Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques.

Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013-2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease.IMPORTANCE Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis.

Inherited Chromosomally Integrated Human Herpesvirus 6 Demonstrates Tissue-Specific RNA Expression In Vivo That Correlates with an Increased Antibody Immune Response.

Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carries one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response are still unclear. In this study, we screened DNA sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A- and 4 iciHHV-6B-positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for HHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A- and iciHHV-6B-positive individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B+ subjects were identified. Plasma samples from iciHHV-6A/B+ and age- and sex-matched controls were analyzed for antibodies to control antigens (cytomegalovirus [CMV], Epstein-Barr virus [EBV], and influenza virus [FLU]) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B+ subjects have significantly more antibodies against the U90 gene product (IE1) than do non-iciHHV-6-positive individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57, and U72, were identical between controls and iciHHV-6A/B+ subjects. CMV-seropositive individuals with iciHHV-6A/B+ have more antibodies against CMV pp150 than do CMV-seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B-specific antibody response.IMPORTANCE HHV-6A and -6B are human herpesviruses that have the unique property of being able to integrate into the telomeric regions of human chromosomes. Approximately 1% of the world's population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression data set, we showed that the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate data sets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B+ subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks, and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production.

Inflammatory Cell Infiltration of Adrenals in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was originated in November-December 2019 in Wuhan, China, and has rapidly spread around the world causing severe health and socioeconomical damage to the entire civilization. The key feature of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is upper respiratory tract infection, which may be complicated by bilateral pneumonia. Angiotensin converting enzyme 2 (ACE2) has been identified as a key host factor, required for virus entry into cells. Interestingly, ACE2 is expressed not only in the respiratory system, but also in the other organs and systems including adrenal glands. Here we provide the first description of the pathomorphological changes in adrenal glands in patients with severe COVID-19 characterized by perivascular infiltration of CD3+ and CD8+ T-lymphocytes. Due to the central role of the adrenals in the stress response of the organism, this finding is of potential clinical relevance, because infection with the SARS-CoV-2 virus might critically impair adrenal function under pathophysiological conditions.

Frequency of varicella zoster virus DNA in human adrenal glands.

Varicella zoster virus (VZV) becomes latent in ganglionic neurons derived from neural crest cells. Because the adrenal gland also contains medullary chromaffin cells of neural crest origin, we examined human adrenal glands and medullary chromaffin cell tumors (pheochromocytomas) for VZV and herpes simplex virus type 1 (HSV-1). We found VZV, but not HSV-1, DNA in 4/63 (6 %) normal adrenal glands. No VZV transcripts or antigens were detected in the 4 VZV DNA-positive samples. No VZV or HSV-1 DNA was found in 21 pheochromocytomas.

Renal and Adrenal Ultrasonography: a valuable diagnostic tool in the salt-wasting infant.

UNLABELLED: The major differential diagnosis in 'salt-wasting' infants (characterised by hyponatraemia and hyperkalaemia) is that of an adrenal or renal disorder. Appropriate management relies on rapid diagnosis, but existing guidelines do not highlight the role of ultrasonography. We describe how ultrasound may lead to a more rapid diagnosis in disorders of sex development (DSD) and other potential 'salt-wasting' infants. CONCLUSION: Ultrasonography as a diagnostic tool in infants with salt-wasting or DSD needs to be more widely recognised.

Clinical features and pathobiology of Ebolavirus infection.

