Evaluation of a New Viral Vaccine Vector in Mice and Rhesus Macaques: J Paramyxovirus Expressing Hemagglutinin of Influenza A Virus H5N1.

H5N1, an avian influenza virus, is known to circulate in many Asian countries, such as Bangladesh, China, Cambodia, Indonesia, and Vietnam. The current FDA-approved H5N1 vaccine has a moderate level of efficacy. A safe and effective vaccine is needed to prevent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in humans. Nonsegmented negative-sense single-stranded viruses (NNSVs) are widely used as a vector to develop vaccines for humans, animals, and poultry. NNSVs stably express foreign genes without integrating with the host genome. J paramyxovirus (JPV) is a nonsegmented negative-strand RNA virus and a member of the proposed genus Jeilongvirus in the family Paramyxoviridae. JPV-specific antibodies have been detected in rodents, bats, humans, and pigs, but the virus is not associated with disease in any species other than mice. JPV replicates in the respiratory tract of mice and efficiently expresses the virus-vectored foreign genes in tissue culture cells. In this work, we explored JPV as a vector for developing an H5N1 vaccine using intranasal delivery. We incorporated hemagglutinin (HA) of H5N1 into the JPV genome by replacing the small hydrophobic (SH) gene to generate a recombinant JPV expressing HA (rJPV-ΔSH-H5). A single intranasal administration of rJPV-ΔSH-H5 protected mice from a lethal HPAI H5N1 challenge. Intranasal vaccination of rJPV-ΔSH-H5 in rhesus macaques elicited antigen-specific humoral and cell-mediated immune responses. This work demonstrates that JPV is a promising vaccine vector. IMPORTANCE A highly pathogenic avian influenza (HPAI) H5N1 outbreak in Southeast Asia destroyed millions of birds. Transmission of H5N1 into humans resulted in deaths in many countries. In this work, we developed a novel H5N1 vaccine candidate using J paramyxovirus (JPV) as a vector and demonstrated that JPV is an efficacious vaccine vector in animals. Nonsegmented negative-sense single-stranded viruses (NNSVs) stably express foreign genes without integrating into the host genome. JPV, an NNSV, replicates efficiently in the respiratory tract and induces robust immune responses.

Rapid Response Subunit Vaccine Design in the Absence of Structural Information.

Prior to 2020, the threat of a novel viral pandemic was omnipresent but largely ignored. Just 12 months prior to the Coronavirus disease 2019 (COVID-19) pandemic our team received funding from the Coalition for Epidemic Preparedness Innovations (CEPI) to establish and validate a rapid response pipeline for subunit vaccine development based on our proprietary Molecular Clamp platform. Throughout the course of 2019 we conducted two mock tests of our system for rapid antigen production against two potential, emerging viral pathogens, Achimota paramyxovirus and Wenzhou mammarenavirus. For each virus we expressed a small panel of recombinant variants of the membrane fusion protein and screened for expression level, product homogeneity, and the presence of the expected trimeric pre-fusion conformation. Lessons learned from this exercise paved the way for our response to COVID-19, for which our candidate antigen is currently in phase I clinical trial.

Human Paramyxovirus Infections Induce T Cells That Cross-React with Zoonotic Henipaviruses.

Humans are infected with paramyxoviruses of different genera early in life, which induce cytotoxic T cells that may recognize conserved epitopes. This raises the question of whether cross-reactive T cells induced by antecedent paramyxovirus infections provide partial protection against highly lethal zoonotic Nipah virus infections. By characterizing a measles virus-specific but paramyxovirus cross-reactive human T cell clone, we discovered a highly conserved HLA-B*1501-restricted T cell epitope in the fusion protein. Using peptides, tetramers, and single cell sorting, we isolated a parainfluenza virus-specific T cell clone from a healthy adult and showed that both clones cleared Nipah virus-infected cells. We identified multiple conserved hot spots in paramyxovirus proteomes that contain other potentially cross-reactive epitopes. Our data suggest that, depending on HLA haplotype and history of paramyxovirus exposures, humans may have cross-reactive T cells that provide protection against Nipah virus. The effect of preferential boosting of these cross-reactive epitopes needs to be further studied in light of paramyxovirus vaccination studies.IMPORTANCE Humans encounter multiple paramyxoviruses early in life. This study shows that infection with common paramyxoviruses can induce T cells cross-reactive with the highly pathogenic Nipah virus. This demonstrates that the combination of paramyxovirus infection history and HLA haplotype affects immunity to phylogenetically related zoonotic paramyxoviruses.

