Isolation and Full-Genome Characterization of Nipah Viruses from Bats, Bangladesh.

Despite molecular and serologic evidence of Nipah virus in bats from various locations, attempts to isolate live virus have been largely unsuccessful. We report isolation and full-genome characterization of 10 Nipah virus isolates from Pteropus medius bats sampled in Bangladesh during 2013 and 2014.

Experimental Infection and Response to Rechallenge of Alpacas with Middle East Respiratory Syndrome Coronavirus.

We conducted a challenge/rechallenge trial in which 3 alpacas were infected with Middle East respiratory syndrome coronavirus. The alpacas shed virus at challenge but were refractory to further shedding at rechallenge on day 21. The trial indicates that alpacas may be suitable models for infection and shedding dynamics of this virus.

Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction.

BACKGROUND: Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 2.3.4.4 (previously provisionally referred to as clade 2.3.4.6), and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms. FINDINGS: Fluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade 2.3.4.4 viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade 2.3.4.4 from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes. CONCLUSIONS: Laos influenza A(H5N6) virus representative of proposed clade 2.3.4.4, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.

Molecular evidence of Ebola Reston virus infection in Philippine bats.

BACKGROUND: In 2008-09, evidence of Reston ebolavirus (RESTV) infection was found in domestic pigs and pig workers in the Philippines. With species of bats having been shown to be the cryptic reservoir of filoviruses elsewhere, the Philippine government, in conjunction with the Food and Agriculture Organization of the United Nations, assembled a multi-disciplinary and multi-institutional team to investigate Philippine bats as the possible reservoir of RESTV. METHODS: The team undertook surveillance of bat populations at multiple locations during 2010 using both serology and molecular assays. RESULTS: A total of 464 bats from 21 species were sampled. We found both molecular and serologic evidence of RESTV infection in multiple bat species. RNA was detected with quantitative PCR (qPCR) in oropharyngeal swabs taken from Miniopterus schreibersii, with three samples yielding a product on conventional hemi-nested PCR whose sequences differed from a Philippine pig isolate by a single nucleotide. Uncorroborated qPCR detections may indicate RESTV nucleic acid in several additional bat species (M. australis, C. brachyotis and Ch. plicata). We also detected anti-RESTV antibodies in three bats (Acerodon jubatus) using both Western blot and ELISA. CONCLUSIONS: The findings suggest that ebolavirus infection is taxonomically widespread in Philippine bats, but the evident low prevalence and low viral load warrants expanded surveillance to elaborate the findings, and more broadly, to determine the taxonomic and geographic occurrence of ebolaviruses in bats in the region.

Hendra virus vaccine, a one health approach to protecting horse, human, and environmental health.

In recent years, the emergence of several highly pathogenic zoonotic diseases in humans has led to a renewed emphasis on the interconnectedness of human, animal, and environmental health, otherwise known as One Health. For example, Hendra virus (HeV), a zoonotic paramyxovirus, was discovered in 1994, and since then, infections have occurred in 7 humans, each of whom had a strong epidemiologic link to similarly affected horses. As a consequence of these outbreaks, eradication of bat populations was discussed, despite their crucial environmental roles in pollination and reduction of the insect population. We describe the development and evaluation of a vaccine for horses with the potential for breaking the chain of HeV transmission from bats to horses to humans, thereby protecting horse, human, and environmental health. The HeV vaccine for horses is a key example of a One Health approach to the control of human disease.

Promotion of Hendra virus replication by microRNA 146a.

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

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-ß response than HeV.

Ebola Reston virus infection of pigs: clinical significance and transmission potential.

In 2008, Reston ebolavirus (REBOV) was isolated from pigs during a disease investigation in the Philippines. Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2) infections were also confirmed in affected herds and the contribution of REBOV to the disease outbreak remains uncertain. We have conducted experimental challenge studies in 5-week-old pigs, with exposure of animals to 10(6) TCID(50) of a 2008 swine isolate of REBOV via either the oronasal or subcutaneous route. Replication of virus in internal organs and viral shedding from the nasopharynx were documented in the absence of clinical signs of disease in infected pigs. These observations confirm not only that asymptomatic infection of pigs with REBOV occurs, but that animals so affected pose a transmission risk to farm, veterinary, and abattoir workers.

Experimental infection of horses with Hendra virus/Australia/horse/2008/Redlands.

Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus harbored by Australian flying foxes with sporadic spillovers directly to horses. Although the mode and critical control points of HeV spillover to horses from flying foxes, and the risk for transmission from infected horses to other horses and humans, are poorly understood, we successfully established systemic HeV disease in 3 horses exposed to Hendra virus/Australia/Horse/2008/Redlands by the oronasal route, a plausible route for natural infection. In 2 of the 3 animals, HeV RNA was detected continually in nasal swabs from as early as 2 days postexposure, indicating that systemic spread of the virus may be preceded by local viral replication in the nasal cavity or nasopharynx. Our data suggest that a critical factor for reducing HeV exposure risk to humans includes early consideration of HeV in the differential diagnosis and institution of appropriate infection control procedures.

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.

Chloroquine administration does not prevent Nipah virus infection and disease in ferrets.

Hendra virus and Nipah virus, two zoonotic paramyxoviruses in the genus Henipavirus, have recently emerged and continue to cause sporadic disease outbreaks in humans and animals. Mortality rates of up to 75% have been reported in humans, but there are presently no clinically licensed therapeutics for treating henipavirus-induced disease. A recent report indicated that chloroquine, used in malaria therapy for over 70 years, prevented infection with Nipah virus in vitro. Chloroquine was assessed using a ferret model of lethal Nipah virus infection and found to be ineffective against Nipah virus infection in vivo.

Reagents for detection of Rift Valley fever virus infection in sheep.

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

Real-time RT-PCR for detection of equine influenza and evaluation using samples from horses infected with A/equine/Sydney/2007 (H3N8).

Equine influenza (EI) virus (H3N8) was identified in the Australian horse population for the first time in August 2007. The principal molecular diagnostic tool used for detection was a TaqMan real-time reverse transcription-polymerase chain reactions (RT-PCR) assay specific for the matrix (MA) gene of influenza virus type A (IVA). As this assay is not specific for EI, we developed a new EI H3-specific TaqMan assay targeting the haemagglutinin (HA) gene of all recent EI H3 strains. The IVA and the EI H3 TaqMan assays were assessed using in vitro transcribed RNA template, virus culture, diagnostic samples from the outbreak and samples from experimentally infected horses. The EI H3 TaqMan assay had a higher diagnostic sensitivity (DSe) when compared to the IVA TaqMan assay and also when using a conventional PCR for EI H3 as a standard of comparison. The performance of both TaqMan assays was compared with an antigen detection ELISA and virus isolation using nasal swabs collected daily from horses experimentally infected with the outbreak strain A/equine/Sydney/2888-8/2007. The EI H3 TaqMan assay was the most sensitive of the assays, able to detect EI from day 1 or 2 post-challenge, as early as virus isolation, and before clinical signs of disease were observed.

Animal infection studies of two recently discovered African bat paramyxoviruses, Achimota 1 and Achimota 2.

Bats are implicated as the natural reservoirs for several highly pathogenic viruses that can infect other animal species, including man. Here, we investigate the potential for two recently discovered bat rubulaviruses, Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2), isolated from urine collected under urban bat (Eidolon helvum) roosts in Ghana, West Africa, to infect small laboratory animals. AchPV1 and AchPV2 are classified in the family Paramyxoviridae and cluster with other bat derived zoonotic rubulaviruses (i.e. Sosuga, Menangle and Tioman viruses). To assess the susceptibility of AchPV1 and AchPV2 in animals, infection studies were conducted in ferrets, guinea pigs and mice. Seroconversion, immunohistological evidence of infection, and viral shedding were identified in ferrets and guinea pigs, but not in mice. Infection was associated with respiratory disease in ferrets. Viral genome was detected in a range of tissues from ferrets and guinea pigs, however virus isolation was only achieved from ferret tissues. The results from this study indicate Achimota viruses (AchPVs) are able to cross the species barrier. Consequently, vigilance for infection with and disease caused by these viruses in people and domesticated animals is warranted in sub-Saharan Africa and the Arabian Peninsula where the reservoir hosts are present.

Production and application of recombinant antibodies to foot-and-mouth disease virus non-structural protein 3ABC.

