Adenovirus: Epidemiology, Global Spread of Novel Types, and Approach to Treatment

Adenoviruses (AdVs) are DNA viruses that typically cause mild infections involving the upper or lower respiratory tract, gastrointestinal tract, or conjunctiva. Rare manifestations of AdV infections include hemorrhagic cystitis, hepatitis, hemorrhagic colitis, pancreatitis, nephritis, or meningoencephalitis. AdV infections are more common in young children, due to lack of humoral immunity. Epidemics of AdV infection may occur in healthy children or adults in closed or crowded settings (particularly military recruits). The vast majority of cases are self-limited. However, the clinical spectrum is broad and fatalities may occur. Dissemination is more likely in patients with impaired immunity (e.g., organ transplant recipients, human immunodeficiency virus infection). Fatality rates for untreated severe AdV pneumonia or disseminated disease may exceed 50%. More than 100 genotypes and 52 serotypes of AdV have been identified and classified into seven species designated HAdV-A through -G. Different types display different tissue tropisms that correlate with clinical manifestations of infection. The predominant types circulating at a given time differ among countries or regions, and change over time. Transmission of novel strains between countries or across continents and replacement of dominant viruses by new strains may occur. Treatment of AdV infections is controversial, as prospective, randomized therapeutic trials have not been done. Cidofovir has been the drug of choice for severe AdV infections, but not all patients require treatment. Live oral vaccines are highly efficacious in reducing the risk of respiratory AdV infection and are in routine use in the military in the United States but currently are not available to civilians.

Deep Parallel Characterization of AAV Tropism and AAV-Mediated Transcriptional Changes via Single-Cell RNA Sequencing

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at high resolution. Using this platform, we have both corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Transcriptomic analysis revealed that this neuronal bias is due mainly to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific transitory responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. The presented experimental and computational approaches for parallel characterization of AAV tropism will facilitate the advancement of safe and precise gene delivery vehicles, and showcase the power of understanding responses to gene therapies at the single-cell level.

Mycoplasma genitalium and M. pneumoniae Regulate a Distinct Set of Protein-Coding Genes in Epithelial Cells

Mycoplasma genitalium and M. pneumoniae are two significant mycoplasmas that infect the urogenital and respiratory tracts of humans. Despite distinct tissue tropisms, they both have similar pathogenic mechanisms and infect/invade epithelial cells in the respective regions and persist within these cells. However, the pathogenic mechanisms of these species in terms of bacterium-host interactions are poorly understood. To gain insights on this, we infected HeLa cells independently with M. genitalium and M. pneumoniae and assessed gene expression by whole transcriptome sequencing (RNA-seq) approach. The results revealed that HeLa cells respond to M. genitalium and M. pneumoniae differently by regulating various protein-coding genes. Though there is a significant overlap between the genes regulated by these species, many of the differentially expressed genes were specific to each species. KEGG pathway and signaling network analyses revealed that the genes specific to M. genitalium are more related to cellular processes. In contrast, the genes specific to M. pneumoniae infection are correlated with immune response and inflammation, possibly suggesting that M. pneumoniae has some inherent ability to modulate host immune pathways.

Extended in vivo transcriptomes of two ascoviruses with different tissue tropisms reveal alternative mechanisms for enhancing virus reproduction in hemolymph

Ascoviruses are large dsDNA viruses characterized by the extraordinary changes they induce in cellular pathogenesis and architecture whereby after nuclear lysis and extensive hypertrophy, each cell is cleaved into numerous vesicles for virion reproduction. However, the level of viral replication and transcription in vesicles compared to other host tissues remains uncertain. Therefore, we applied RNA-Sequencing to compare the temporal transcriptome of Spodoptera frugiperda ascovirus (SfAV) and Trichoplusia ni ascovirus (TnAV) at 7, 14, and 21 days post-infection (dpi). We found most transcription occurred in viral vesicles, not in initial tissues infected, a remarkably novel reproduction mechanism compared to all other viruses and most other intracellular pathogens. Specifically, the highest level of viral gene expression occurred in hemolymph, for TnAV at 7 dpi, and SfAV at 14 dpi. Moreover, we found that host immune genes were partially down-regulated in hemolymph, where most viral replication occurred in highly dense accumulations of vesicles.

Viral receptor profiles of masked palm civet revealed by single-cell transcriptomics

Civets are small mammals belonging to the family Viverridae. The masked palm civets (Paguma larvata) served as an intermediate host in the bat-to-human transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003. Because of their unique role in the SARS outbreak, civets were suspected as a potential intermediate host of SARS-CoV-2, the etiological pathogen of the COVID-19 pandemic. Besides their susceptibility to coronaviruses, civets can also be infected by other viruses, such as canine distemper viruses, parvoviruses, influenza viruses, etc. Regarding the ecological and economical role of civets, it is vital to evaluate the potential threats from different pathogens to these animals. Receptor binding is a necessary step for virus entry into host cells. Understanding the distribution of receptors of various viruses provides hints to their potential tissue tropisms. Herein, we characterized the cell atlas of five important organs (the frontal lobe, lung, liver, spleen and kidney) of masked palm civets (Paguma larvata) and described the expression profiles of receptor associated genes of 132 viruses from 25 families, including 16 viruses from 10 families reported before that can attack civets and 116 viruses with little infection record.

Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing

Engineered variants of recombinant adeno-associated viruses (rAAVs) are being developed rapidly to meet the need for gene-therapy delivery vehicles with particular cell-type and tissue tropisms. While high-throughput AAV engineering and selection methods have generated numerous variants, subsequent tropism and response characterization have remained low throughput and lack resolution across the many relevant cell and tissue types. To fully leverage the output of these large screening paradigms across multiple targets, we have developed an experimental and computational single-cell RNA sequencing (scRNA-seq) pipeline for in vivo characterization of barcoded rAAV pools at unprecedented resolution. Using our platform, we have corroborated previously reported viral tropisms and discovered unidentified AAV capsid targeting biases. As expected, we observed that the tropism profile of AAV.CAP-B10 in mice was shifted toward neurons and away from astrocytes when compared with AAV-PHP.eB. Our transcriptomic analysis revealed that this neuronal bias is mainly due to increased targeting efficiency for glutamatergic neurons, which we confirmed by RNA fluorescence in situ hybridization. We further uncovered cell subtype tropisms of AAV variants in vascular and glial cells, such as low transduction of pericytes and Myoc+ astrocytes. Additionally, we have observed cell-type-specific responses to systemic AAV-PHP.eB administration, such as upregulation of genes involved in p53 signaling in endothelial cells three days post-injection, which return to control levels by day twenty-five. Such ability to parallelize the characterization of AAV tropism and simultaneously measure the transcriptional response of transduction will facilitate the advancement of safe and precise gene delivery vehicles.

Beyond the Gastrointestinal Tract: The Emerging and Diverse Tissue Tropisms of Astroviruses

Astroviruses are single stranded, positive-sense RNA viruses that have been historically associated with diseases of the gastrointestinal tract of vertebrates, including humans. However, there is now a multitude of evidence demonstrating the capacity of these viruses to cause extraintestinal diseases. The most striking causal relationship is neurological diseases in humans, cattle, pigs, and other mammals, caused by astrovirus infection. Astroviruses have also been associated with disseminated infections, localized disease of the liver or kidneys, and there is increasing evidence suggesting a potential tropism to the respiratory tract. This review will discuss the current understanding of the tissue tropisms for astroviruses and their emerging capacity to cause disease in multiple organ systems.