Viral Diversity of Microbats within the South West Botanical Province of Western Australia.

Bats are known reservoirs of a wide variety of viruses that rarely result in overt clinical disease in the bat host. However, anthropogenic influences on the landscape and climate can change species assemblages and interactions, as well as undermine host-resilience. The cumulative result is a disturbance of bat-pathogen dynamics, which facilitate spillover events to sympatric species, and may threaten bat communities already facing synergistic stressors through ecological change. Therefore, characterisation of viral pathogens in bat communities provides important basal information to monitor and predict the emergence of diseases relevant to conservation and public health. This study used targeted molecular techniques, serological assays and next generation sequencing to characterise adenoviruses, coronaviruses and paramyxoviruses from 11 species of insectivorous bats within the South West Botanical Province of Western Australia. Phylogenetic analysis indicated complex ecological interactions including virus-host associations, cross-species infections, and multiple viral strains circulating concurrently within selected bat populations. Additionally, we describe the entire coding sequences for five alphacoronaviruses (representing four putative new species), and one novel adenovirus. Results indicate that viral burden (both prevalence and richness) is not homogeneous among species, with Chalinolobus gouldii identified as a key epidemiological element within the studied communities.

Hervey virus: Study on co-circulation with Henipaviruses in Pteropid bats within their distribution range from Australia to Africa.

In 2011, an unusually large number of independent Hendra virus outbreaks were recorded on horse properties in Queensland and New South Wales, Australia. Urine from bat colonies adjacent to the outbreak sites were sampled and screened for Hendra and other viruses. Several novel paramyxoviruses were also isolated at different locations. Here one of the novel viruses, named Hervey virus (HerPV), is fully characterized by genome sequencing, annotation, phylogeny and in vitro host range, and its serological cross-reactivity and neutralization patterns are examined. HerPV may have ecological and spatial and temporal patterns similar to Hendra virus and could serve as a sentinel virus for the surveillance of this highly pathogenic virus. The suitability of HerPV as potential sentinel virus is further assessed by determining the serological prevalence of HerPV antibodies in fruit-eating bats from Australia, Indonesia, Papua New Guinea, Tanzania and the Gulf of Guinea, indicating the presence of similar viruses in regions beyond the Australian border.

Vaccines for the Paramyxoviruses and Pneumoviruses: Successes, Candidates, and Hurdles.

Human parainfluenza viruses (family Paramyxoviridae), human metapneumovirus, and respiratory syncytial virus (family Pneumoviridae) infect most infants and children within the first few years of life and are the etiologic agents for many serious acute respiratory illnesses. These virus infections are also associated with long-term diseases that impact quality of life, including asthma. Despite over a half-century of vaccine research, development, and clinical trials, no vaccine has been licensed to date for the paramyxoviruses or pneumoviruses for the youngest infants. In this study, we describe the recent reclassification of paramyxoviruses and pneumoviruses into distinct families by the International Committee on the Taxonomy of Viruses. We also discuss some past unsuccessful vaccine trials and some currently preferred vaccine strategies. Finally, we discuss hurdles that must be overcome to support successful respiratory virus vaccine development for the youngest children.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

Hendra Virus Infection Dynamics in the Grey-Headed Flying Fox (Pteropus poliocephalus) at the Southern-Most Extent of Its Range: Further Evidence This Species Does Not Readily Transmit the Virus to Horses.

