Jennifer Harper

Novel Paramyxoviruses in Free-Ranging European Bats

The zoonotic potential of paramyxoviruses is particularly demonstrated by their broad host range like the highly pathogenic Hendra and Nipah viruses originating from bats. But while so far all bat-borne paramyxoviruses have been identified in fruit bats across Africa, Australia, South America, and Asia, we describe the detection and characterization of the first paramyxoviruses in free-ranging European bats. Moreover, we examined the possible impact of paramyxovirus infection on individual animals by comparing histo-pathological findings and virological results. Organs from deceased insectivorous bats of various species were sampled in Germany and tested for paramyxovirus RNA in parallel to a histo-pathological examination. Nucleic acids of three novel paramyxoviruses were detected, two viruses in phylogenetic relationship to the recently proposed genus Jeilongvirus and one closely related to the genus Rubulavirus. Two infected animals revealed subclinical pathological changes within their kidneys, suggestive of a similar pathogenesis as the one described in fruit bats experimentally infected with Hendra virus. Our findings indicate the presence of bat-born paramyxoviruses in geographic areas free of fruit bat species and therefore emphasize a possible virus–host co-evolution in European bats. Since these novel viruses are related to the very distinct genera Rubulavirus and Jeilongvirus, a similarly broad genetic diversity among paramyxoviruses in other Microchiroptera compared to Megachiroptera can be assumed. Given that the infected bats were either found in close proximity to heavily populated human habitation or areas of intensive agricultural use, a potential risk of the emergence of zoonotic paramyxoviruses in Europe needs to be considered.

The pathobiology of two Indonesian H5N1 avian influenza viruses representing different clade 2.1 sublineages in chickens and ducks

To determine the pathobiology of Indonesian H5N1 highly pathogenic avian influenza, two viruses representing clades 2.1.1 and 2.1.3 were inoculated into broiler chickens and Pekin ducks via the eyes, nostrils and oropharynx. In chickens, both viruses produced fulminant disease; tissue tropism was broad but predominantly endothelial and viral loads in tissues were high. Except for one case of meningoencephalitis, the infection in ducks was sub-clinical, leading only to seroconversion. In these ducks, virus and viral antigen occurred in lower amounts, mainly in the respiratory tract (airsac and sinuses), prior to day 7 after inoculation. During clinical disease, chickens shed high virus titres orally and cloacally. Ducks intermittently shed low virus titres from the oral route for up to 8 days post-inoculation. We discuss the significance of the data for understanding the pathogenesis and pathobiology of Indonesian H5N1 in chickens and ducks.

Transcriptome analysis of pigeon milk production - role of cornification and triglyceride synthesis genes.

BackgroundThe pigeon crop is specially adapted to produce milk that is fed to newly hatched young. The process of pigeon milk production begins when the germinal cell layer of the crop rapidly proliferates in response to prolactin, which results in a mass of epithelial cells that are sloughed from the crop and regurgitated to the young. We proposed that the evolution of pigeon milk built upon the ability of avian keratinocytes to accumulate intracellular neutral lipids during the cornification of the epidermis. However, this cornification process in the pigeon crop has not been characterised.ResultsWe identified the epidermal differentiation complex in the draft pigeon genome scaffold and found that, like the chicken, it contained beta-keratin genes. These beta-keratin genes can be classified, based on sequence similarity, into several clusters including feather, scale and claw keratins. The cornified cells of the pigeon crop express several cornification-associated genes including cornulin, S100-A9 and A16-like, transglutaminase 6-like and the pigeon ‘lactating’ crop-specific annexin cp35. Beta-keratins play an important role in ‘lactating’ crop, with several claw and scale keratins up-regulated. Additionally, transglutaminase 5 and differential splice variants of transglutaminase 4 are up-regulated along with S100-A10.ConclusionsThis study of global gene expression in the crop has expanded our knowledge of pigeon milk production, in particular, the mechanism of cornification and lipid production. It is a highly specialised process that utilises the normal keratinocyte cellular processes to produce a targeted nutrient solution for the young at a very high turnover.

Australian bat lyssavirus infection in two horses

In May 2013, the first cases of Australian bat lyssavirus infections in domestic animals were identified in Australia. Two horses (filly-H1 and gelding-H2) were infected with the Yellow-bellied sheathtail bat (YBST) variant of Australian bat lyssavirus (ABLV). The horses presented with neurological signs, pyrexia and progressing ataxia. Intra-cytoplasmic inclusion bodies (Negri bodies) were detected in some Purkinje neurons in haematoxylin and eosin (H&E) stained sections from the brain of one of the two infected horses (H2) by histological examination. A morphological diagnosis of sub-acute moderate non-suppurative, predominantly angiocentric, meningo-encephalomyelitis of viral aetiology was made. The presumptive diagnosis of ABLV infection was confirmed by the positive testing of the affected brain tissue from (H2) in a range of laboratory tests including fluorescent antibody test (FAT) and real-time PCR targeting the nucleocapsid (N) gene. Retrospective testing of the oral swab from (H1) in the real-time PCR also returned a positive result. The FAT and immunohistochemistry (IHC) revealed an abundance of ABLV antigen throughout the examined brain sections. ABLV was isolated from the brain (H2) and oral swab/saliva (H1) in the neuroblastoma cell line (MNA). Alignment of the genome sequence revealed a 97.7% identity with the YBST ABLV strain.