Cyclic AMP signalling and glucose metabolism mediate pH taxis by African trypanosomes

More than a decade ago it was proposed that the collective migration of African trypanosomes on semi-solid surfaces could be explained by a combination of migration factors and repellents released by the parasites, but the identity of these molecules was unknown. Here we show that procyclic (insect midgut) forms acidify their environment as a consequence of glucose metabolism, generating pH gradients by diffusion. Early and late procyclic forms exhibit self-organising properties on surfaces. Both forms are attracted to alkali, but while early procyclic forms are repelled by acid and migrate outwards, late procyclic forms remain at the inoculation site. pH taxis relies on cyclic AMP signalling. Acid sensing requires a flagellar adenylate cyclase, ACP5, and a cyclic AMP response protein, CARP3, that interacts with ACP5. Deletion of the flagellar phosphodiesterase PDEB1 abolishes pH taxis completely. Trypanosomes can also respond to exogenously formed gradients. pH sensing is likely to be biologically relevant as trypanosomes experience large differences in pH as they progress through their tsetse fly host. In addition, self-generated gradients may help reinforce directionality. Moreover, since trypanosomes encode a large family of adenylate cyclases, these may govern other chemotactic responses and tissue tropisms in both the mammal and the fly.

Tissue Tropisms of Avian Influenza A Viruses Affect Their Spillovers from Wild Birds to Pigs

ABSTRACT Wild aquatic birds maintain a large, genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and, ultimately, humans. Through phenotypic analyses of viral replication efficiency, only a small set of avian IAVs were found to replicate well in epithelial cells of the swine upper respiratory tract, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait of the viral replication efficiency appears to emerge randomly and is distributed among IAVs across multiple avian species and geographic and temporal orders. It is not determined by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that phenotypic variants of viral replication efficiency exist among avian IAVs but that only a few of these may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become adapted to pigs, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations. IMPORTANCE Swine serve as a mixing vessel for generating pandemic strains of human influenza virus. All hemagglutinin subtypes of IAVs can infect swine; however, only sporadic cases of infection with avian IAVs are reported in domestic swine. The molecular mechanisms affecting the ability of avian IAVs to infect swine are still not fully understood. From the findings of phenotypic analyses, this study suggests that the tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs. It was found that this phenotype is determined not by receptor binding preference but is determined by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results show that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species and geographic and temporal orders. This study suggests an efficient way for assessment of the risk posed by avian IAVs, such as in evaluating their potentials to be transmitted from birds to pigs.

Tissue Tropisms and Transstadial Transmission of a Rickettsia Endosymbiont in the Highland Midge, Culicoides impunctatus (Diptera: Ceratopogonidae)

ABSTRACT Rickettsia is a genus of intracellular bacteria which can manipulate host reproduction and alter sensitivity to natural enemy attack in a diverse range of arthropods. The maintenance of Rickettsia endosymbionts in insect populations can be achieved through both vertical and horizontal transmission routes. For example, the presence of the symbiont in the follicle cells and salivary glands of Bemisia whiteflies allows Belli group Rickettsia transmission via the germ line and plants, respectively. However, the transmission routes of other Rickettsia bacteria, such as those in the Torix group of the genus, remain underexplored. Through fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) screening, this study describes the pattern of Torix Rickettsia tissue tropisms in the highland midge, Culicoides impunctatus (Diptera: Ceratopogonidae). Of note is the high intensity of infection of the ovarian suspensory ligament, suggestive of a novel germ line targeting strategy. Additionally, localization of the symbiont in tissues of several developmental stages suggests transstadial transmission is a major route for ensuring maintenance of Rickettsia within C. impunctatus populations. Aside from providing insights into transmission strategies, the presence of Rickettsia bacteria in the fat body of larvae indicates potential host fitness and vector capacity impacts to be investigated in the future. IMPORTANCE Microbial symbionts of disease vectors have garnered recent attention due to their ability to alter vectorial capacity. Their consideration as a means of arbovirus control depends on symbiont vertical transmission, which leads to spread of the bacteria through a population. Previous work has identified a Rickettsia symbiont present in several species of biting midges (Culicoides spp.), which transmit bluetongue and Schmallenberg arboviruses. However, symbiont transmission strategies and host effects remain underexplored. In this study, we describe the presence of Rickettsia in the ovarian suspensory ligament of Culicoides impunctatus. Infection of this organ suggests the connective tissue surrounding developing eggs is important for ensuring vertical transmission of the symbiont in midges and possibly other insects. Additionally, our results indicate Rickettsia localization in the fat body of Culicoides impunctatus. As the arboviruses spread by midges often replicate in the fat body, this location implies possible symbiont-virus interactions to be further investigated.