A recent gibbon ape leukemia virus germline integration in a rodent from New Guinea.

Germline colonization by retroviruses results in the formation of endogenous retroviruses (ERVs). Most colonization's occurred millions of years ago. However, in the Australo-Papuan region (Australia and New Guinea), several recent germline colonization events have been discovered. The Wallace Line separates much of Southeast Asia from the Australo-Papuan region restricting faunal and pathogen dispersion. West of the Wallace Line, gibbon ape leukemia viruses (GALVs) have been isolated from captive gibbons. Two microbat species from China appear to have been infected naturally. East of Wallace's Line, the woolly monkey virus (a GALV) and the closely related koala retrovirus (KoRV) have been detected in eutherians and marsupials in the Australo-Papuan region, often vertically transmitted. The detected vertically transmitted GALV-like viruses in Australo-Papuan fauna compared to sporadic horizontal transmission in Southeast Asia and China suggest the GALV-KoRV clade originates in the former region and further models of early-stage genome colonization may be found. We screened 278 samples, seven bat and one rodent family endemic to the Australo-Papuan region and bat and rodent species found on both sides of the Wallace Line. We identified two rodents (Melomys) from Australia and Papua New Guinea and no bat species harboring GALV-like retroviruses. Melomys leucogaster from New Guinea harbored a genomically complete replication-competent retrovirus with a shared integration site among individuals. The integration was only present in some individuals of the species indicating this retrovirus is at the earliest stages of germline colonization of the Melomys genome, providing a new small wild mammal model of early-stage genome colonization.

Analysis of Sporulation in Bacillus cereus Biovar anthracis Which Contains an Insertion in the Gene for the Sporulation Factor σK.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction-modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

The domed architecture of Giardias ventral disc is necessary for attachment and host pathogenesis.

After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 um in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened crescent or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.

First virological and pathological study of Göttingen Minipigs with Dippity Pig Syndrome (DPS).

Dippity Pig Syndrome (DPS) is a well-known but rare complex of clinical signs affecting minipigs, which has not been thoroughly investigated yet. Clinically affected animals show acute appearance of red, exudating lesions across the spine. The lesions are painful, evidenced by arching of the back (dipping), and the onset of clinical signs is generally sudden. In order to understand the pathogenesis, histological and virological investigations were performed in affected and unaffected Göttingen Minipigs (GöMPs). The following DNA viruses were screened for using PCR-based methods: Porcine cytomegalovirus (PCMV), which is a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3), porcine circoviruses (PCV1, PCV2, PCV3, PCV4), porcine parvovirus 1 (PPV1), and Torque Teno sus viruses (TTSuV1, TTSuV2). Screening was also performed for integrated porcine endogenous retroviruses (PERV-A, PERV-B, PERV-C) and recombinant PERV-A/C and their expression as well as for the RNA viruses hepatitis E virus (HEV) and SARS-CoV-2. Eight clinically affected and one unaffected GöMPs were analyzed. Additional unaffected minipigs had been analyzed in the past. The analyzed GöMPs contained PERV-A and PERV-B integrated in the genome, which are present in all pigs and PERV-C, which is present in most, but not all pigs. In one affected GöMPs recombinant PERV-A/C was detected in blood. In this animal a very high expression of PERV mRNA was observed. PCMV/PRV was found in three affected animals, PCV1 was found in three animals with DPS and in the unaffected minipig, and PCV3 was detected in two animals with DPS and in the unaffected minipig. Most importantly, in one animal only PLHV-3 was detected. It was found in the affected and unaffected skin, and in other organs. Unfortunately, PLHV-3 could not be studied in all other affected minipigs. None of the other viruses were detected and using electron microscopy, no virus particles were found in the affected skin. No porcine virus RNA with exception of PERV and astrovirus RNA were detected in the affected skin by next generation sequencing. This data identified some virus infections in GöMPs with DPS and assign a special role to PLHV-3. Since PCMV/PRV, PCV1, PCV3 and PLHV-3 were also found in unaffected animals, a multifactorial cause of DPS is suggested. However, elimination of the viruses from GöMPs may prevent DPS.

A Family of Spore Lipoproteins Stabilizes the Germination Apparatus by Altering Inner Spore Membrane Fluidity in Bacillus subtilis Spores.

