Immunocompetent hamsters as a model for orthobunyavirus-induced neuroinvasion and neuropathology.

BACKGROUND: Bunyavirus infections, including those caused by Bunyamwera serogroup orthobunyaviruses, represent a significant and yet likely still vastly underappreciated cause of mild to moderate human febrile infections. In severe cases, these infections can also cause neurological disease, particularly meningitis and encephalitis, and infection can even be fatal. However, with a few exceptions, information regarding the mechanisms underlying the neuroinvasion and neuropathogenesis of such infections is limited. This is due in part to a lack of animal models to facilitate such studies. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to develop an immunocompetent model of infection with Bunyamwera serogroup orthobunyaviruses, we infected 4-6-week-old female hamsters via either the intraperitoneal or subcutaneous route with 106 pfu/animal of Bunyamwera virus (BUNV), Batai virus or Ngari virus. Only BUNV infection resulted in clinical disease, which was characterized by weight loss, lethargy and neurological signs (i.e. tremor of the head or limbs, loss of righting reflex, "waltzing"). While symptoms were of similar severity for both routes, they occurred more frequently following subcutaneous inoculation. Consistent with these clinical signs, both antigen staining and histopathological abnormalities were found extensively throughout the brain. CONCLUSIONS/SIGNIFICANCE: The reported hamster model of BUNV infection provides a new tool for studying orthobunyavirus infection, and particularly neuroinvasion and the development of neuropathology. This model is particularly significant because it makes use of immunologically competent animals and relies on a subcutaneous inoculation route that more closely mimics the natural infection route for arboviruses, thereby providing a more authentic cellular and immunological context at the initial site of infection.

Favipiravir (T-705) Protects IFNAR-/- Mice against Lethal Zika Virus Infection in a Sex-Dependent Manner.

Zika virus (ZIKV), a member of the Flaviviridae family, is an important human pathogen that has caused epidemics in Africa, Southeast Asia, and the Americas. No licensed treatments for ZIKV disease are currently available. Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) and ribavirin (1-(ß-D-Ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide) are nucleoside analogs that have exhibited antiviral activity against a broad spectrum of RNA viruses, including some flaviviruses. In this study, we strengthened evidence for favipiravir and ribavirin inhibition of ZIKV replication in vitro. Testing in IFNAR-/- mice revealed that daily treatments of favipiravir were sufficient to provide protection against lethal ZIKV challenge in a dose-dependent manner but did not completely abrogate disease. Ribavirin, on the other hand, had no beneficial effect against ZIKV infection in this model and under the conditions examined. Combined treatment of ribavirin and favipiravir did not show improved outcomes over ribavirin alone. Surprisingly, outcome of favipiravir treatment was sex-dependent, with 87% of female but only 25% of male mice surviving lethal ZIKV infection. Since virus mutations were not associated with outcome, a sex-specific host response likely explains the observed sex difference.

Influenza virus A/Anhui/1/2013 (H7N9) replicates efficiently in the upper and lower respiratory tracts of cynomolgus macaques.

UNLABELLED: In March 2013, three fatal human cases of infection with influenza A virus (H7N9) were reported in China. Since then, human cases have been accumulating. Given the public health importance of this virus, we performed a pathogenicity study of the H7N9 virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection. Cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. Virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at 6 days after inoculation. Virus shedding occurred mainly via the throat. Histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. Analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during H7N9 infection as well as leads for development of therapeutics targeting host responses rather than virus replication. Overall, H7N9 infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human H7N9 infection (H. N. Gao et al., N. Engl. J. Med. 368:2277-2285, 2013, doi:10.1056/NEJMoa1305584). IMPORTANCE: Influenza A virus H7N9 emerged early in 2013, and human cases have continued to emerge since then. Although H7N9 virus-induced disease in humans is often very severe and even lethal, the majority of reported H7N9 cases occurred in older people and people with underlying medical conditions. To better understand the pathogenicity of this virus, healthy cynomolgus macaques were inoculated with influenza A virus H7N9. Cynomolgus macaques were used as a model because the receptor distribution for H7N9 virus in macaques was recently shown to be more similar to that in humans than that of other frequently used animal models. From comparison with previous studies, we conclude that the emerging H7N9 influenza virus was more pathogenic in cynomolgus macaques than seasonal influenza A viruses and most isolates of the pandemic H1N1 virus but less pathogenic than the 1918 Spanish influenza virus or highly pathogenic avian influenza (HPAI) H5N1 virus.

