A Novel Model of Lethal Hendra Virus Infection in African Green Monkeys and the Effectiveness of Ribavirin Treatment

The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are emerging zoonotic paramyxoviruses that can cause severe and often lethal neurologic and/or respiratory disease in a wide variety of mammalian hosts, including humans. There are presently no licensed vaccines or treatment options approved for human or veterinarian use. Guinea pigs, hamsters, cats, and ferrets, have been evaluated as animal models of human HeV infection, but studies in nonhuman primates (NHP) have not been reported, and the development and approval of any vaccine or antiviral for human use will likely require efficacy studies in an NHP model. Here, we examined the pathogenesis of HeV in the African green monkey (AGM) following intratracheal inoculation. Exposure of AGMs to HeV produced a uniformly lethal infection, and the observed clinical signs and pathology were highly consistent with HeV-mediated disease seen in humans. Ribavirin has been used to treat patients infected with either HeV or NiV; however, its utility in improving outcome remains, at best, uncertain. We examined the antiviral effect of ribavirin in a cohort of nine AGMs before or after exposure to HeV. Ribavirin treatment delayed disease onset by 1 to 2 days, with no significant benefit for disease progression and outcome. Together our findings introduce a new disease model of acute HeV infection suitable for testing antiviral strategies and also demonstrate that, while ribavirin may have some antiviral activity against the henipaviruses, its use as an effective standalone therapy for HeV infection is questionable.Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus (family Paramyxoviridae) that can cause severe respiratory illness and/or encephalitis in a wide variety of mammals, including horses, pigs, and humans (7, 23). HeV was identified as the causative agent of an acute respiratory disease in horses in 1994 in Queensland, Australia (23), and to date there have been 14 outbreaks in Australia since, with at least one occurrence per year since 2006, most recently in May 2010 (ProMed-mail no. 20100522.1699 [International Society for Infectious Diseases, http://www.promedmail.org]). Every outbreak of HeV has involved horses as the initial infected host, and there have been a total of seven human cases arising from exposure to infected horses. Four human fatalities have occurred (22), with the most recent occurring in August of 2009 (ProMed-mail no. 20090826.2998 and 20090903.3098). All patients initially presented with influenza-like illnesses (ILIs) after an incubation period of 7 to 16 days. While two individuals recovered from ILI, one patient developed pneumonitis and died from multiorgan failure. Three of the lethal cases developed encephalitic manifestations (mild confusion and ataxia), with two patients experiencing seizures (22, 23, 27).Data on the histopathology of fatal human HeV cases are limited, but the pathology includes small necrotic plaques in the cerebrum and cerebellum, in addition to mild parenchymal inflammation (21, 27). Severe parenchymal inflammation and necrosis were observed in the lungs. More extensive histopathologic data are available from 32 autopsies of fatal human NiV cases (28). Similarly to the HeV cases, pathology was characterized by systemic vasculitis and parenchymal necrosis in the central nervous system (CNS), while in the lung, pathological findings mainly included vasculitis, fibrinoid necrosis, alveolar hemorrhage, pulmonary edema, and aspiration pneumonia. Other organs that were affected included heart, kidney, and spleen and showed generally mild or focal inflammation. The development of syncytial multinucleated endothelial cells is characteristic of both HeV and NiV (27, 28). At present, the details of the pathogenesis and histopathological changes mediated by either HeV or NiV infection in humans are naturally derived from only the late phases of the disease course, and therefore a relevant animal model is needed that mimics the disease progression seen in humans.Pteropid fruit bats, commonly known as flying foxes in the family Pteropodidae, are the principle natural reservoirs for both NiV and HeV (reviewed in reference 3). However, these henipaviruses display a broad species tropism, and in addition to bats, horses and humans, natural and/or experimental infection of HeV has been demonstrated in guinea pigs, hamsters, pigs, cats, and ferrets (25). Experimental infections of Syrian hamsters with HeV is lethal, and animals show disease similar to that of human cases, including respiratory and neurological symptoms, depending on the dose (11; unpublished data). In this model, viral RNA can be detected in various organs of infected hamsters, including brain, lung, kidney, heart, liver, and spleen. The main histopathological findings included parenchymal infection in various organs, including the brain, with vasculitis and syncytial multinucleated endothelial cells in many blood vessels (11). While this model is useful in studying pathogenesis, it is limited in the availability of reagents to do so.There are currently no vaccines or treatments licensed for human use. Several in vitro studies have shown that ribavirin is effective against both HeV and NiV infection (1, 2, 29). An open-label ribavirin treatment trial was run during an outbreak of NiV in Malaysia in 1998 and reported to reduce mortality by 36% (6). Of the seven recorded human HeV cases, three patients were treated with ribavirin, one of whom survived (22). In the most recent outbreak of HeV in Australia, three additional people received ribavirin treatment in combination with chloroquine after suspected exposure to HeV-contaminated secretions from infected horses. While all three individuals survived, infection was not confirmed, and therefore it remains unknown whether the treatment had any beneficiary effect (ProMed-mail no. 20090826.2998). In addition, two animal studies in hamsters showed that ribavirin treatment delays but does not prevent death from NiV or HeV infection (8, 10). Therefore, an animal model with greater relevance to humans and that recapitulates the disease processes seen in human cases of HeV is needed to get a better answer to whether ribavirin might be effective against henipavirus infections. In addition, the U.S. FDA implemented the “Animal Efficacy Rule,” which specifically applies to the development of therapeutic products when human efficacy studies are not possible or ethical, such as is often the case with highly virulent pathogens like HeV (24). Essentially, this rule allows for the evaluation of vaccines or therapeutics using data derived from studies carried out in at least two animal models. The licensure of any therapeutic modalities for HeV will require a thorough evaluation of HeV pathogenesis in nonhuman primates (NHPs).In the present study, we report the development and characterization of a new nonhuman primate (NHP) model of lethal HeV infection in the African green monkey (AGM). The pathogenesis and disease progression in the AGM upon HeV infection essentially mirrored the lethal disease episodes seen among human cases of HeV. Using this new model, the efficacy of ribavirin treatment against lethal challenge with HeV was examined. Here we have shown that ribavirin treatment can significantly delay but not prevent death of AGMs from lethal HeV infection. In addition to severe respiratory symptoms in all animals, prolonged disease progression in ribavirin-treated animals was also marked by the appearance of neurological symptoms.     