Hendra virus (HeV) is an important emergent virus in Australia known to infect horses and humans in certain regions of the east coast. Whilst pteropid bats ("flying foxes") are considered the natural reservoir of HeV, which of the four mainland species is the principal reservoir has been a source of ongoing debate, particularly as shared roosting is common. To help resolve this, we sampled a colony consisting of just one of these species, the grey-headed flying fox, (Pteropus poliocephalus), at the southernmost extent of its range. Using the pooled urine sampling technique at approximately weekly intervals over a two year period, we determined the prevalence of HeV and related paramyxoviruses using a novel multiplex (Luminex) platform. Whilst all the pooled urine samples were negative for HeV nucleic acid, we successfully identified four other paramyxoviruses, including Cedar virus; a henipavirus closely related to HeV. Collection of serum from individually caught bats from the colony showed that antibodies to HeV, as estimated by a serological Luminex assay, were present in between 14.6% and 44.5% of animals. The wide range of the estimate reflects uncertainties in interpreting intermediate results. Interpreting the study in the context of HeV studies from states to the north, we add support for an arising consensus that it is the black flying fox and not the grey-headed flying fox that is the principal source of HeV in spillover events to horses.

Human and Mouse Eosinophils Have Antiviral Activity against Parainfluenza Virus.

Respiratory viruses cause asthma exacerbations. Because eosinophils are the prominent leukocytes in the airways of 60-70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice after ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726) but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein eosinophil peroxidase, using eosinophil peroxidase-deficient transgenic mice, did not reduce eosinophils' antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor, Toll-like receptor (TLR) 7. IFNγ increased expression of eosinophil TLR7 and potentiated TLR7-induced nitric oxide production. These results suggest that eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.

Antigenic Drift of A/H3N2/Virus and Circulation of Influenza-Like Viruses During the 2014/2015 Influenza Season in Poland.

Morbidity rates of influenza could be greatly reduced due to vaccination. However, the virus is able to evolve through genetic mutations, which is why vaccines with updated composition are necessary every season. Their effectiveness depends on whether there is a good antigenic match between circulating viruses and vaccine strains. In Poland, the 2014/2015 influenza epidemic started in week 5 (January/February) of 2015 and continued until week 17 (April) of 2015. The influenza activity was moderate with the highest incidence of influence-like illness at week 10/2015 (March). During that season, antigenic drift of influenza virus A/H3N2/ occurred causing higher rates of A/H3N2/ infections. Among the 2416 tested specimens, 22.6 % of influenza cases were positive for A/H3N2/, while A/H1N1/pdm09 constituted 14.6 % cases. Influenza A viruses were detected in co-circulation with influenza B viruses; the latter amounted to 34.1 % of all influenza detections. Other detected causes of influenza-like illness consisted of respiratory syncytial virus (RSV), being predominant, and, sporadically, human coronavirus, parainfluenza 1-3, rhinovirus, and adenovirus. Despite low vaccine effectiveness of solely one component, A/H3N2/, the vaccine could mitigate or shorten the length of influenza infection and reduce the number of severe outcomes and mortality. Thus, vaccination against influenza remains the most effective way to prevent illness and possibly fatal outcomes.

The immune evasion function of J and Beilong virus V proteins is distinct from that of other paramyxoviruses, consistent with their inclusion in the proposed genus Jeilongvirus.