There has clearly been a deluge of international press coverage of the recent outbreak of Ebolavirus in Africa and is partly related to the "fear factor" that comes across when one is confronted with the fact that once infected, not only is the speed of death in a majority of cases rapid but also the images of the cause of death such as bleeding from various orifices gruesome and frightening. The fact that it leads to infection and death of health care providers (10% during the current epidemic) and the visualization of protective gear worn by these individuals to contain such infection adds to this "fear factor". Finally, there is a clear perceived notion that such an agent can be utilized as a bioterrorism agent that adds to the apprehension. Thus, in efforts to gain an objective view of the growing threat Ebolavirus poses to the general public, it is important to provide some basic understanding for the lethality of Ebolavirus infection that is highlighted in Fig. 1. This virus infection first appears to disable the immune system (the very system needed to fight the infection) and subsequently disables the vascular system that leads to blood leakage (hemorrhage), hypotension, drop in blood pressure, followed by shock and death. The virus appears to sequentially infect dendritic cells disabling the interferon system (one of the major host anti-viral immune systems) then macrophages (that trigger the formation of blood clots, release of inflammatory proteins and nitric oxide damaging the lining of blood vessels leading to blood leakage) and finally endothelial cells that contribute to blood leakage. The virus also affects organs such as the liver (that dysregulates the formation of coagulation proteins), the adrenal gland (that destroys the ability of the patient to synthesize steroids and leads to circulation failure and disabling of regulators of blood pressure) and the gastro-intestinal tract (leading to diarrhea). The ability of the virus to disable such major mechanisms in the body facilitates the ability of the virus to replicate in an uncontrolled fashion leading to the rapidity by which the virus can cause lethality. Various laboratories have been working on defining such mechanisms utilizing in vitro culture systems, a variety of animal models including inbred strains of normal and select gene knock out mice, guinea pigs and nonhuman primates that have led to a better understanding of the potential mechanisms involved. There have also been some major advances made in the identification of therapies from the very simple (major supportive type of therapy), to the identification of a number of highly effective chemotherapeutic agents, a variety of highly effective preventive (demonstrating 100% effectiveness in nonhuman primate models) recombinant formulations (adenovirus based, VSV-based, rabies virus based), therapeutic candidate vaccines (cocktail of monoclonal antibodies such as ZMAPP) and alternate approaches (RNAi-based such as TKM-Ebola and antisense based such as AVI-7537) that show great promise and at an unprecedented rate of discovery that speaks well for the scientific research community at large.

Bilateral adrenal EBV-associated smooth muscle tumors in a child with a natural killer cell deficiency.

EBV-associated smooth muscle tumors are found in immunocompromised patients, most commonly HIV/AIDS. We present a 12-year-old girl with the first documented case of EBV-related smooth muscle tumors in the presence of a rare classic NK cell deficiency. This sheds light on the role of NK cells in controlling EBV-related smooth muscle tumors.

Pathogenesis of highly pathogenic avian influenza A virus (H7N1) infection in chickens inoculated with three different doses.

To study the pathogenesis of a H7N1 highly pathogenic avian influenza virus strain, specific pathogen free chickens were inoculated with decreasing concentrations of virus: 10(5.5) median embryo lethal dose (ELD(50)) (G1), 10(3.5) ELD(50) (G2) and 10(1.5) ELD(50) (G3). Disease progression was monitored over a period of 16 days and sequential necropsies and tissue samples were collected for histological and immunohistochemical examination. Viral RNA loads were also quantified in different tissues, blood, oropharyngeal swabs, and cloacal swabs using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Clinical signs of depression, apathy, listlessness, huddling and ruffled feathers were recorded in G1 and a few G2 birds, whilst neurological signs were only observed in chickens inoculated with the highest dose. Gross lesions of haemorrhages were observed in the unfeathered skin of the comb and legs, and skeletal muscle, lung, pancreas and kidneys of birds inoculated with 10(5.5) ELD(50) and 10(3.5) ELD(50) doses. Microscopic lesions and viral antigen were demonstrated in cells of the nasal cavity, lung, heart, skeletal muscle, brain, spinal cord, gastrointestinal tract, pancreas, liver, bone marrow, thymus, bursa of Fabricius, spleen, kidney, adrenal gland and skin. Viral RNA was detected by RT-qPCR in kidney, lung, intestine, and brain samples of G1 and G2 birds. However, in birds infected with the lowest dose, viral RNA was detected only in brain and lung samples in low amounts at 5 and 7 days post infection. Interestingly, viral shedding was observed in oropharyngeal and cloacal swabs with proportionate decrease with the inoculation dose. We conclude that although an adequate infectious dose is critical in reproducing the clinical infection, chickens exposed to lower doses can be infected and shed virus representing a risk for the dissemination of the viral agent.

