NiV) (Genus Henipavirus) is a recently emerged zoonotic virus that causes severe disease in humans and has been found in bats of the genus Pteropus. Whilst NiV has not been detected in Australia, evidence for NiV-infection has been found in pteropid bats in some of Australia’s closest neighbours. The aim of this study was to determine the occurrence of henipaviruses in fruit bat (Family Pteropodidae) populations to the north of Australia. In particular we tested the hypothesis that Nipah virus is restricted to west of Wallace’s Line. Fruit bats from Australia, Papua New Guinea, East Timor and Indonesia were tested for the presence of antibodies to Hendra virus (HeV) and Nipah virus, and tested for the presence of HeV, NiV or henipavirus RNA by PCR. Evidence was found for the presence of Nipah virus in both Pteropus vampyrus and Rousettus amplexicaudatus populations from East Timor. Serology and PCR also suggested the presence of a henipavirus that was neither HeV nor NiV in Pteropus alecto and Acerodon celebensis. The results demonstrate the presence of NiV in the fruit bat populations on the eastern side of Wallace’s Line and within 500 km of Australia. They indicate the presence of non-NiV, non-HeV henipaviruses in fruit bat populations of Sulawesi and Sumba and possibly in Papua New Guinea. It appears that NiV is present where P. vampyrus occurs, such as in the fruit bat populations of Timor, but where this bat species is absent other henipaviruses may be present, as on Sulawesi and Sumba. Evidence was obtained for the presence henipaviruses in the non-Pteropid species R. amplexicaudatus and in A. celebensis. The findings of this work fill some gaps in knowledge in geographical and species distribution of henipaviruses in Australasia which will contribute to planning of risk management and surveillance activities.     

Interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines

Bats are natural reservoirs for a spectrum of infectious zoonotic diseases including the recently emerged henipaviruses (Hendra and Nipah viruses). Henipaviruses have been observed both naturally and experimentally to cause serious and often fatal disease in many different mammal species, including humans. Interestingly, infection of the flying fox with henipaviruses occurs in the absence of clinical disease. The extreme variation in the disease pattern between humans and bats has led to an investigation into the effects of henipavirus infection on the innate immune response in bat cell lines. We report that henipavirus infection does not result in the induction of interferon expression, and the viruses also inhibit interferon signaling. We also confirm that the interferon production and signaling block in bat cells is not due to differing viral protein expression levels between human and bat hosts. This information, in addition to the known lack of clinical signs in bats following henipavirus infection, suggests that bats control henipavirus infection by an as yet unidentified mechanism, not via the interferon response. This is the first report of henipavirus infection in bat cells specifically investigating aspects of the innate immune system.     

Hendra and Nipah viruses: pathogenesis and therapeutics

Within the past decade a number of new zoonotic paramyxoviruses emerged from flying foxes to cause serious disease outbreaks in man and livestock. Hendra virus was the cause of fatal infections of horses and man in Australia in 1994, 1999 and 2004. Nipah virus caused encephalitis in humans both in Malaysia in 1998/99, following silent spread of the virus in the pig population, and in Bangladesh from 2001 to 2004 probably as a result of direct bat to human transmission and spread within the human population. Hendra and Nipah viruses are highly pathogenic in humans with case fatality rates of 40% to 70%. Their genetic constitution, virulence and wide host range make them unique paramyxoviruses and they have been given Biosecurity Level 4 status in a new genus Henipavirus within the family Paramyxoviridae. Recent studies on the virulence, host range and cell tropisms of henipaviruses provide insights into the unique biological properties of these emerging human pathogens and suggest approaches for vaccine development and therapeutic countermeasures.     

