Serological Evidence for the Circulation of Ebolaviruses in Pigs From Sierra Leone.

Many human ebolavirus outbreaks have been linked to contact with wildlife including nonhuman primates and bats, which are assumed to serve as host species. However, it is largely unknown to what extent other animal species, particularly livestock, are involved in the transmission cycle or act as additional hosts for filoviruses. Pigs were identified as a susceptible host for Reston virus with subsequent transmission to humans reported in the Philippines. To date, there is no evidence of natural Ebola virus (EBOV) infection in pigs, although pigs were shown to be susceptible to EBOV infection under experimental settings. To investigate the potential role of pigs in the ecology of EBOV, we analyzed 400 porcine serum samples from Sierra Leone for the presence of ebolavirus-specific antibodies. Three samples reacted with ebolavirus nucleoproteins but had no neutralizing antibodies. Our results (1) suggest the circulation of ebolaviruses in swine in Sierra Leone that are antigenically related but not identical to EBOV and (2) could represent undiscovered ebolaviruses with unknown pathogenic and/or zoonotic potential.

Nipah virus entry and egress from polarized epithelial cells.

Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal.

Replication, pathogenicity, shedding, and transmission of Zaire ebolavirus in pigs.

UNLABELLED: (See the editorial commentary by Bausch, on pages 179-81.) BACKGROUND: Reston ebolavirus was recently detected in pigs in the Philippines. Specific antibodies were found in pig farmers, indicating exposure to the virus. This important observation raises the possibility that pigs may be susceptible to Ebola virus infection, including from other species, such as Zaire ebolavirus (ZEBOV), and can transmit to other susceptible hosts. METHODS: This study investigated whether ZEBOV, a species commonly reemerging in central Africa, can replicate and induce disease in pigs and can be transmitted to naive animals. Domesticated Landrace pigs were challenged through mucosal exposure with a total of 1 ×10(6) plaque-forming units of ZEBOV and monitored for virus replication, shedding, and pathogenesis. Using similar conditions, virus transmission from infected to naive animals was evaluated in a second set of pigs. RESULTS: Following mucosal exposure, pigs replicated ZEBOV to high titers (reaching 10(7) median tissue culture infective doses/mL), mainly in the respiratory tract, and developed severe lung pathology. Shedding from the oronasal mucosa was detected for up to 14 days after infection, and transmission was confirmed in all naive pigs cohabiting with inoculated animals. CONCLUSIONS: These results shed light on the susceptibility of pigs to ZEBOV infection and identify an unexpected site of virus amplification and shedding linked to transmission of infectious virus.

Treatment with Ad5-Porcine Interferon-α Attenuates Ebolavirus Disease in Pigs.

Under experimental conditions, pigs infected with Ebola Virus (EBOV) develop disease and can readily transmit the virus to non-human primates or pigs. In the event of accidental or intentional EBOV infection of domestic pigs, complex and time-consuming safe depopulation and carcass disposal are expected. Delaying or preventing transmission through a reduction in viral shedding is an absolute necessity to limit the spread of the virus. In this study, we tested whether porcine interferon-α or λ3 (porIFNα or porIFNλ3) delivered by a replication-defective human type 5 adenovirus vector (Ad5-porIFNα or Ad5-porIFNλ3) could limit EBOV replication and shedding in domestic pigs. Our results show that pigs pre-treated with Ad5-porIFNα did not develop measurable clinical signs, did not shed virus RNA, and displayed strongly reduced viral RNA load in tissues. A microarray analysis of peripheral blood mononuclear cells indicated that Ad5-porIFNα treatment led to clear upregulation in immune and inflammatory responses probably involved in protection against disease. Our results indicate that administration of Ad5-porIFNα can potentially be used to limit the spread of EBOV in pigs.

Genetic and pathobiologic characterization of pandemic H1N1 2009 influenza viruses from a naturally infected swine herd.