Dormant bacterial spores undergo the process of germination to return to a vegetative state. In most species, germination involves the sensing of nutrient germinants, the release of various cations and a calcium-dipicolinic acid (DPA) complex, spore cortex degradation, and full rehydration of the spore core. These steps are mediated by membrane-associated proteins, and all these proteins have exposure on the outer surface of the membrane, a hydrated environment where they are potentially subject to damage during dormancy. A family of lipoproteins, including YlaJ, which is expressed from the sleB operon in some species, are present in all sequenced Bacillus and Clostridium genomes that contain sleB. B. subtilis possesses four proteins in this family, and prior studies have demonstrated two of these are required for efficient spore germination and these proteins contain a multimerization domain. Genetic studies of strains lacking all combinations of these four genes now reveal all four play roles in ensuring efficient germination, and affect multiple steps in this process. Electron microscopy does not reveal significant changes in spore morphology in strains lacking lipoproteins. Generalized polarization measurements of a membrane dye probe indicate the lipoproteins decrease spore membrane fluidity. These data suggest a model in which the lipoproteins form a macromolecular structure on the outer surface of the inner spore membrane, where they act to stabilize the membrane and potentially interact with other germination proteins, and thus stabilize the function of multiple components of the germination machinery. IMPORTANCE Bacterial spores exhibit extreme longevity and resistance to many killing agents, and are thus problematic agents of several diseases and of food spoilage. However, to cause disease or spoilage, germination of the spore and return to the vegetative state is necessary. The proteins responsible for initiation and progression of germination are thus potential targets for spore-killing processes. A family of membrane-bound lipoproteins that are conserved across most spore-forming species was studied in the model organism Bacillus subtilis. The results indicate that these proteins reduce the membrane fluidity and increase the stability of other membrane associated proteins that are required for germination. Further understanding of such protein interactions on the spore membrane surface will enhance our understanding of the germination process and its potential as a decontamination method target.

Facile modification of phosphole-based aggregation-induced emission luminogens with sulfonyl isocyanates.

Phosphole oxides undergo a highly chemoselective reaction with sulfonyl isocyanates forming sulfonylimino phospholes in high yields. This facile modification proved to be a powerful tool for obtaining new phosphole-based aggregation-induced emission (AIE) luminogens with high fluorescence quantum yields in the solid state. Changing the chemical environment of the phosphorus atom of the phosphole framework results in a significant shift of the fluorescence maximum to longer wavelengths.

Distinct tissue niches direct lung immunopathology via CCL18 and CCL21 in severe COVID-19.

Prolonged lung pathology has been associated with COVID-19, yet the cellular and molecular mechanisms behind this chronic inflammatory disease are poorly understood. In this study, we combine advanced imaging and spatial transcriptomics to shed light on the local immune response in severe COVID-19. We show that activated adventitial niches are crucial microenvironments contributing to the orchestration of prolonged lung immunopathology. Up-regulation of the chemokines CCL21 and CCL18 associates to endothelial-to-mesenchymal transition and tissue fibrosis within these niches. CCL21 over-expression additionally links to the local accumulation of T cells expressing the cognate receptor CCR7. These T cells are imprinted with an exhausted phenotype and form lymphoid aggregates that can organize in ectopic lymphoid structures. Our work proposes immune-stromal interaction mechanisms promoting a self-sustained and non-resolving local immune response that extends beyond active viral infection and perpetuates tissue remodeling.

Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy.

BACKGROUND: SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. METHODS: Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. RESULTS: Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. CONCLUSIONS: The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.

SARS-CoV-2 Omicron variant is attenuated for replication in a polarized human lung epithelial cell model.

SARS-CoV-2 and its emerging variants of concern remain a major threat for global health. Here we introduce an infection model based upon polarized human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells grown at the air-liquid interface to estimate replication and epidemic potential of respiratory viruses in the human lower respiratory tract. hAELVI cultures are highly permissive for different human coronaviruses and seasonal influenza A virus and upregulate various mediators following virus infection. Our analysis revealed a significantly reduced capacity of SARS-CoV-2 Omicron BA.1 and BA.2 variants to propagate in this human model compared to earlier D614G and Delta variants, which extends early risk assessments from epidemiological and animal studies suggesting a reduced pathogenicity of Omicron.

Colony growth and biofilm formation of Aspergillus niger under simulated microgravity.

The biotechnology- and medicine-relevant fungus Aspergillus niger is a common colonizer of indoor habitats such as the International Space Station (ISS). Being able to colonize and biodegrade a wide range of surfaces, A. niger can ultimately impact human health and habitat safety. Surface contamination relies on two key-features of the fungal colony: the fungal spores, and the vegetative mycelium, also known as biofilm. Aboard the ISS, microorganisms and astronauts are shielded from extreme temperatures and radiation, but are inevitably affected by spaceflight microgravity. Knowing how microgravity affects A. niger colony growth, in particular regarding the vegetative mycelium (biofilm) and spore production, will help prevent and control fungal contaminations in indoor habitats on Earth and in space. Because fungal colonies grown on agar can be considered analogs for surface contamination, we investigated A. niger colony growth on agar in normal gravity (Ground) and simulated microgravity (SMG) conditions by fast-clinorotation. Three strains were included: a wild-type strain, a pigmentation mutant (ΔfwnA), and a hyperbranching mutant (ΔracA). Our study presents never before seen scanning electron microscopy (SEM) images of A. niger colonies that reveal a complex ultrastructure and biofilm architecture, and provide insights into fungal colony development, both on ground and in simulated microgravity. