Cell-Type–Specific Transcriptional Profiles of the Dimorphic Pathogen Penicillium marneffei Reflect Distinct Reproductive, Morphological, and Environmental Demands

Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify “phase or cell-state–specific” gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase–encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity

Influenza A viral loads in respiratory samples collected from patients infected with pandemic H1N1, seasonal H1N1 and H3N2 viruses

BACKGROUND: Nasopharyngeal aspirate (NPA), nasal swab (NS), and throat swab (TS) are common specimens used for diagnosis of respiratory virus infections based on the detection of viral genomes, viral antigens and viral isolation. However, there is no documented data regarding the type of specimen that yields the best result of viral detection. In this study, quantitative real time RT-PCR specific for M gene was used to determine influenza A viral loads present in NS, NPA and TS samples collected from patients infected with the 2009 pandemic H1N1, seasonal H1N1 and H3N2 viruses. Various copy numbers of RNA transcripts derived from recombinant plasmids containing complete M gene insert of each virus strain were assayed by RT-PCR. A standard curve for viral RNA quantification was constructed by plotting each Ct value against the log quantity of each standard RNA copy number. RESULTS: Copy numbers of M gene were obtained through the extrapolation of Ct values of the test samples against the corresponding standard curve. Among a total of 29 patients with severe influenza enrolled in this study (12 cases of the 2009 pandemic influenza, 5 cases of seasonal H1N1 and 12 cases of seasonal H3N2 virus), NPA was found to contain significantly highest amount of viral loads and followed in order by NS and TS specimen. Viral loads among patients infected with those viruses were comparable regarding type of specimen analyzed. CONCLUSION: Based on M gene copy numbers, we conclude that NPA is the best specimen for detection of influenza A viruses, and followed in order by NS and TS

Complex Genetic Architecture Underlies Regulation of Influenza-A-Virus-Specific Antibody Responses in the Collaborative Cross

Noll et al. use the Collaborative Cross, a mouse genetic reference population, to map genetic loci associated with variation in the humoral response to influenza virus infection. Cross-dataset comparison shows that mapped loci are important for antibody response to multiple pathogens, and candidate genes with likely translational relevance are identified. © 2020 The Author(s)Host genetic factors play a fundamental role in regulating humoral immunity to viral infection, including influenza A virus (IAV). Here, we utilize the Collaborative Cross (CC), a mouse genetic reference population, to study genetic regulation of variation in antibody response following IAV infection. CC mice show significant heritable variation in the magnitude, kinetics, and composition of IAV-specific antibody response. We map 23 genetic loci associated with this variation. Analysis of a subset of these loci finds that they broadly affect the antibody response to IAV as well as other viruses. Candidate genes are identified based on predicted variant consequences and haplotype-specific expression patterns, and several show overlap with genes identified in human mapping studies. These findings demonstrate that the host antibody response to IAV infection is under complex genetic control and highlight the utility of the CC in modeling and identifying genetic factors with translational relevance to human health and disease

A Novel Pathogenic Mechanism of Highly Pathogenic Avian Influenza H5N1 Viruses Involves Hemagglutinin Mediated Resistance to Serum Innate Inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes

Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008–2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100%(CI 94%,100%) and negative predictive value = 96%(CI 88%,98%)). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96%(CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49%(CI 38%,61%) sensitivity and 98%(CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 103 copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability

Influenza A viral loads in respiratory samples collected from patients infected with pandemic H1N1, seasonal H1N1 and H3N2 viruses

Abstract Background Nasopharyngeal aspirate (NPA), nasal swab (NS), and throat swab (TS) are common specimens used for diagnosis of respiratory virus infections based on the detection of viral genomes, viral antigens and viral isolation. However, there is no documented data regarding the type of specimen that yields the best result of viral detection. In this study, quantitative real time RT-PCR specific for M gene was used to determine influenza A viral loads present in NS, NPA and TS samples collected from patients infected with the 2009 pandemic H1N1, seasonal H1N1 and H3N2 viruses. Various copy numbers of RNA transcripts derived from recombinant plasmids containing complete M gene insert of each virus strain were assayed by RT-PCR. A standard curve for viral RNA quantification was constructed by plotting each Ct value against the log quantity of each standard RNA copy number. Results Copy numbers of M gene were obtained through the extrapolation of Ct values of the test samples against the corresponding standard curve. Among a total of 29 patients with severe influenza enrolled in this study (12 cases of the 2009 pandemic influenza, 5 cases of seasonal H1N1 and 12 cases of seasonal H3N2 virus), NPA was found to contain significantly highest amount of viral loads and followed in order by NS and TS specimen. Viral loads among patients infected with those viruses were comparable regarding type of specimen analyzed. Conclusion Based on M gene copy numbers, we conclude that NPA is the best specimen for detection of influenza A viruses, and followed in order by NS and TS.</p