Since its initial identification in Mexico and the United States, concerns have been raised that the novel H1N1 influenza virus might cause a pandemic of severity comparable to that of the 1918 pandemic. In late April 2009, viruses phylogenetically related to pandemic H1N1 influenza virus were isolated from an outbreak on a Canadian pig farm. This outbreak also had epidemiological links to a suspected human case. Experimental infections carried out in pigs using one of the swine isolates from this outbreak and the human isolate A/Mexico/InDRE4487/2009 showed differences in virus recovery from the lower respiratory tract. Virus was consistently isolated from the lungs of pigs infected with A/Mexico/InDRE4487/2009, while only one pig infected with A/swine/Alberta/OTH-33-8/2008 yielded live virus from the lung, despite comparable amounts of viral RNA and antigen in both groups of pigs. Clinical disease resembled other influenza virus infections in swine, albeit with somewhat prolonged virus antigen detection and delayed viral-RNA clearance from the lungs. There was also a noteworthy amount of genotypic variability among the viruses isolated from the pigs on the farm. This, along with the somewhat irregular pathobiological characteristics observed in experimentally infected animals, suggests that although the virus may be of swine origin, significant viral evolution may still be ongoing.

Investigation of Ebolavirus exposure in pigs presented for slaughter in Uganda.

In 2008, an outbreak of Reston ebolavirus (RESTV) in pigs in the Philippines expanded our understanding of the host range of ebolaviruses. Subsequent experimental infections with the human-pathogenic species Zaire ebolavirus (EBOV) confirmed that pigs are susceptible to African species of ebolaviruses. Pig keeping has become an increasingly important livelihood strategy throughout parts of sub-Saharan Africa, driven by increasing demand for pork. The growth in pig keeping is particularly rapid in Uganda, which has the highest per capita pork consumption in East Africa and a history of sporadic human outbreaks of Ebola virus disease (EVD). Using a systematic sampling protocol, we collected sera from 658 pigs presented for slaughter in Uganda between December 2015 and October 2016. Forty-six pigs (7%) were seropositive based on ELISA tests at two different institutions. Seropositive pigs had antibodies that bound to Sudan NP (n = 27), Zaire NP (Kikwit; n = 8) or both NPs (n = 11). Sera from 4 of the ELISA-positive pigs reacted in Western blot (EBOV NP = 1; RESTV NP = 2; both NPs = 2), and one sample had full neutralizing antibody against Sudan ebolavirus (SUDV) in virus neutralization tests. Pigs sampled in June 2016 were significantly more likely to be seropositive than pigs sampled in October 2016 (p = .03). Seropositive pigs were sourced from all regions except Western region. These observed temporal and spatial variations are suggestive of multiple introductions of ebolaviruses into the pig population in Uganda. This is the first report of exposure of pigs in Uganda to ebolaviruses and the first to employ systematic abattoir sampling for ebolavirus surveillance during a non-outbreak period. Future studies will be necessary to further define the role pigs play (if any) in ebolavirus maintenance and transmission so that potential risks can be mitigated.

1918 and 2009 H1N1 influenza viruses are not pathogenic in birds.

The susceptibility of chickens to both 1918 and 2009 H1N1 influenza virus was evaluated. The intravenous pathogenicity index of 1918 and 2009 H1N1 viruses in chickens was 0. Chickens did not develop clinical signs following experimental inoculation simulating natural infection. No gross pathological changes were observed in any tissues of chickens between 2 and 18 days post-infection (p.i.) and viral RNA was not detected by real-time RT-PCR in mucosal secretions or tissues. Seroconversion was not detected in any of the chickens following inoculation with H1N1 2009 virus, whereas half the chickens developed influenza-specific antibodies at 28 days p.i. with 1918 influenza, suggesting limited infection. Viral RNA was detected by real-time RT-PCR in mallard ducks following inoculation with 1918 influenza virus at 3 days p.i. in cloacal swabs, but not in tissues, and all ducks seroconverted by 28 days p.i. Both 1918 and 2009 H1N1 influenza viruses behave as LPAI in gallinaceous poultry.

Experimental infection of pigs with the human 1918 pandemic influenza virus.