IFN-antagonist function is a major determinant of pathogenicity and cross-species infection by viruses, but remains poorly defined for many potentially zoonotic viruses resident in animal species. The paramyxovirus family contains several zoonotic viruses, including highly pathogenic viruses such as Nipah virus and Hendra virus, and an increasing number of largely uncharacterized animal viruses. Here, we report the characterization of IFN antagonism by the rodent viruses J virus (JPV) and Beilong virus (BeiPV) of the proposed genus Jeilongvirus of the paramyxoviruses. Infection of cells by JPV and BeiPV was found to inhibit IFN-activated nuclear translocation of signal transducer and activator of transcription 1 (STAT1). However, in contrast to most other paramyxoviruses, the JPV and BeiPV V proteins did not interact with or inhibit signalling by STAT1 or STAT2, suggesting that JPV/BeiPV use an atypical V protein-independent strategy to target STATs, consistent with their inclusion in a separate genus. Nevertheless, the V proteins of both viruses interacted with melanoma differentiation-associated protein 5 (MDA5) and robustly inhibited MDA5-dependent activation of the IFN-ß promoter. This supports a growing body of evidence that MDA5 is a universal target of paramyxovirus V proteins, such that the V-MDA5 interaction represents a potential target for broad-spectrum antiviral approaches.

Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization.

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.

Viral counterattack against the host innate immune system.

Paramyxoviruses evade antiviral immune response using a small nonstructural protein, V, which binds to the host dsRNA sensor MDA5 and prevents it from activating the interferon signaling pathway. A recent crystal structure of the V protein in complex with MDA5, published in Science, revealed that V disrupts the structure of MDA5 and is integrated into the MDA5 protein fold, providing an intriguing new example of viral mimicry as a countermeasure against the host immune system.

Parainfluenza virus 5-based vaccine vectors expressing vaccinia virus (VACV) antigens provide long-term protection in mice from lethal intranasal VACV challenge.

To test the potential for parainfluenza virus 5 (PIV5)-based vectors to provide protection from vaccinia virus (VACV) infection, PIV5 was engineered to express secreted VACV L1R and B5R proteins, two important antigens for neutralization of intracellular mature (IMV) and extracellular enveloped (EEV) virions, respectively. Protection of mice from lethal intranasal VACV challenge required intranasal immunization with PIV5-L1R/B5R in a prime-boost protocol, and correlated with low VACV-induced pathology in the respiratory tract and anti-VACV neutralizing antibody. Mice immunized with PIV5-L1R/B5R showed some disease symptoms following VACV challenge such as loss of weight and hunching, but these symptoms were delayed and less severe than with unimmunized control mice. While immunization with PIV5 expressing B5R alone conferred at least some protection, the most effective immunization included the PIV5 vector expressing L1R alone or in combination with PIV5-B5R. PIV5-L1R/B5R vectors elicited protection from VACV challenge even when CD8+ cells were depleted, but not in the case of mice that were defective in B cell production. Mice were protected from VACV challenge out to at least 1.5 years after immunization with PIV5-L1R/B5R vectors, and showed significant levels of anti-VACV neutralizing antibodies. These results demonstrate the potential for PIV5-based vectors to provide long lasting protection against complex human respiratory pathogens such as VACV, but also highlight the need to understand mechanisms for the generation of strong immune responses against poorly immunogenic viral proteins.

Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference.

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins.

Novel monoclonal antibodies against Menangle virus nucleocapsid protein.

Menangle virus (MenV) is a member of the family Paramyxoviridae isolated in Australia that causes a reproductive disease of pigs. There is a need for specific immunoassays for virus detection to facilitate the diagnosis of MenV infection. Three novel monoclonal antibodies (MAbs) of the IgG1 subtype were generated by immunizing mice with recombinant yeast-expressed MenV nucleocapsid (N) protein self-assembled to nucleocapsid-like structures. One MAb was cross-reactive with recombinant N protein of Tioman virus. The epitopes of MAbs were mapped using a series of truncated MenV N proteins lacking the 29-119 carboxy-terminal amino acid (aa) residues. The epitopes of two MAbs were mapped to aa 430-460 of the MenV N protein, whilst the epitope of one MAb was mapped to residues 460-490. All three MAbs specifically recognized MenV, as indicated by immunohistochemical staining of brain tissue isolated from a field case (a stillborn piglet) of MenV infection. The MAbs against MenV N protein may be a useful tool for immunohistological diagnosis of MenV infection.