Histopathological Analysis of Adrenal Glands after Simian Varicella Virus Infection.

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with ßIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with "long-hauler" symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

Is there impact of the SARS-CoV-2 pandemic on steroidogenesis and fertility?

In December of 2019, several cases of unknown atypical respiratory diseases emerged in Wuhan, Hubei Province in China. After preliminary research, it was stated that the disease is transmittable between humans and was named COVID-19. Over the course of next months, it spread all over the world by air and sea transport and caused a global pandemic which affects life of everyone now-a-days. A large number of countries, have since been forced to take precautions such as curfews, lockdowns, wearing facemasks etc. Even with vaccines being produced in mass numbers, lack of targeted therapy continues to be a major problem. According to studies so far it seems that elderly people are more vulnerable to severe symptoms while children tend to by asymptomatic or have milder form the disease. In our review, we focused on gathering data about the virus itself, its characteristics, paths of transmission, and its effect on hormone production and secretion. In such, there is insufficient information in the literature worldwide, especially the ones that focus on the effect of COVID-19 on individual organs systems within the human body. Hence, the present evidence-based study focused on the possible effects of COVID-19 on adrenal gland and gonads i.e. on the process of steroidogenesis and fertility.

Apoptosis induction after herpes simplex virus infection differs according to cell type in vivo.

We compared apoptosis induction in mice following three routes of infection. After intravenous infection, wild-type herpes simplex virus (HSV) types 1 and 2 and US3Delta mutants infected the adrenal gland and caused apoptosis. Corneal infection with wild-type virus resulted in apoptosis in a fraction of infected epithelium cells. Interestingly, many uninfected cells were apoptotic in the retina. Although neurons in the trigeminal ganglion were heavily infected, no apoptotic neurons were observed. Intracranial infection with wild-type virus resulted in HSV-infected cells inside the brain; however, most of the infected neurons escaped apoptosis. In contrast, infection with US3Delta and gamma(1)34.5Delta mutants caused apoptosis in infected neurons. Cleaved caspase-8 and p53 were detected in apoptotic cells in the adrenal gland and the brain; however, phospho-JNK was detected only in apoptotic cells of the brain. These results suggest that the activation of apoptotic signaling proteins differs depending on the host cell type and modulates the induction of apoptosis in HSV-infected cells.

Differential Growth Characteristics of Crimean-Congo Hemorrhagic Fever Virus in Kidney Cells of Human and Bovine Origin.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a lethal tick-borne zoonotic disease with severe clinical manifestation in humans but does not produce symptomatic disease in wild or domestic animals. The factors contributing to differential outcomes of infection between species are not yet understood. Since CCHFV is known to have tropism to kidney tissue and cattle play an important role as an amplifying host for CCHFV, in this study, we assessed in vitro cell susceptibility to CCHFV infection in immortalized and primary kidney and adrenal gland cell lines of human and bovine origin. Based on our indirect fluorescent focus assay (IFFA), we suggest a cell-to-cell CCHF viral spread process in bovine kidney cells but not in human cells. Over the course of seven days post-infection (dpi), infected bovine kidney cells are found in restricted islet-like areas. In contrast, three dpi infected human kidney or adrenal cells were noted in areas distant from one another yet progressed to up to 100% infection of the monolayer. Pronounced CCHFV replication, measured by quantitative real-time RT-PCR (qRT-PCR) of both intra- and extracellular viral RNA, was documented only in human kidney cells, supporting restrictive infection in cells of bovine origin. To further investigate the differences, lactate dehydrogenase activity and cytopathic effects were measured at different time points in all mentioned cells. In vitro assays indicated that CCHFV infection affects human and bovine kidney cells differently, where human cell lines seem to be markedly permissive. This is the initial reporting of CCHFV susceptibility and replication patterns in bovine cells and the first report to compare human and animal cell permissiveness in vitro. Further investigations will help to understand the impact of different cell types of various origins on the virus-host interaction.