Swine influenza was first recognized as a disease entity during the 1918 "Spanish flu" pandemic. The aim of this work was to determine the virulence of a plasmid-derived human 1918 pandemic H1N1 influenza virus (reconstructed 1918, or 1918/rec, virus) in swine using a plasmid-derived A/swine/Iowa/15/1930 H1N1 virus (1930/rec virus), representing the first isolated influenza virus, as a reference. Four-week-old piglets were inoculated intratracheally with either the 1930/rec or the 1918/rec virus or intranasally with the 1918/rec virus. A transient increase in temperature and mild respiratory signs developed postinoculation in all virus-inoculated groups. In contrast to other mammalian hosts (mice, ferrets, and macaques) where infection with the 1918/rec virus was lethal, the pigs did not develop severe respiratory distress or become moribund. Virus titers in the lower respiratory tract as well as macro- and microscopic lesions at 3 and 5 days postinfection (dpi) were comparable between the 1930/rec and 1918/rec virus-inoculated animals. In contrast to the 1930/rec virus-infected animals, at 7 dpi prominent lung lesions were present in only the 1918/rec virus-infected animals, and all the piglets developed antibodies at 7 dpi. Presented data support the hypothesis that the 1918 pandemic influenza virus was able to infect and replicate in swine, causing a respiratory disease, and that the virus was likely introduced into the pig population during the 1918 pandemic, resulting in the current lineage of the classical H1N1 swine influenza viruses.

Animal models of henipavirus infection: a review.

Hendra virus (HeV) and Nipah virus (NiV) form a separate genus Henipavirus within the family Paramyxoviridae, and are classified as biosafety level four pathogens due to their high case fatality rate following human infection and because of the lack of effective vaccines or therapy. Both viruses emerged from their natural reservoir during the last decade of the 20th century, causing severe disease in humans, horses and swine, and infecting a number of other mammalian species. The current review summarises current published data relating to experimental infection of small and large animals, including the natural reservoir species, the Pteropus bat, with HeV or NiV. Susceptibility to infection and virus distribution in the individual species is discussed, along with the pathogenesis, pathological changes, and potential routes of transmission.

Evidence of exposure to henipaviruses in domestic pigs in Uganda.

Hendra virus (HeV) and Nipah virus (NiV), belonging to the genus Henipavirus, are among the most pathogenic of viruses in humans. Old World fruit bats (family Pteropodidae) are the natural reservoir hosts. Molecular and serological studies found evidence of henipavirus infection in fruit bats from several African countries. However, little is known about the potential for spillover into domestic animals in East Africa, particularly pigs, which served as amplifying hosts during the first outbreak of NiV in Malaysia and Singapore. We collected sera from 661 pigs presented for slaughter in Uganda between December 2015 and October 2016. Using HeV G and NiV G indirect ELISAs, 14 pigs (2%) were seroreactive in at least one ELISA. Seroprevalence increased to 5.4% in October 2016, when pigs were 9.5 times more likely to be seroreactive than pigs sampled in December 2015 (p = 0.04). Eight of the 14 ELISA-positive samples reacted with HeV N antigen in Western blot. None of the sera neutralized HeV or NiV in plaque reduction neutralization tests. Although we did not detect neutralizing antibodies, our results suggest that pigs in Uganda are exposed to henipaviruses or henipa-like viruses. Pigs in this study were sourced from many farms throughout Uganda, suggesting multiple (albeit rare) introductions of henipaviruses into the pig population. We postulate that given the widespread distribution of Old World fruit bats in Africa, spillover of henipaviruses from fruit bats to pigs in Uganda could result in exposure of pigs at multiple locations. A higher risk of a spillover event at the end of the dry season might be explained by higher densities of bats and contact with pigs at this time of the year, exacerbated by nutritional stress in bat populations and their reproductive cycle. Future studies should prioritize determining the risk of spillover of henipaviruses from pigs to people, so that potential risks can be mitigated.

Framework for leadership and training of Biosafety Level 4 laboratory workers.

Construction of several new Biosafety Level 4 (BSL-4) laboratories and expansion of existing operations have created an increased international demand for well-trained staff and facility leaders. Directors of most North American BSL-4 laboratories met and agreed upon a framework for leadership and training of biocontainment research and operations staff. They agreed on essential preparation and training that includes theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experiences relevant to positional responsibilities as essential preparation before a person's independent access to a BSL-4 facility. They also agreed that the BSL-4 laboratory director is the key person most responsible for ensuring that staff members are appropriately prepared for BSL-4 operations. Although standardized certification of training does not formally exist, the directors agreed that facility-specific, time-limited documentation to recognize specific skills and experiences of trained persons is needed.

Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species.

Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.

Use of a SLAM transfected Vero cell line to isolate and characterize marine mammal morbilliviruses using an experimental ferret model.