Experimental Andes virus infection in deer mice: characteristics of infection and clearance in a heterologous rodent host.

New World hantaviruses can cause hantavirus cardiopulmonary syndrome with high mortality in humans. Distinct virus species are hosted by specific rodent reservoirs, which also serve as the vectors. Although regional spillover has been documented, it is unknown whether rodent reservoirs are competent for infection by hantaviruses that are geographically separated, and known to have related, but distinct rodent reservoir hosts. We show that Andes virus (ANDV) of South America, carried by the long tailed pygmy rice rat (Oligoryzomys longicaudatus), infects and replicates in vitro and in vivo in the deer mouse (Peromyscus maniculatus), the reservoir host of Sin Nombre virus (SNV), found in North America. In experimentally infected deer mice, viral RNA was detected in the blood, lung, heart and spleen, but virus was cleared by 56 days post inoculation (dpi). All of the inoculated deer mice mounted a humoral immune response by 14 dpi, and produced measurable amounts of neutralizing antibodies by 21 dpi. An up-regulation of Ccl3, Ccl4, Ccl5, and Tgfb, a strong CD4⁺ T-cell response, and down-regulation of Il17, Il21 and Il23 occurred during infection. Infection was transient with an absence of clinical signs or histopathological changes. This is the first evidence that ANDV asymptomatically infects, and is immunogenic in deer mice, a non-natural host species of ANDV. Comparing the immune response in this model to that of the immune response in the natural hosts upon infection with their co-adapted hantaviruses may help clarify the mechanisms governing persistent infection in the natural hosts of hantaviruses.

The Ebola virus glycoprotein contributes to but is not sufficient for virulence in vivo.

Among the Ebola viruses most species cause severe hemorrhagic fever in humans; however, Reston ebolavirus (REBOV) has not been associated with human disease despite numerous documented infections. While the molecular basis for this difference remains unclear, in vitro evidence has suggested a role for the glycoprotein (GP) as a major filovirus pathogenicity factor, but direct evidence for such a role in the context of virus infection has been notably lacking. In order to assess the role of GP in EBOV virulence, we have developed a novel reverse genetics system for REBOV, which we report here. Together with a previously published full-length clone for Zaire ebolavirus (ZEBOV), this provides a unique possibility to directly investigate the role of an entire filovirus protein in pathogenesis. To this end we have generated recombinant ZEBOV (rZEBOV) and REBOV (rREBOV), as well as chimeric viruses in which the glycoproteins from these two virus species have been exchanged (rZEBOV-RGP and rREBOV-ZGP). All of these viruses could be rescued and the chimeras replicated with kinetics similar to their parent virus in tissue culture, indicating that the exchange of GP in these chimeric viruses is well tolerated. However, in a mouse model of infection rZEBOV-RGP demonstrated markedly decreased lethality and prolonged time to death when compared to rZEBOV, confirming that GP does indeed contribute to the full expression of virulence by ZEBOV. In contrast, rREBOV-ZGP did not show any signs of virulence, and was in fact slightly attenuated compared to rREBOV, demonstrating that GP alone is not sufficient to confer a lethal phenotype or exacerbate disease in this model. Thus, while these findings provide direct evidence that GP contributes to filovirus virulence in vivo, they also clearly indicate that other factors are needed for the acquisition of full virulence.

Recently emerged swine influenza A virus (H2N3) causes severe pneumonia in Cynomolgus macaques.

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.

Host response dynamics following lethal infection of rhesus macaques with Zaire ebolavirus.