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP.     

Neural substrate and allatostatin-like innervation of the gut of Locusta migratoria

Allatostatin-like immunoreactivity (ALI) is widely distributed in processes and varicosities on the fore-, mid-, and hindgut of the locust, and within midgut open-type endocrine-like cells. ALI is also observed in cells and processes in all ganglia of the central nervous system (CNS) and the stomatogastric nervous system (SNS). Ventral unpaired median neurons (VUMs) contained ALI within abdominal ganglia IV-VII. Neurobiotin retrograde fills of the branches of the 11th sternal nerve that innervate the hindgut revealed 2-4 VUMs in abdominal ganglia IV-VIIth, which also contain ALI. The VIIIth abdominal ganglion contained three ventral medial groups of neurons that filled with neurobiotin and contained ALI. The co-localization of ALI in the identified neurons suggests that these cells are the source of ALI on the hindgut. A retrograde fill of the nerves of the ingluvial ganglia that innervate the foregut revealed numerous neurons within the frontal ganglion and an extensive neuropile in the hypocerebral ganglion, but there seems to be no apparent co-localization of neurobiotin and ALI in these neurons, indicating the source of ALI on the foregut comes via the brain, through the SNS.     

Quantifying conversion of linoleic to arachidonic and other n-6 polyunsaturated fatty acids in unanesthetized rats

Isotope feeding studies report a wide range of conversion fractions of dietary shorter-chain polyunsaturated fatty acids (PUFAs) to long-chain PUFAs, which limits assessing nutritional requirements and organ effects of arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-3) acids. In this study, whole-body (largely liver) steady-state conversion coefficients and rates of circulating unesterified linoleic acid (LA, 18:2n-6) to esterified AA and other elongated n-6 PUFAs were quantified directly using operational equations, in unanesthetized adult rats on a high-DHA but AA-free diet, using 2 h of intravenous [U-¹³C]LA infusion. Unesterified LA was converted to esterified LA in plasma at a greater rate than to esterified γ-linolenic (γ-LNA, 18:3n-6), eicosatrienoic acid (ETA, 20:3n-6), or AA. The steady-state whole-body synthesis-secretion (conversion) coefficient to AA equaled 5.4 × 10⁻³ min⁻¹, while the conversion rate (coefficient × concentration) equaled 16.1 μmol/day. This rate exceeds the reported brain AA consumption rate by 27-fold. As brain and heart cannot synthesize significant AA from circulating LA, liver synthesis is necessary to maintain their homeostatic AA concentrations in the absence of dietary AA. The heavy-isotope intravenous infusion method could be used to quantify steady-state liver synthesis-secretion of AA from LA under different conditions in rodents and in humans.     

The Solution Structure of the C-Terminal Ig-like Domain of the Bacteriophage λ Tail Tube Protein

Immunoglobulin (Ig)-like domains are found frequently on the surface of tailed double-stranded DNA bacteriophages, yet their functional role remains obscure. Here, we have investigated the structure and function of the C-terminal Ig-like domain of gpV (gpV_C), the tail tube protein of phage λ. This domain has been predicted through sequence similarity to be a member of the bacterial Ig-like domain 2 (Big_2) family, which is composed of more than 1300 phage and bacterial sequences. Using trypsin proteolysis, we have delineated the boundaries of gpV_C and have shown that its removal reduces the biological activity of gpV by 100-fold; thus providing a definitive demonstration of a functional role for this domain. Determination of the solution structure of gpV_C by NMR spectroscopy showed that it adopts a canonical Ig-like fold of the I-set class. This represents the first structure of a phage-encoded Ig-like domain and only the second structure of a Big_2 domain. Structural and sequence comparisons indicate that the gpV_C structure is more representative of both the phage-encoded Big_2 domains and Big_2 domains in general than the other available Big_2 structure. Bioinformatics analyses have identified two conserved clusters of residues on the surface of gpV_C that may be important in mediating the function of this domain.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.