The stamping out of animals to control a foot-and-mouth disease (FMD) outbreak results in enormous livestock losses. The implementation of vaccination strategies can reduce these losses; however it complicates the process of establishing freedom from disease following an outbreak. The availability of quality diagnostic tests to differentiate infected from vaccinated animals (DIVA) is crucial to prove freedom from disease and allow for the resumption of trade in livestock products. All current foot-and-mouth disease virus (FMDV) DIVA tests rely on polyclonal or monoclonal hybridoma derived antibody reagents, which can be difficult to prepare and maintain in a quality-assured manner and in the quantities required for post-outbreak surveillance. Recombinant antibodies can be produced in large quantities at low cost in bacteria to guarantee the supply of a consistent and well-characterised reagent. The production of recombinant antibodies does not rely on animal immunisation and does not require the maintenance of viable hybridoma cell lines. In this study, phage display libraries of recombinant antibody single chain variable fragments (scFv) against FMDV were generated from chickens immunised with recombinant non-structural protein (NSP) 3ABC. A total of 32 positive clones were obtained that represented three distinctive genetic sequences, Chicken Recombinant Antibody-Foot-and-Mouth disease (CRAb-FM) 26, -FM27 and -FM29. Each was shown to bind the 3B region of the 3ABC protein. When evaluated in a C-ELISA format using sera derived from cattle, sheep and pigs representing naïve, FMDV-vaccinated or FMDV-infected animals, CRAb-FM27 gave the best performance when paired with an E. coli-derived recombinant 3ABC, demonstrating the potential to be used as a species- and serotype-independent FMDV DIVA test. To our knowledge, this is the first FMDV DIVA test that uses both recombinant antibody and antigen derived from bacterial expression systems.

Flavivirus detection and differentiation by a microsphere array assay.

Flaviviruses of the Japanese encephalitis virus (JEV) serocomplex include major human and animal pathogens that have a propensity to spread and emerge in new geographic areas. Different genotypes or genetic lineages have been defined for many of these viruses, and they are distributed worldwide. Tools enabling rapid detection of new or emerging flaviviruses and differentiation of important subgroups have widespread application for arbovirus diagnosis and surveillance, and are crucial for detecting virus incursions, tracking virus emergence and for disease control. A microsphere suspension array assay was developed to identify JEV serocomplex flaviviruses of medical and veterinary importance. Assay performance was evaluated using representative virus strains as well as clinical and surveillance samples. The assay detected all JEV serocomplex viruses tested in this study with an apparent analytical sensitivity equal or better than the reference real-time or conventional RT-PCR assays and was able to identify mixed virus populations. The ability to identify mixed virus populations at a high analytical sensitivity would be pertinent in the Australian context when attempting to detect exotic JEV or West Nile virus (WNV), and differentiate from endemic Murray Valley encephalitis virus and WNV-Kunjin virus. The relatively low cost, the ability to identify mixed virus populations and the multiplex nature makes this assay valuable for a wide range of applications including diagnostic investigations, virus exclusions, and surveillance programs.

Microsphere suspension array assays for detection and differentiation of Hendra and Nipah viruses.

Microsphere suspension array systems enable the simultaneous fluorescent identification of multiple separate nucleotide targets in a single reaction. We have utilized commercially available oligo-tagged microspheres (Luminex MagPlex-TAG) to construct and evaluate multiplexed assays for the detection and differentiation of Hendra virus (HeV) and Nipah virus (NiV). Both these agents are bat-borne zoonotic paramyxoviruses of increasing concern for veterinary and human health. Assays were developed targeting multiple sites within the nucleoprotein (N) and phosphoprotein (P) encoding genes. The relative specificities and sensitivities of the assays were determined using reference isolates of each virus type, samples from experimentally infected horses, and archival veterinary diagnostic submissions. Results were assessed in direct comparison with an established qPCR. The microsphere array assays achieved unequivocal differentiation of HeV and NiV and the sensitivity of HeV detection was comparable to qPCR, indicating high analytical and diagnostic specificity and sensitivity.

Hendra virus detection using Loop-Mediated Isothermal Amplification.

Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical.

Improvement of a recombinant antibody-based serological assay for foot-and-mouth disease virus.

Differentiating foot-and-mouth disease virus (FMDV) antibodies generated during a natural infection from those due to vaccination (DIVA) is crucial for proving freedom from disease after an outbreak and allowing resumption of trade in livestock products. The World Organisation for Animal Health (OIE) recommends that FMDV vaccines are composed of inactivated virus that has been purified to remove non-structural viral proteins. Such purified vaccines primarily induce antibodies to viral structural proteins, whereas replicating virus stimulates host antibodies specific for both structural and non-structural proteins. The current preferred FMDV DIVA test is a competitive ELISA (C-ELISA) designed to detect antibodies to the non-structural protein 3ABC. Previously, we described the development of an FMDV DIVA test based entirely on recombinant proteins (the recombinant detecting antibody and the 3ABC coating antigen) produced in Escherichia coli. In this study, we have determined the precise binding site of the recombinant detecting antibody to a conserved sequence within the 3B region of the 3ABC protein, replaced the original E-tag of the detecting antibody with two in-house tags and engineered a direct antibody-reporting enzyme (alkaline phosphatase) fusion protein. These modifications have further improved the DIVA test, providing great potential for large scale production and uptake due to its simplicity, reproducibility and low cost.