Temporal Variation in Physiological Biomarkers in Black Flying-Foxes (<Emphasis Type="Italic">Pteropus alecto</Emphasis>), Australia

Bats of the genus Pteropus (Pteropodidae) are recognised as the natural host of multiple emerging pathogenic viruses of animal and human health significance, including henipaviruses, lyssaviruses and ebolaviruses. Some studies have suggested that physiological and ecological factors may be associated with Hendra virus infection in flying-foxes in Australia; however, it is essential to understand the normal range and seasonal variability of physiological biomarkers before seeking physiological associations with infection status. We aimed to measure a suite of physiological biomarkers in P. alecto over time to identify any seasonal fluctuations and to examine possible associations with life-cycle and environmental stressors. We sampled 839 adult P. alecto in the Australian state of Queensland over a 12-month period. The adjusted population means of every assessed hematologic and biochemical parameter were within the reported reference range on every sampling occasion. However, within this range, we identified significant temporal variation in these parameters, in urinary parameters and body condition, which primarily reflected the normal annual life cycle. We found no evident effect of remarkable physiological demands or nutritional stress, and no indication of clinical disease driving any parameter values outside the normal species reference range. Our findings identify underlying temporal physiological changes at the population level that inform epidemiological studies and assessment of putative physiological risk factors driving Hendra virus infection in P. alecto. More broadly, the findings add to the knowledge of Pteropus populations in terms of their relative resistance and resilience to emerging infectious disease.     

Henipavirus neutralising antibodies in an isolated island population of African fruit bats

Isolated islands provide valuable opportunities to study the persistence of viruses in wildlife populations, including population size thresholds such as the critical community size. The straw-coloured fruit bat, Eidolon helvum, has been identified as a reservoir for henipaviruses (serological evidence) and Lagos bat virus (LBV; virus isolation and serological evidence) in continental Africa. Here, we sampled from a remote population of E. helvum annobonensis fruit bats on Annobón island in the Gulf of Guinea to investigate whether antibodies to these viruses also exist in this isolated subspecies. Henipavirus serological analyses (Luminex multiplexed binding and inhibition assays, virus neutralisation tests and western blots) and lyssavirus serological analyses (LBV: modified Fluorescent Antibody Virus Neutralisation test, LBV and Mokola virus: lentivirus pseudovirus neutralisation assay) were undertaken on 73 and 70 samples respectively. Given the isolation of fruit bats on Annobón and their lack of connectivity with other populations, it was expected that the population size on the island would be too small to allow persistence of viruses that are thought to cause acute and immunising infections. However, the presence of antibodies against henipaviruses was detected using the Luminex binding assay and confirmed using alternative assays. Neutralising antibodies to LBV were detected in one bat using both assays. We demonstrate clear evidence for exposure of multiple individuals to henipaviruses in this remote population of E. helvum annobonensis fruit bats on Annobón island. The situation is less clear for LBV. Seroprevalences to henipaviruses and LBV in Annobón are notably different to those in E. helvum in continental locations studied using the same sampling techniques and assays. Whilst cross-sectional serological studies in wildlife populations cannot provide details on viral dynamics within populations, valuable information on the presence or absence of viruses may be obtained and utilised for informing future studies.     

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.     

Antibodies to henipavirus or henipa-like viruses in domestic pigs in Ghana, West Africa

Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), have Pteropid bats as their known natural reservoirs. Antibodies against henipaviruses have been found in Eidolon helvum, an old world fruit bat species, and henipavirus-like nucleic acid has been detected in faecal samples from E. helvum in Ghana. The initial outbreak of NiV in Malaysia led to over 265 human encephalitis cases, including 105 deaths, with infected pigs acting as amplifier hosts for NiV during the outbreak. We detected non-neutralizing antibodies against viruses of the genus Henipavirus in approximately 5% of pig sera (N = 97) tested in Ghana, but not in a small sample of other domestic species sampled under a E. helvum roost. Although we did not detect neutralizing antibody, our results suggest prior exposure of the Ghana pig population to henipavirus(es). Because a wide diversity of henipavirus-like nucleic acid sequences have been found in Ghanaian E. helvum, we hypothesise that these pigs might have been infected by henipavirus(es) sufficiently divergent enough from HeVor NiV to produce cross-reactive, but not cross-neutralizing antibodies to HeV or NiV.     

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.     

Viral infections in renal allograft recipients treated with long-term immunosuppression.

Thirty-nine renal allograft recipients who had received continuous immunosuppression for six to 13 years were examined clinically and virologically for evidence of past or present viral infection. Twenty-five had common warts, usually on the hands. In most the warts had appeared about one year after transplantation; once present, they never disappeared. Six patients had had a zoster rash from two months to four years after transplantation. None had had jaundice, and there was no change in the frequency of colds or non-specific fibrile illness. Four patients had no cytomegalovirus complement-fixing antibodies throughout the observation period; in the other 35 the antibody titre had risen appreciably during the first three to four months after transplantation. Antibody titres were high (mean 64) at follow-up, being only slightly lower than the highest titres achieved during the immediate postoperative period. None of the patients had had symptomatic cytomegalovirus infection, and in only two was the virus isolated from the urine at follow-up; the titres were extremely low. No changes occurred in the frequency of herpes simplex eruptions. Although all patients had herpes simplex humoral antibody, none excreted the virus. Although cytomegalovirus antibody titres were high, virus excretion was rare, indicating that chronic cytomegalovirus infection in these patients is immunologically well controlled.     

Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus)

Hendra virus (HeV) is a lethal paramyxovirus which emerged in humans in 1994. Poor understanding of HeV dynamics in Pteropus spp. (flying fox or fruit bat) reservoir hosts has limited our ability to determine factors driving its emergence. We initiated a longitudinal field study of HeV in little red flying foxes (LRFF; Pteropus scapulatus) and examined individual and population risk factors for infection, to determine probable modes of intraspecific transmission. We also investigated whether seasonal changes in host behaviour, physiology and demography affect host-pathogen dynamics. Data showed that pregnant and lactating females had significantly higher risk of infection, which may explain previously observed temporal associations between HeV outbreaks and flying fox birthing periods. Age-specific seroprevalence curves generated from field data imply that HeV is transmitted horizontally via faeces, urine or saliva. Rapidly declining seroprevalence between two field seasons suggests that immunity wanes faster in LRFF than in other flying fox species, and highlights the potentially critical role of this species in interspecific viral persistence. The highest seroprevalence was observed when animals showed evidence of nutritional stress, suggesting that environmental processes that alter flying fox food sources, such as habitat loss and climate change, may increase HeV infection and transmission. These insights into the ecology of HeV in flying fox populations suggest causal links between anthropogenic environmental change and HeV emergence.     

Cedar Virus: A Novel Henipavirus Isolated from Australian Bats

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.     

Ecological conditions predict the intensity of Hendra virus excretion over space and time from bat reservoir hosts

The ecological conditions experienced by wildlife reservoirs affect infection dynamics and thus the distribution of pathogen excreted into the environment. This spatial and temporal distribution of shed pathogen has been hypothesised to shape risks of zoonotic spillover. However, few systems have data on both long-term ecological conditions and pathogen excretion to advance mechanistic understanding and test environmental drivers of spillover risk. We here analyse three years of Hendra virus data from nine Australian flying fox roosts with covariates derived from long-term studies of bat ecology. We show that the magnitude of winter pulses of viral excretion, previously considered idiosyncratic, are most pronounced after recent food shortages and in bat populations displaced to novel habitats. We further show that cumulative pathogen excretion over time is shaped by bat ecology and positively predicts spillover frequency. Our work emphasises the role of reservoir host ecology in shaping pathogen excretion and provides a new approach to estimate spillover risk.     

Recrudescent Infection Supports Hendra Virus Persistence in Australian Flying-Fox Populations

Zoonoses from wildlife threaten global public health. Hendra virus is one of several zoonotic viral diseases that have recently emerged from Pteropus species fruit-bats (flying-foxes). Most hypotheses regarding persistence of Hendra virus within flying-fox populations emphasize horizontal transmission within local populations (colonies) via urine and other secretions, and transmission among colonies via migration. As an alternative hypothesis, we explore the role of recrudescence in persistence of Hendra virus in flying-fox populations via computer simulation using a model that integrates published information on the ecology of flying-foxes, and the ecology and epidemiology of Hendra virus. Simulated infection patterns agree with infection patterns observed in the field and suggest that Hendra virus could be maintained in an isolated flying-fox population indefinitely via periodic recrudescence in a manner indistinguishable from maintenance via periodic immigration of infected individuals. Further, post-recrudescence pulses of infectious flying-foxes provide a plausible basis for the observed seasonal clustering of equine cases. Correct understanding of the infection dynamics of Hendra virus in flying-foxes is fundamental to effectively managing risk of infection in horses and humans. Given the lack of clear empirical evidence on how the virus is maintained within populations, the role of recrudescence merits increased attention.     