To gain further insight into the interdependent pathogenic processes in Ebola hemorrhagic fever (EHF), we have examined the dynamics of host responses in individual rhesus macaques infected with Zaire ebolavirus over the entire disease course. Examination of coagulation parameters revealed that decreased coagulation inhibitor activity triggered severe coagulopathy as indicated by prolonged coagulation times and decreased fibrinogen levels. This has been proposed as one of the significant mechanisms underlying disseminated intravascular coagulation in EHF patients. Furthermore, monitoring of expression levels for cytokines/chemokines suggested a mixed anti-inflammatory response syndrome (MARS), which indicates that a catastrophic uncontrolled immunological status contributes to the development of fatal hemorrhagic fever. These results highlight the pathological analogies between EHF and severe sepsis and not only contribute to our understanding of the pathogenic process, but will also help to establish novel postexposure treatment modalities.

Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the burkholderia pseudomallei capsule mutant.

Pneumonia is a common manifestation of the potentially fatal disease melioidosis, caused by the select agent bacteria Burkholderia pseudomallei. In this study we describe a new model system to investigate pulmonary melioidosis in vivo using bioluminescent-engineered bacteria in a murine respiratory disease model. Studies were performed to validate that the stable, light producing B. pseudomallei strain JW280 constitutively produced light in biologically relevant host-pathogen interactions. Hairless outbred SKH1 mice were used to enhance the ability to monitor B. pseudomallei respiratory disease, and were found to be similarly susceptible to respiratory melioidosis as BALB/c mice. This represents the first demonstration of in vivo diagnostic imaging of pulmonary melioidosis permitting the detection of B. pseudomallei less than 24 h post-infection. Diagnostic imaging of pulmonary melioidosis revealed distinct temporal patterns of bacterial colonization unique to both BALB/c and SKH1 mice. Validation of these model systems included the use of the previously characterized capsule mutant, which was found to colonize the upper respiratory tract at significantly higher levels than the wild type strain. These model systems allow for high resolution detection of bacterial pulmonary disease which will facilitate studies of therapeutics and basic science evaluation of melioidosis.

Comparative pathogenesis of three human and zoonotic SARS-CoV strains in cynomolgus macaques.

The severe acute respiratory syndrome (SARS) epidemic was characterized by increased pathogenicity in the elderly due to an early exacerbated innate host response. SARS-CoV is a zoonotic pathogen that entered the human population through an intermediate host like the palm civet. To prevent future introductions of zoonotic SARS-CoV strains and subsequent transmission into the human population, heterologous disease models are needed to test the efficacy of vaccines and therapeutics against both late human and zoonotic isolates. Here we show that both human and zoonotic SARS-CoV strains can infect cynomolgus macaques and resulted in radiological as well as histopathological changes similar to those seen in mild human cases. Viral replication was higher in animals infected with a late human phase isolate compared to a zoonotic isolate. While there were significant differences in the number of host genes differentially regulated during the host responses between the three SARS-CoV strains, the top pathways and functions were similar and only apparent early during infection with the majority of genes associated with interferon signaling pathways. This study characterizes critical disease models in the evaluation and licensure of therapeutic strategies against SARS-CoV for human use.

Pandemic swine-origin H1N1 influenza A virus isolates show heterogeneous virulence in macaques.

