Michelle Nelson

Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis

Dengue virus (DV) is the most widespread arbovirus, being endemic in over 100 countries, and is estimated to cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain can play a role in determining the virus virulence and subsequent clinical disease severity. Virus vector competence plays an integral role in virus transmission and is a critical factor in determining the severity and impact of DV outbreaks. Host genetic variations in immune-related genes, including the human leukocyte antigen, have also been shown to correlate with clinical disease and thus may play a role in regulating disease severity. The hosts immune system, however, appears to be the primary factor in DV pathogenesis with the delicate interplay of innate and acquired immunity playing a crucial role. Although current research of DV pathogenesis has been limited by the lack of an appropriate animal model, the development of DV therapeutics has been a primary focus of research groups around the world. In the past decade advances in both the development of vaccines and anti-virals have increased in dramatically. This review summarises the current understanding of viral, vector and host factors which contribute to dengue virus pathogenesis and how this knowledge is critically important in the development of pharmaceutical interventions.

Pulmonary infection of mice with human metapneumovirus induces local cytotoxic T-cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory-tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory-tract infection with hMPV, we demonstrate the accumulation of gamma interferon (IFN-gamma)-producing CD8+ T cells in the airways and lungs at day 7 post-infection (p.i.), associated with cytotoxic T lymphocytes (CTLs) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th1 cytokines IFN-gamma and interleukin (IL)-12 and antiviral cytokines (IFN-beta), as well as chemokines Mip-1alpha, Mip-1beta, Mig, IP-10 and CX3CL1. There was also a moderate increase in Th2-type cytokines IL-4 and IL-10 compared with uninfected mice. At 21 days p.i., a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response to the two related pathogens.

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus

ABSTRACT The alphaviral 6k gene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the 6k proteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel 6k in-frame deletion mutant. Comprehensive microscopic analysis revealed that the 6k proteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the 6k proteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the 6k deletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequent in vivo studies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the 6k proteins may contribute to alphaviral disease manifestations and suggest that manipulation of the 6k gene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV 6k deletion mutant. The absence of the 6k gene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

Recovery and identification of bacterial DNA from illicit drugs

Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002-2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated. Methods based on the ChargeSwitch(®)gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS). An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch(®) method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed. This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug users as well as enabling forensic links between seizures to be examined.

Dual Proinflammatory and Antiviral Properties of Pulmonary Eosinophils in Respiratory Syncytial Virus Vaccine-Enhanced Disease

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCE This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.

Role of envelope N-linked glycosylation in Ross River virus virulence and transmission.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αβ) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

Radical sequestration by protein-bound 3,4-dihydroxyphenylalanine

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA), a redox-active product of protein oxidation, is capable of functioning as both a pro- and antioxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Being the major treatment available for Parkinsons disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. We hypothesise that the generation of PB-DOPA can trigger an enhancement of the cellular antioxidant defence system, thus enabling PB-DOPA to restrict and potentially terminate the initiating oxidative stress, minimising the level of oxidative damage. Using luminol-enhanced chemiluminescence, we demonstrate that free DOPA is capable of direct peroxyl radical scavenging, even in the presence of competing scavengers, and has a different effect to that of the parent amino acid, tyrosine. Furthermore, we show that both free and PB-DOPA, in combination or individually, were able to protect monocytes and macrophages from peroxyl radical-induced oxidative stress in vitro. These results confirm a role for both free and PB-DOPA in cellular antioxidant defences and suggest the possibility of using DOPA as a potential therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage.

An overview of biosecurity in Australia

Threats of natural and deliberate biological agent release are very real and are of serious concern within Australia and internationally, having the potential to substantially impact human, plant and animal health as well as agriculture, trade relations, infrastructure and the economy. Biosecurity programmes and policies are essential for counteracting these threats and enabling a nation to effectively prevent and/or respond to the natural, accidental and deliberate release of a biological agent. The major biosecurity focus in Australia is the protection against invasive animal, plant and insect species; however, more recently, the threat of microbiological agents has become a priority for Australia’s national security. Australia’s biosecurity capabilities are maintained through a coordinated effort, involving national, state and local government and non-government agencies. Communication and collaboration between all parties, at both a national and international level, is the key to an effective response. The threat from biological agents is a very real concern and Australia must maintain its preparedness in order to ensure its capacity to respond to biological threats. This review provides a contemporary overview of biosecurity in Australia and the role of forensic science in the investigation of biosecurity incidents.

Mechanisms of Chikungunya virus disease informed by Ross River virus research

Chikungunya virus (CHIKV) was first isolated in Tanzania in 1953 [1]. Since then, CHIKV outbreaks have occurred sporadically in SubSaharan Africa [2], southeast Asia [3] and India [4]. Most recently, in 2005/2006, a CHIKV outbreak was reported in a number of islands in the western Indian Ocean, which has since spread into India, giving rise to the largest CHIKV epidemic on record [5]. Reports of CHIKV cases in Italy, a more temperate environment, have suggested the potential for this virus to spread worldwide [6]. CHIKV, an Alphavirus belonging to the Togaviridae family, is maintained by transmission cycles between mosquito vectors and vertebrate hosts. Whilst CHIKV disease is generally not fatal, it can cause debilitating symptoms including fever, rash, headache, myalgia and arthralgia. Some more severe cases have been described, for example, in neonates where infection has been found to involve the CNS [7]. As yet, there are no specific treatments available for CHIKV disease, although analgesics and nonsteroidal anti-inflammatory drugs can be given for symptomatic relief.