Quantifying dose-, strain-, and tissue-specific kinetics of parainfluenza virus infection

Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory infection hospitalization in children, yet little is known about how dose, strain, tissue tropism, and individual heterogeneity affects the processes driving growth and clearance kinetics. Longitudinal measurements are possible by using reporter Sendai viruses, the murine counterpart of HPIV 1, that express luciferase, where the insertion location yields a wild-type (rSeV-luc(M-F*)) or attenuated (rSeV-luc(P-M)) phenotype. Bioluminescence from individual animals suggests that there is a rapid increase in expression followed by a peak, biphasic clearance, and resolution. However, these kinetics vary between individuals and with dose, strain, and whether the infection was initiated in the upper and/or lower respiratory tract. To quantify the differences, we translated the bioluminescence measurements from the nasopharynx, trachea, and lung into viral loads and used a mathematical model together a nonlinear mixed effects approach to define the mechanisms distinguishing each scenario. The results confirmed a higher rate of virus production with the rSeV-luc(M-F*) virus compared to its attenuated counterpart, and suggested that low doses result in disproportionately fewer infected cells. The analyses indicated faster infectivity and infected cell clearance rates in the lung and that higher viral doses, and concomitantly higher infected cell numbers, resulted in more rapid clearance. This parameter was also highly variable amongst individuals, which was particularly evident during infection in the lung. These critical differences provide important insight into distinct HPIV dynamics, and show how bioluminescence data can be combined with quantitative analyses to dissect host-, virus-, and dose-dependent effects.     

Feline immunodeficiency virus in puma: Estimation of force of infection reveals insights into transmission

相似文献   

Mechanism of interference mediated by human parainfluenza virus type 3 infection

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.     

GFP in living animals reveals dynamic developmental responses to ecdysone during Drosophila metamorphosis

Studies of Drosophila metamorphosis have been hampered by our inability to visualize many of the remarkable changes that occur within the puparium. To circumvent this problem, we have expressed GFP in specific tissues of living prepupae and pupae and compiled images of these animals into time-lapse movies. These studies reveal, for the first time, the dynamics and coordination of morphogenetic movements that could only be inferred from earlier studies of dissected staged animals. We also identify responses that have not been described previously. These include an unexpected variation in some wild-type animals, where one of the first pairs of legs elongates in the wrong position relative to the second pair of legs and then relocates to its appropriate location. At later stages, the antennal imaginal discs migrate from a lateral position in the head to their final location at the anterior end, as leg and mouth structures are refined and the wings begin to fold. The larval salivary glands translocate toward the dorsal aspect of the animal and undergo massive cell death following head eversion, in synchrony with death of the abdominal muscles. These death responses fail to occur in rbp(5) mutants of the Broad-Complex (BR-C), and imaginal disc elongation and eversion is abolished in br(5) mutants of the BR-C. Leg malformations associated with the crol(3) mutation can be seen to arise from defects in imaginal disc morphogenesis during prepupal stages. This approach provides a new tool for characterizing the dynamic morphological changes that occur during metamorphosis in both wild-type and mutant animals.     

Cryo-EM reveals a previously unrecognized structural protein of a dsRNA virus implicated in its extracellular transmission

Mosquito viruses cause unpredictable outbreaks of disease. Recently, several unassigned viruses isolated from mosquitoes, including the Omono River virus (OmRV), were identified as totivirus-like viruses, with features similar to those of the Totiviridae family. Most reported members of this family infect fungi or protozoans and lack an extracellular life cycle stage. Here, we identified a new strain of OmRV and determined high-resolution structures for this virus using single-particle cryo-electron microscopy. The structures feature an unexpected protrusion at the five-fold vertex of the capsid. Disassociation of the protrusion could result in several conformational changes in the major capsid. All these structures, together with some biological results, suggest the protrusions’ associations with the extracellular transmission of OmRV.     

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.     

The infection of mouse preimplantation embryos to Sendai virus (parainfluenza I)

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.     

Rapid diagnosis and management of parainfluenza I virus infection in common marmosets (Callithrix jacchus)

During 1983 a severe episode of respiratory infection occurred in a marmoset colony at these laboratories. Of 91 marmosets, 69 showed clinical signs of disease, one died and nine were so ill that euthanasia was necessary. Eight were examined post mortem and all showed consolidation of the lungs. Laboratory studies were carried out in an attempt to establish the cause of the outbreak and an interstitial pneumonia was found in seven animals which were examined histologically. Direct electron microscopy of nasal swabs and lung samples revealed the presence of a high titre of a paramyxovirus, and subsequent immunofluorescence studies established that the particular paramyxovirus involved was parainfluenza virus type I. Subsequent studies showed that surviving affected animals had seroconverted to parainfluenza I virus while animals that had not been implicated in the outbreak had not.     

Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.     

Characterization of a novel parainfluenza virus,caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus)

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.     

Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.