The first influenza pandemic of the new millennium was caused by a newly emerged swine-origin influenza virus (SOIV) (H1N1). This new virus is characterized by a previously unknown constellation of gene segments derived from North American and Eurasian swine lineages and the absence of common markers predictive of human adaptation. Overall, human infections appeared to be mild, but an alarming number of young individuals presented with symptoms atypical for seasonal influenza. The new SOIV also showed a sustained human-to-human transmissibility and higher reproduction ratio than common seasonal viruses, altogether indicating a higher pathogenic potential for this newly emerged virus. To study the virulence of the SOIV, we used a recently established cynomolgus macaque model and compared parameters of clinical disease, virology, host responses, and pathology/histopathology with a current seasonal H1N1 virus. We here show that infection of macaques with two genetically similar but clinically distinct SOIV isolates from the early stage of the pandemic (A/Mexico/4108/2009 and A/Mexico/InDRE4487/2009) resulted in upper and lower respiratory tract infections and clinical disease ranging from mild to severe pneumonia that was clearly advanced over the mild infection caused by A/Kawasaki/UTK-4/2009, a current seasonal strain. Unexpectedly, we observed heterogeneity among the two SOIV isolates in virus replication, host transcriptional and cytokine responses, and disease progression, demonstrating a higher pathogenic potential for A/Mexico/InDRE4487/2009. Differences in virulence may explain more severe disease, as was seen with certain individuals infected with the emerged pandemic influenza virus. Thus, the nonhuman primate model closely mimics influenza in humans.

Role for the Burkholderia pseudomallei capsular polysaccharide encoded by the wcb operon in acute disseminated melioidosis.

The capsular polysaccharide of Burkholderia pseudomallei is an essential virulence determinant that is required for protection from host serum cidal activity and opsonophagocytosis. In this study, the immune response directed against a B. pseudomallei capsule mutant (JW270) was investigated in an acute respiratory murine model. JW270 was significantly attenuated in this model ( approximately 2 logs) to levels resembling those of avirulent Burkholderia thailandensis. At lethal doses, JW270 colonized the lung, liver, and spleen at levels similar to the wild-type strain levels and was found to trigger reduced pathology in the liver and spleen. Several cytokine responses were altered in these tissues, and importantly, the levels of gamma interferon were reduced in the livers and spleens of JW270-infected mice but not in the lungs. These results suggest that the capsular polysaccharide of B. pseudomallei is a critical virulence determinant in respiratory tract infections and that it is an important antigen for generating the Th1 immune response commonly observed in systemic melioidosis. Furthermore, the data suggest that host recognition of B. pseudomallei capsular polysaccharide in the lungs may not be as important to the disease outcome as the innate immune response in the peripheral organs.

The heart is an early target of anthrax lethal toxin in mice: a protective role for neuronal nitric oxide synthase (nNOS).

Anthrax lethal toxin (LT) induces vascular insufficiency in experimental animals through unknown mechanisms. In this study, we show that neuronal nitric oxide synthase (nNOS) deficiency in mice causes strikingly increased sensitivity to LT, while deficiencies in the two other NOS enzymes (iNOS and eNOS) have no effect on LT-mediated mortality. The increased sensitivity of nNOS-/- mice was independent of macrophage sensitivity to toxin, or cytokine responses, and could be replicated in nNOS-sufficient wild-type (WT) mice through pharmacological inhibition of the enzyme with 7-nitroindazole. Histopathological analyses showed that LT induced architectural changes in heart morphology of nNOS-/- mice, with rapid appearance of novel inter-fiber spaces but no associated apoptosis of cardiomyocytes. LT-treated WT mice had no histopathology observed at the light microscopy level. Electron microscopic analyses of LT-treated mice, however, revealed striking pathological changes in the hearts of both nNOS-/- and WT mice, varying only in severity and timing. Endothelial/capillary necrosis and degeneration, inter-myocyte edema, myofilament and mitochondrial degeneration, and altered sarcoplasmic reticulum cisternae were observed in both LT-treated WT and nNOS-/- mice. Furthermore, multiple biomarkers of cardiac injury (myoglobin, cardiac troponin-I, and heart fatty acid binding protein) were elevated in LT-treated mice very rapidly (by 6 h after LT injection) and reached concentrations rarely reported in mice. Cardiac protective nitrite therapy and allopurinol therapy did not have beneficial effects in LT-treated mice. Surprisingly, the potent nitric oxide scavenger, carboxy-PTIO, showed some protective effect against LT. Echocardiography on LT-treated mice indicated an average reduction in ejection fraction following LT treatment in both nNOS-/- and WT mice, indicative of decreased contractile function in the heart. We report the heart as an early target of LT in mice and discuss a protective role for nNOS against LT-mediated cardiac damage.