Cross-infection enhancement among African flaviviruses by immune mouse ascitic fluids

Cross-infection enhancement of seven African flaviviruses by subneutralising concentrations of antibody in immune ascitic fluids was investigated in P388D1 cell culture. Infection by all the seven flaviviruses tested was enhanced by homologous and at least one of six heterologous immune mouse ascitic fluids (IMAF) tested. Enhancement ratios and enhancing antibody titres were higher in homologous than in heterologous enhancement. Zika, Wesselsbron, Uganda S and West Nile viruses were enhanced in culture by all the IMAF tested. Enhancement of Dakar bat and Yellow fever viruses was produced by five heterologous IMAF, but Potiskum virus was enhanced by one heterologous flavivirus antibody. The antibody to Potiskum virus was the most potent mediator of heterologous infection enhancement; all six heterologous flaviviruses were markedly enhanced by this antibody.     

Evidence of henipavirus infection in West African fruit bats

Henipaviruses are emerging RNA viruses of fruit bat origin that can cause fatal encephalitis in man. Ghanaian fruit bats (megachiroptera) were tested for antibodies to henipaviruses. Using a Luminex multiplexed microsphere assay, antibodies were detected in sera of Eidolon helvum to both Nipah (39%, 95% confidence interval: 27-51%) and Hendra (22%, 95% CI: 11-33%) viruses. Virus neutralization tests further confirmed seropositivity for 30% (7/23) of Luminex positive serum samples. Our results indicate that henipavirus is present within West Africa.     

Synthetic protocells interact with viral nanomachinery and inactivate pathogenic human virus

We present a new antiviral strategy and research tool that could be applied to a wide range of enveloped viruses that infect human beings via membrane fusion. We test this strategy on two emerging zoonotic henipaviruses that cause fatal encephalitis in humans, Nipah (NiV) and Hendra (HeV) viruses. In the new approach, artificial cell-like particles (protocells) presenting membrane receptors in a biomimetic manner were developed and found to attract and inactivate henipavirus envelope glycoprotein pseudovirus particles, preventing infection. The protocells do not accumulate virus during the inactivation process. The use of protocells that interact with, but do not accumulate, viruses may provide significant advantages over current antiviral drugs, and this general approach may have wide potential for antiviral development.     

Avian influenza virus infection dynamics in shorebird hosts

To gain insight into avian influenza virus (AIV) transmission, exposure, and maintenance patterns in shorebirds at Delaware Bay during spring migration, we examined temporal AIV prevalence trends in four Charadriiformes species with the use of serial cross-sectional data from 2000 through 2008 and generalized linear and additive models. Prevalence of AIV in Ruddy Turnstones (Arenaria interpres morinella) increased after arrival, peaked in mid-late May, and decreased prior to departure. Antibody prevalence also increased over this period; together, these results suggested local infection and recovery prior to departure. Red Knots (Calidris canutus rufa), Sanderlings (Calidris alba), and Laughing Gulls (Leucophaeus atricilla) were rarely infected, but dynamic changes in antibody prevalence differed among species. In Red Knots, declining antibody prevalence over the stopover period suggested AIV exposure prior to arrival at Delaware Bay with limited infection at this site. Antibody prevalence was consistently high in Laughing Gulls and low in Sanderlings. Both viral prevalence and antibody prevalence in Sanderlings varied directly with those in turnstones, suggesting virus spillover to Sanderlings. Results indicate that, although hundreds of thousands of birds concentrate at Delaware Bay during spring, dynamics of AIV infection differ among species, perhaps due to differences in susceptibility, potential for contact with AIV at this site, or prior exposure. Additionally, Ruddy Turnstones possibly act as a local AIV amplifying host rather than a reservoir.