Rapid, sensitive, and specific, low-resource molecular detection of Hendra virus.

Efficient and accurate diagnosis of Hendra virus (HeV), a biosafety level 4 (BSL-4) pathogen and zoonotic disease, is of primary importance for surveillance and outbreak control in the Australian equine industry. Sporadic HeV spillover events pose a serious public health concern and are predicted to expand geographically, aligning with the moving distribution of the main reservoir hosts, the flying-foxes. Here we describe the development of a low-resource rapid Hendra test. The test used a fast and simple sample processing protocol followed by reverse transcription isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results were obtained in 30 min and required only a heating block, ice, and pipettes for liquid handling. The one-step sample processing protocol inactivated HeV in 2 min, providing a simple protocol that could enable safe testing outside of a laboratory. Analytical sensitivity testing demonstrated a detection limit of 1000 copies/µL of synthetic HeV RNA, and analytical specificity testing indicated assays did not detect other pathogens. Gamma-irradiated HeV-spiked in viral transport medium was detected with high sensitivity, down to 10,000 TCID50/mL, the equivalent of 18 RNA copies per reaction. Collectively, our data suggests that our rapid Hendra test offers a potential first-line screening on-site alternative to gold-standard RT-PCR detection, which requires samples to be shipped to central containment laboratories, thermocyclers and labour-intensive viral RNA purification, with testing time of approximately four hours. Our rapid Hendra test provided performance and speed without compromising sensitivity and specificity, and could become a promising more accessible tool for testing under resource-limited conditions for the veterinary community and thoroughbred industry.

Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam.

Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health.

Henipaviruses: an updated review focusing on the pteropid reservoir and features of transmission.

The henipaviruses, Hendra virus and Nipah virus, are pathogens that have emerged from flying foxes in Australia and South-east Asia to infect both livestock and humans, often fatally. Since the emergence of Hendra virus in Australia in 1994 and the identification of Australian flying foxes as hosts to this virus, our appreciation of bats as reservoir hosts of henipaviruses has expanded globally to include much of Asia and areas of Africa. Despite this, little is currently known of the mechanisms by which bats harbour viruses capable of causing such severe disease in other terrestrial mammals. Pteropid bat ecology, henipavirus virology, therapeutic developments and features of henipavirus infection, pathology and disease in humans and other mammals are reviewed elsewhere in detail. This review focuses on bats as reservoir hosts to henipaviruses and features of transmission of Hendra virus and Nipah virus following spillover from these reservoir hosts.

Conservation of a unique mechanism of immune evasion across the Lyssavirus genus.

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.

Design and evaluation of consensus PCR assays for henipaviruses.

Henipaviruses were first discovered in the 1990s, and their potential threat to public health is of increasing concern with increasing knowledge. Old-world fruit bats are the reservoir hosts for these viruses, and spill-over events cause lethal infections in a wide range of mammalian species, including humans. In anticipation of these spill-over events, and to investigate further the geographical range of these genetically diverse viruses, assays for detection of known and potentially novel strains of henipaviruses are required. The development of multiple consensus PCR assays for the detection of henipaviruses, including both SYBR Green and TaqMan real-time PCRs and a conventional heminested PCR is described. The assays are highly sensitive and have defined specificity. In addition to being useful tools for detection of known and novel henipaviruses, evaluation of assay efficiency and sensitivity across both biological and synthetic templates has provided valuable insight into consensus PCR design and use.

The role of reverse genetics in the development of vaccines against respiratory viruses.

Despite their significance, the only available vaccines against respiratory viruses are those for the prevention of influenza. Attempts have been made to produce vaccines against other respiratory viruses using traditional techniques, but have met with little success. Reverse genetics, although still a relatively new tool for the manipulation of negative-strand RNA viruses, has great potential for the preparation of vaccines against many of the common respiratory viruses. In the preparation of live vaccines, reverse genetics systems allow the direct modification of the specific regions in the genomes of negative-stranded RNA viruses concerned with attenuation; the ultimate goal is the introduction of site-specific mutations through a cDNA intermediate in order to develop strains with the requisite attenuation, antigenic and growth properties needed in a vaccine. These techniques can also be used to disarm potentially highly pathogenic viruses, such as emerging H5N1 avian influenza viruses, in order to facilitate large-scale preparation of viruses for use in inactivated vaccines under conditions of manufacturing safety. Before these vaccines become available, residual issues concerned with intellectual property rights to the technology and its application will need to be resolved.

An evaluation of the genetic stability and pathogenicity of the Russian cold-adapted influenza A donor strains A/Leningrad/134/17/57 and A/Leningrad/134/47/57 in ferrets.

The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.

Preparation and characterisation of attenuated cold-adapted influenza A reassortants derived from the A/Leningrad/134/17/57 donor strain.

The development of a rapid cell culture method for the preparation of cold-adapted (ca) influenza A reassortant viruses is described and compared with a currently used egg method. Mixtures of the ca donor A/Leningrad/134/17/57-ca (A/Len/17) and A/Beijing/32/92 (A/Beij/32), a recent H3N2 epidemic strain, were used to co-infect chicken embryo kidney (CEK) cell cultures; reassortant progeny were selected using an infectious centre assay. The assay was capable of detecting interference where the infectivity ratio for A/Len/17 and A/Beij/32 was 1:7. Progeny viruses were characterised genetically by amplification of defined regions within each of the six internal genes by PCR and identification of the products by restriction enzyme analysis. Reassortants were also tested for ca and temperature-sensitive (ts) phenotype and the identity of the surface antigens. The infectious centre assay was shown to be an effective method for isolating reassortant progeny that possessed the haemagglutinin and neuraminidase surface antigens of A/Beij/32. Reassortants with the six internal genes derived from the donor strain and possessing the ca and ts phenotype were readily obtained when (a) an infectivity ratio of 1:49 was used and (b) two plaque-to-plaque isolations of progeny virus were made after growth at 25 degrees C and one at 34 degrees C, both in the presence of antiserum to the donor strain.

Effectiveness of low sodium diets for recycling of egg production type hens.

Egg type hens were recycled by the use of low sodium diet treatments compared to a conventional forced-molt procedure and an unrecycled control. Use of a low sodium diet containing .02 to .06% sodium for 6 weeks with reduction in daily photoperiod resulted in improvements in egg production, egg specific gravity, and albumen thickness similar to those of a forced-molt group in three separate experiments. Egg production was increased 11 to 13%, egg specific gravity was increased by .002 to .004, and albumen thickness was increased by 2 to 8 Haugh units over the 32-week posttreatment period for both treatments. Hens fed the low sodium diet for 3.5 or 4 weeks did not respond as favorably as hens fed this diet for 6 weeks. Eight weeks on the low sodium diet did not further improve performance. Results comparable to the forced-molt procedure were achieved with a decline in egg production at .03 to .07% sodium in the diet, a decline in feed intake at .03 to .07% sodium, a loss in body weight at .03 to .10% sodium, and an increase in molt score at .03 to .11% sodium during the experimental period. During the posttreatment period, results comparable to the forced-molt procedure were obtained for egg production increase at .03 to .08% sodium, for egg specific gravity increase at .03 to .12% sodium, and for egg albumen thickness increase at .03 to .12% dietary sodium. Mortality was unchanged.