Characteristics of 263K scrapie agent in multiple hamster species.

Transmissible spongiform encephalopathy (TSE) diseases are known to cross species barriers, but the pathologic and biochemical changes that occur during transmission are not well understood. To better understand these changes, we infected 6 hamster species with 263K hamster scrapie strain and, after each of 3 successive passages in the new species, analyzed abnormal proteinase K (PK)-resistant prion protein (PrPres) glycoform ratios, PrPres PK sensitivity, incubation periods, and lesion profiles. Unique 263K molecular and biochemical profiles evolved in each of the infected hamster species. Characteristics of 263K in the new hamster species seemed to correlate best with host factors rather than agent strain. Furthermore, 2 polymorphic regions of the prion protein amino acid sequence correlated with profile differences in these TSE-infected hamster species.

A haptic-robotic platform for upper-limb reaching stroke therapy: preliminary design and evaluation results.

BACKGROUND: It has been shown that intense training can significantly improve post-stroke upper-limb functionality. However, opportunities for stroke survivors to practice rehabilitation exercises can be limited because of the finite availability of therapists and equipment. This paper presents a haptic-enabled exercise platform intended to assist therapists and moderate-level stroke survivors perform upper-limb reaching motion therapy. This work extends on existing knowledge by presenting: 1) an anthropometrically-inspired design that maximizes elbow and shoulder range of motions during exercise; 2) an unobtrusive upper body postural sensing system; and 3) a vibratory elbow stimulation device to encourage muscle movement. METHODS: A multi-disciplinary team of professionals were involved in identifying the rehabilitation needs of stroke survivors incorporating these into a prototype device. The prototype system consisted of an exercise device, postural sensors, and a elbow stimulation to encourage the reaching movement. Eight experienced physical and occupational therapists participated in a pilot study exploring the usability of the prototype. Each therapist attended two sessions of one hour each to test and evaluate the proposed system. Feedback about the device was obtained through an administered questionnaire and combined with quantitative data. RESULTS: Seven of the nine questions regarding the haptic exercise device scored higher than 3.0 (somewhat good) out of 4.0 (good). The postural sensors detected 93 of 96 (97%) therapist-simulated abnormal postures and correctly ignored 90 of 96 (94%) of normal postures. The elbow stimulation device had a score lower than 2.5 (neutral) for all aspects that were surveyed, however the therapists felt the rehabilitation system was sufficient for use without the elbow stimulation device. CONCLUSION: All eight therapists felt the exercise platform could be a good tool to use in upper-limb rehabilitation as the prototype was considered to be generally well designed and capable of delivering reaching task therapy. The next stage of this project is to proceed to clinical trials with stroke patients.

Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.

Identification of the genetic events that contribute to host-pathogen interactions is important for understanding the natural history of infectious diseases and developing therapeutics. Transcriptome studies conducted on pathogens have been central to this goal in recent years. However, most of these investigations have focused on specific end points or disease phases, rather than analysis of the entire time course of infection. To gain a more complete understanding of how bacterial gene expression changes over time in a primate host, the transcriptome of group A Streptococcus (GAS) was analyzed during an 86-day infection protocol in 20 cynomolgus macaques with experimental pharyngitis. The study used 260 custom Affymetrix (Santa Clara, CA) chips, and data were confirmed by TaqMan analysis. Colonization, acute, and asymptomatic phases of disease were identified. Successful colonization and severe inflammation were significantly correlated with an early onset of superantigen gene expression. The differential expression of two-component regulators covR and spy0680 (M1_spy0874) was significantly associated with GAS colony-forming units, inflammation, and phases of disease. Prophage virulence gene expression and prophage induction occurred predominantly during high pathogen cell densities and acute inflammation. We discovered that temporal changes in the GAS transcriptome were integrally linked to the phase of clinical disease and host-defense response. Knowledge of the gene expression patterns characterizing each phase of pathogen-host interaction provides avenues for targeted investigation of proven and putative virulence factors and genes of unknown function and will assist vaccine research.