Two ferrets (Mustela putorius furo) were experimentally infected with phocine distemper virus (PDV), from the 1988 seal epizootic in Europe, in order to determine whether the stable transfected Vero cell line (Vero.DogSLAMtag) expressing canine "signaling lymphocyte activation molecules" (SLAM; CD150) receptors, was more suitable for isolating and characterizing PDV when compared with Vero (American Type Culture Collection # C1008) and primary seal kidney (PSK) cells. Both ferrets displayed characteristic clinical signs of distemper, including fever and rash, 10 days postinoculation (dpi) and, due to increased morbidity, they were euthanized 12 dpi. Histologic lesions, suggestive of infection with morbilliviruses, were observed in tissues from both ferrets, and the tissues stained positive using immunohistochemistry. Isolation of PDV from isolated peripheral blood lymphocytes (PBLs), taken at 5 and 10 dpi, was achieved by cocultivation with Vero and PSK cells, following several passages. Cytopathic effects (CPE) were observed in Vero cell cultures at 29 dpi and in PSK cell cultures at 22 dpi. Phocine distemper virus was isolated from frozen infected ferret lung tissue within 48 hr, when isolation was attempted using the Vero.DogSLAMtag cell line. In addition, a reverse transcriptase polymerase chain reaction (RT-PCR) test was developed to detect a 114 base pair (bp) portion of the nucleocapsid gene found only in PDV. This RT-PCR methodology was used to confirm the identity of the virus subsequently isolated from the ferrets. Viral isolates from the infected ferrets, as well as cultures of virus originally isolated from a dolphin and a porpoise and maintained in Vero cells, also replicated faster and produced higher titers of virus when propagated in Vero.DogSLAMtag cells. These results indicate that Vero.DogSLAMtag cells offer a substantial improvement (including faster viral replication resulting in primary viral isolation in a shorter period of time, and higher yield of virus finally obtained) over traditional cell culture methodologies for isolation and characterization of marine mammal morbilliviruses.

RVFV Infection in Goats by Different Routes of Inoculation.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus of the Phenuiviridae family. Infection causes abortions in pregnant animals, high mortality in neonate animals, and mild to severe symptoms in both people and animals. There is currently an ongoing effort to produce safe and efficacious veterinary vaccines against RVFV in livestock to protect against both primary infection in animals and zoonotic infections in people. To test the efficacy of these vaccines, it is essential to have a reliable challenge model in relevant target species, including ruminants. We evaluated two goat breeds (Nubian and LaMancha), three routes of inoculation (intranasal, mosquito-primed subcutaneous, and subcutaneous) using an infectious dose of 107 pfu/mL, a virus strain from the 2006⁻2007 Kenyan/Sudan outbreak and compared the effect of using virus stocks produced in either mammalian or mosquito cells. Our results demonstrated that the highest and longest viremia titers were achieved in Nubian goats. The Nubian breed was also efficient at producing clinical signs, consistent viremia (peak viremia: 1.2 × 10³â»1.0 × 105 pfu/mL serum), nasal and oral shedding of viral RNA (1.5 × 10¹â»8 × 106 genome copies/swab), a systemic infection of tissues, and robust antibody responses regardless of the inoculation route. The Nubian goat breed and a needle-free intranasal inoculation technique could both be utilized in future vaccine and challenge studies. These studies are important for preventing the spread and outbreak of zoonotic viruses like RVFV and are supported by the Canadian-led BSL4ZNet network.

Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs.

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus in the family Paramyxoviridae. Henipavirus infections were first reported in the 1990's causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus Pteropus have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G) proteins as well as the full-length NiV nucleocapsid (N) protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA) were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses.

Interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus.

Macrophages are an important cellular component of the innate immune system and are normally rapidly recruited and/or activated at the site of virus infection. They can participate in the antiviral response by killing infected cells, by producing antiviral cytokines such as nitric oxide and by producing chemokines and immunoregulatory cytokines that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions. Probiotics, as a part of the normal gut intestinal flora, are important in supporting a functional yet balanced immune system. Improving our understanding of their role in the activation of macrophages and their stimulation of proinflammatory cytokine production in early viral infection was the main goal of this study. Our in vitro model study showed that probiotic bacteria, either from the species Lactobacillus or Bifidobacteria have the ability to decrease viral infection by establishing the antiviral state in macrophages, by production of NO and inflammatory cytokines such as interleukin 6 and interferon-gamma. These effects correlated with the mitochondrial activity of infected macrophages, therefore, the measurements of mitochondrial dehydrogenases activity could be implied as the first indicator of potential inhibitory effects of the probiotics on virus replication. The interactions between probiotic bacteria, macrophages and vesicular stomatitis virus (VSV), markedly depended on the bacterial strain studied.

A novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria.

As shown in many intervention studies, probiotic bacteria can have a beneficial effect on rotavirus and HIV-induced diarrhoea. In spite of that fact, antiviral effects of probiotic bacteria have not been systematically studied yet. Non-tumorigenic porcine intestinal epithelial cells (IPEC-J2) and alveolar macrophages (3D4/2) were treated in different experimental designs with probiotic and other lactic bacteria and their metabolic products. Vesicular stomatitis virus (VSV) was used in the study as a model virus. Cell survival and viral inhibition were determined by antiviral assay and confirmed by immunofluorescence. Pre-incubation of cell monolayers with probiotic bacteria reduced viral infectivity up to 60%. All bacteria used prevented VSV binding to the cell monolayers by direct binding of VSV to their surface. Probiotic and other lactic bacteria prevented viral infection also by establishment of the antiviral state in pre-treated cell monolayers. Probiotic and other lactic bacteria secreted antiviral substances during their growth, as the infectivity of the virus was diminished by 68% when bacterial supernatants were tested. It was shown for the first time that probiotic and other lactic bacteria exhibit an antiviral activity in a cell culture model. Possible mechanisms of antiviral activity include: 1) hindering the adsorption and cell internalisation of the VSV due to the direct trapping of the virus by the bacteria, 2) "cross-talk" with the cells in establishing the antiviral protection and 3) production of metabolites with a direct antiviral effect.

Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus.

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.

Swine alveolar macrophage cell model allows optimal replication of influenza A viruses regardless of their origin.

The importance of pigs in interspecies transmission of influenza A viruses has been repeatedly demonstrated over the last century. Eleven influenza A viruses from avian, human and swine hosts were evaluated for replication phenotypes at three physiologically relevant temperatures (41°C, 37°C, 33°C) in an immortalized swine pulmonary alveolar macrophage cell line (IPAM 3D4/31) to determine whether this system would allow for their efficient replication. All isolates replicated well in IPAMs at 37°C while clear distinctions were observed at 41°C and 33°C, correlating to species of origin of the PB2, reflected in distinct amino acid residue profiles rather than in one particular PB2 residue. A strong TNF-α response was induced by some mammalian but not avian IAVs, while other selected cytokines remained below detection levels. Porcine IPAMs represent a natural host cell model for influenza virus replication where the only condition requiring modification for optimal IAV replication, regardless of virus origin.

Protection against henipaviruses in swine requires both, cell-mediated and humoral immune response.

Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus, within the family Paramyxoviridae. Nipah virus has caused outbreaks of human disease in Bangladesh, Malaysia, Singapore, India and Philippines, in addition to a large outbreak in swine in Malaysia in 1998/1999. Recently, NiV was suspected to be a causative agent of an outbreak in horses in 2014 in the Philippines, while HeV has caused multiple human and equine outbreaks in Australia since 1994. A swine vaccine able to prevent shedding of infectious virus is of veterinary and human health importance, and correlates of protection against henipavirus infection in swine need to be better understood. In the present study, three groups of animals were employed. Pigs vaccinated with adjuvanted recombinant soluble HeV G protein (sGHEV) and challenged with HeV, developed antibody levels considered to be protective prior to the challenge (titers of 320). However, activation of the cell-mediated immune response was not detected, and the animals were only partially protected against challenge with 5×10(5) PFU of HeV per animal. In the second group, cross-neutralizing antibody levels against NiV in the sGHEV vaccinated animals did not reach protective levels, and with no activation of cellular immune memory, these animals were not protected against NiV. Only pigs orally infected with 5×10(4) PFU of NiV per animal were protected against nasal challenge with 5×10(5) PFU of NiV per animal. This group of pigs developed protective antibody levels, as well as cell-mediated immune memory. Peripheral blood mononuclear cells restimulated with UV-inactivated NiV upregulated IFN-gamma, IL-10 and the CD25 activation marker on CD4(+)CD8(+) T memory helper cells and to lesser extent on CD4(-)CD8(+) T cells. In conclusion, both humoral and cellular immune responses were required for protection of swine against henipaviruses.