Role for the paramyxovirus genomic promoter in limiting host cell antiviral responses and cell killing.

The parainfluenza virus simian virus 5 (SV5) is a poor inducer of innate immune responses. In contrast, the naturally occurring SV5 variant Wake Forest parainfluenza virus (WF-PIV) activates the synthesis of proinflammatory cytokines and beta interferon (IFN-beta). Comparison of SV5 and WF-PIV genome sequences revealed nine nucleotide differences within the viral genomic promoter, including two substitutions (U5C and A14G) in the most highly conserved 3'-end promoter element. To test the consequences of these promoter variations, a recombinant SV5 mutant [Le-(U5C, A14G)] was engineered to harbor the two WF-PIV genomic promoter substitutions in an otherwise wild-type (WT) SV5 background. Human lung epithelial cells infected with the Le-(U5C, A14G) mutant had higher rates of viral protein synthesis and levels of mRNA than cells infected with WT SV5, but levels of genomic RNA were not changed. Unlike WT SV5, the Le-(U5C, A14G) mutant was a potent inducer of interleukin-6 and IFN-beta synthesis, despite expressing a functional V protein antagonist. Cytokine responses to Le-(U5C, A14G) infection were reduced either by small interfering RNA-mediated knockdown of retinoic acid-inducible gene I (RIG-I) or after infection of cells that were engineered to express the reovirus sigma3 double-stranded RNA-binding protein. Le-(U5C, A14G) induced cytopathic effects not seen with WT SV5, and the extent of cell killing correlated with elevated levels of viral F protein and cell-cell fusion. Our results support a model whereby the SV5 promoter has evolved to function at an attenuated level in order to limit (i) synthesis of aberrant RNAs which induce RIG-I-mediated responses and (ii) overproduction of mRNA for potentially toxic gene products, such as the F protein. Control of genomic promoter activity may be particularly important for viruses such as SV5, that express a V protein targeting mda-5 but do not encode antagonists such as the paramyxovirus C proteins, that specifically target RIG-I.

Serological evidence of possible human infection with Tioman virus, a newly described paramyxovirus of bat origin.

Tioman virus, a relatively new paramyxovirus, was isolated from fruit bats (Pteropus species) on Tioman Island, Malaysia, in 2001. The objective of this study was to determine the prevalence of antibodies to T. virus in island inhabitants, by use of comparative ELISA and serum neutralization assays. Of the 169 human sera analyzed, 5 (approximately 3.0%) were positive for T. virus, by comparative ELISA. Of these 5 sera, 3 (1.8% of the total) had neutralizing antibodies against T. virus, suggesting previous infection of this study population by this virus or a similar virus.

An improved method for the recovery of recombinant paramyxovirus vaccine candidates suitable for use in human clinical trials.

We describe a method for the generation of clinical grade, live-attenuated vaccines in Vero cells entirely from cDNA plasmids. The entire electroporation procedure can be completed in less than 15 minutes and this is a significant improvement over previous lipid or electroporation based transfection techniques that also involve a heat-shock step. Importantly, the virus preparations can be generated with a minimal use of animal product derived materials, an important consideration for a vaccine candidate that is to be tested in humans. Since it is likely that all live-attenuated parainfluenza virus and pneumovirus vaccines in the future will be generated using reverse genetics, this simplified method provides guidance on how this can be achieved.

Antibodies to Nipah-like virus in bats (Pteropus lylei), Cambodia.

Serum specimens from fruit bats were obtained at restaurants in Cambodia. We detected antibodies cross-reactive to Nipah virus by enzyme immunoassay in 11 (11.5%) of 96 Lyle's flying foxes (Pteropus lylei). Our study suggests that viruses closely related to Nipah or Hendra viruses are more widespread in Southeast Asia than previously documented.