Evaluation of a mouse model for the West Nile virus group for the purpose of determining viral pathotypes

West Nile virus (WNV; family Flaviviridae; genus Flavivirus) group members are an important cause of viral meningoencephalitis in some areas of the world. They exhibit marked variation in pathogenicity, with some viral lineages (such as those from North America) causing high prevalence of severe neurological disease, whilst others (such as Australian Kunjin virus) rarely cause disease. The aim of this study was to characterize WNV disease in a mouse model and to elucidate the pathogenetic features that distinguish disease variation. Tenfold dilutions of five WNV strains (New York 1999, MRM16 and three horse isolates of WNV-Kunjin: Boort and two isolates from the 2011 Australian outbreak) were inoculated into mice by the intraperitoneal route. All isolates induced meningoencephalitis in different proportions of infected mice. WNVNY99 was the most pathogenic, the three horse isolates were of intermediate pathogenicity and WNVKUNV-MRM16 was the least, causing mostly asymptomatic disease with seroconversion. Infectivity, but not pathogenicity, was related to challenge dose. Using cluster analysis of the recorded clinical signs, histopathological lesions and antigen distribution scores, the cases could be classified into groups corresponding to disease severity. Metrics that were important in determining pathotype included neurological signs (paralysis and seizures), meningoencephalitis, brain antigen scores and replication in extra-neural tissues. Whereas all mice infected with WNVNY99 had extra-neural antigen, those infected with the WNV-Kunjin viruses only occasionally had antigen outside the nervous system. We conclude that the mouse model could be a useful tool for the assessment of pathotype for WNVs.

Menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs

This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin that causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, the virus was only isolated from affected fetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in 6-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of 2-3 days, virus was shed in nasal and oral secretions, faeces and urine, typically for less than 1 week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programmes should it ever re-emerge in the pig population.

Novel Reassortant H5N6 Influenza A Virus from the Lao People’s Democratic Republic Is Highly Pathogenic in Chickens

Avian influenza viruses of H5 subtype can cause highly pathogenic disease in poultry. In March 2014, a new reassortant H5N6 subtype highly pathogenic avian influenza virus emerged in Lao People’s Democratic Republic. We have assessed the pathogenicity, pathobiology and immunological responses associated with this virus in chickens. Infection caused moderate to advanced disease in 6 of 6 chickens within 48 h of mucosal inoculation. High virus titers were observed in blood and tissues (kidney, spleen, liver, duodenum, heart, brain and lung) taken at euthanasia. Viral antigen was detected in endothelium, neurons, myocardium, lymphoid tissues and other cell types. Pro-inflammatory cytokines were elevated compared to non-infected birds. Our study confirmed that this new H5N6 reassortant is highly pathogenic, causing disease in chickens similar to that of Asian H5N1 viruses, and demonstrated the ability of such clade 2.3.4-origin H5 viruses to reassort with non-N1 subtype viruses while maintaining a fit and infectious phenotype. Recent detection of influenza H5N6 poultry infections in Lao PDR, China and Viet Nam, as well as six fatal human infections in China, demonstrate that these emergent highly pathogenic H5N6 viruses may be widely established in several countries and represent an emerging threat to poultry and human populations.

Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes

ABSTRACT Although avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease. IMPORTANCE Risks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated liquid. We also showed that virus could spread from the respiratory tract to the brain, which was associated with end-stage disease, and very early viremia provided a marker for this. With upper respiratory tract exposure, infection of the brain was common but hard to detect, suggesting that human neurological infections might be typically undetected at autopsy. However, viral spread to systemic sites was slower after exposure to virus by this route than when virus was additionally delivered to the lungs, providing a better therapeutic window. In addition to exposure history, early parameters of infection, such as viremia, could help prioritize antiviral treatments for patients most at risk of succumbing to infection.

Evolution of high pathogenicity of H5 avian influenza virus: haemagglutinin cleavage site selection of reverse-genetics mutants during passage in chickens

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.

An Australian Newcastle disease virus with a virulent fusion protein cleavage site produces minimal pathogenicity in chickens

Newcastle disease is an important disease of poultry caused by virulent strains of Newcastle disease virus (NDV). During the 1998 to 2002 outbreaks of Newcastle disease in Australia, it was observed that the mild clinical signs seen in some chickens infected with NDV did not correlate with the viruses’ virulent fusion protein cleavage site motifs or standard pathogenicity indices. The pathogenicity of 2 Australian NDV isolates was evaluated in experimentally challenged chickens based on clinical evaluation, histopathology, immunohistochemistry, and molecular techniques. One of these virus isolates, Meredith/02, was shown to induce only very mild clinical signs with no mortalities in an experimental setting, in contrast to the velogenic Herts 33/56 and Texas GB isolates. This minimal pathogenicity was associated with decreased virus replication and antigen distribution in tissues. This demonstrates that the Australian Meredith/02 NDV, despite possessing a virulent fusion protein cleavage site, did not display a velogenic phenotype.