Novel borna virus in psittacine birds with proventricular dilatation disease.

Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.

Transneuronal retrograde viral labeling in the brain stem and hypothalamus is more intense from the left than from the right adrenal gland.

Previous studies using the viral transneuronal tracing technique demonstrated central autonomic circuits involved in the innervation of the adrenal gland. Since increasing number of data indicate laterality in the neuroendocrine system, we aimed to investigate whether the supraspinal innervation of the adrenal gland exhibits asymmetry or not. The central circuitry involved in the innervation of the left and the right adrenal gland was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (Ba-DupGreen and Ba-Duplac expressing lacZ) of Bartha strain of pseudorabies virus. Viral infection of brain nuclei (dorsal vagal nucleus, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, hypothalamic paraventricular nucleus) from the left adrenal was more severe than that from the right organ. Dual-infected neurons were present both in the brain stem and in the hypothalamus. The results indicate a predominance in the supraspinal innervation of the left adrenal gland, and that each adrenal gland is innervated both by side-specific neurons and by neurons that project to both organs.

Pathobiology of Asian highly pathogenic avian influenza H5N1 virus infections in ducks.

Ducks and other wild aquatic birds are the natural reservoir of type A influenza viruses, which normally are nonpathogenic in these birds. However, the Asian highly pathogenic avian influenza (HPAI) viruses have evolved from producing no disease or mild respiratory infections in ducks to some strains producing severe systemic disease and mortality. To further understand the pathogenicity of these strains in ducks, we studied the gross and histologic lesions and tissue distribution of viral antigen in 2- and 5-wk-old white Pekin ducks infected with different Asian-origin H5N1 AI viruses. Seven of eight 2-wk-old ducks inoculated with A/Egret/HK/757.2/02 developed acute disease, including severe neurological dysfunction and death. However, this virus killed only two of eight 5-wk-old ducks. Two additional viruses, A/Vietnam/1203/04 and A/Crow/Thailand/04, also produced high mortality in 2-wk-old ducks. Microscopic lesions and AI viral antigen were observed most frequently in the nasal cavity, brain, heart, adrenal glands, and pancreas. Another virus, A/Thailand PB/6231/04, killed three of eight 2-wk-old ducks but did not induce neurological signs. Furthermore, older ducks infected with this virus did not present clinical signs or gross lesions, and their tissues showed very few microscopic lesions. All the viruses studied established systemic infections in both younger and older ducks, with viral replication in tissues correlating with the severity of the clinical signs. The differences in mortality induced by HPAI H5N1 viruses in ducks are reflected in the pathological findings and antigen distribution in tissues. However, the observed differences in pathology between ducks infected at different ages is unclear and may be associated with a variety of factors including the virus strain, host immune response, host cell maturation, and capacity to support viral replication.

The supraspinal innervation of the left adrenal is more intense than that of the right one.

BACKGROUND AND PURPOSE: Previous studies using the viral transneuronal tracing technique demonstrated that central autonomic circuits are involved in the innervation of the adrenal gland. Since increasing number of data indicate laterality in the neuroendocrine system, we aimed to investigate whether the supraspinal innervation of the adrenal gland exhibits asymmetry or not. METHODS: The central circuitry involved in the innervation of the left and the right adrenal gland was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (BDG and BDL) of Bartha strain of pseudorabies virus. RESULTS: Viral infection of brain nuclei (dorsal vagal nucleus, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, hypothalamic paraventricular nucleus) from the left adrenal was more severe than that from the right organ. Dual-infected neurons from the two adrenals were also detected both in the brain stem and in the hypothalamus. CONCLUSION: The results indicate a predominance in the supraspinal innervation of the left adrenal gland. Data further suggest that each adrenal gland is innervated both by side-specific neurons and by neurons which project to both organs.

Development of real-time PCRs for detection and quantitation of human MMTV-like (HML) sequences HML expression in human tissues.

The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1-10, also referred to as human endogenous retrovirus "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.