In vivo tropism of attenuated and pathogenic measles virus expressing green fluorescent protein in macaques

The global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation. In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare the in vivo tropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despite in vitro differences, attenuated and pathogenic MV show highly similar in vivo tropism in the lungs. However, systemic spread of attenuated MV is restricted.

Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: Implications for respiratory virus-induced cough reflex sensitivity

ABSTRACT (250 words)
BACKGROUND: The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Up-regulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurones and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression.
METHODS: IMR32 neuroblastoma cells were differentiated in culture to express three TRP channels, TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus- induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1ß, IL-6 and IL-8 levels in response to infection.
RESULTS: Early up-regulation of TRPA1 and TRPV1 expression occurred 2 to4 hours post infection. This was independent of replicating virus as virus induced soluble factors alone were sufficient to increase channel expression 50 and 15 fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 hours (9.6 fold) and required virus replication rather than soluble factors
CONCLUSIONS We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes up-regulation of TRP channels by channel specific mechanisms. Increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.

IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1a, nsp1b, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 30 end of the viral genome encodes four minor and three major structural proteins. The GP2a, GP3 and GP4 (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP5 (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

In vitro and in vivo effects of deoxynivalenol (DON) mycotoxin on porcine reproductive and respiratory syndrome virus (PRRSV) in piglets

Les récoltes de céréales sont souvent contaminées par des moisissures qui se développent pendant la récolte et l’entreposage et produisent des métabolites secondaires appelés mycotoxines. Le porc est reconnu pour être sensible au déoxynivalénol (DON). L’infection virale la plus importante chez le porc est causée par le virus du syndrome reproducteur et respiratoire porcin (VSRRP). Celui-ci provoque un syndrome grippal et des troubles de reproduction. L’objectif du présent projet était de déterminer l'effet in vitro de DON sur la réplication du VSRRP dans de lignées cellulaires permissives, MARC-145 et PAM, et déterminer in vivo l'impact de DON dans des aliments naturellement contaminés sur l’infection au VSRRP chez le porcelet. Tout d’abord, les cellules ont été incubées avec des doses croissantes de DON et ont été infectées avec du VSRRP pour évaluer la viabilité et la mortalité cellulaire, la réplication virale et l’expression de cytokines. Les résultats ont montré que les concentrations de DON de 560ng/ml et plus affectaient significativement la survie des cellules MARC-145 et PAM infectées par le VSRRP. En revanche, il y avait une augmentation significative de la viabilité et une réduction de la mortalité cellulaire à des concentrations de DON de 140 à 280 ng/ml pour les cellules PAM et de 70 à 280 ng/ml pour les cellules MARC-145 avec une réduction de l'effet cytopathique provoqué parle VSRRP. Au niveau in vivo, 30 porcelets divisés en 3 groupes de 10 porcelets et nourris pendant 2 semaines avec 3 différentes diètes naturellement ont été contaminées avec DON (0; 2,5 et 3,5 mg/kg). Les porcelets ont été subdivisés en 6 groupes, 3 groupes de 6 porcelets et ont été exposés au DON pendant 2 semaines et infectés par voie intratrachéale et intramusculaire avec le virus. Les 3 autres groupes de 4 porcelets servaient de contrôle non infectés. Les signes cliniques ont été enregistrés pendant 21 jours. La virémie a été évaluée par PCR. À la fin de l’expérimentation, les porcelets ont été euthanasiés et les lésions pulmonaires ont été évaluées. Les résultats ont montré que l’ingestion de DON à 3,5 mg/kg a augmenté l’effet du VSRRP sur la sévérité des signes cliniques, les lésions pulmonaires et la mortalité. L’ingestion de DON à 2,5 mg/kg a entrainé une augmentation de la virémie au jour 3 après l’infection mais sans impact sur les signes cliniques et les lésions pulmonaires. Mot clés: DON, VSRRP, MARC-145, PAM, effet cytopathique, cytokines, PCR

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Mutations in H5N1 influenza virus hemagglutinin that confer binding to human tracheal airway epithelium

The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary "dead end." We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.

The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus

The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

The raft-promoting property of virion-associated cholesterol, but not the presence of virion-associated brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity

Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally diverse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.

Mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer

The specific impact of mutations that abrogate human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) dimerization on virus replication is not known, as mutations shown previously to inhibit RT dimerization also impact Gag-Pol stability, resulting in pleiotropic effects on HIV-1 replication. We have previously characterized mutations at codon 401 in the HIV-1 RT tryptophan repeat motif that abrogate RT dimerization in vitro, leading to a loss in polymerase activity. The introduction of the RT dimerization-inhibiting mutations W401L and W401A into HIV-1 resulted in the formation of noninfectious viruses with reduced levels of both virion-associated and intracellular RT activity compared to the wild-type virus and the W401F mutant, which does not inhibit RT dimerization in vitro. Steady-state levels of the p66 and p51 RT subunits in viral lysates of the W401L and W401A mutants were reduced, but no significant decrease in Gag-Pol was observed compared to the wild type. In contrast, there was a decrease in processing of p66 to p51 in cell lysates for the dimerization-defective mutants compared to the wild type. The treatment of transfected cells with indinavir suggested that the HIV-1 protease contributed to the degradation of virion-associated RT subunits. These data demonstrate that mutations near the RT dimer interface that abrogate RT dimerization in vitro result in the production of replication-impaired viruses without detectable effects on Gag-Pol stability or virion incorporation. The inhibition of RT activity is most likely due to a defect in RT maturation, suggesting that RT dimerization represents a valid drug target for chemotherapeutic intervention.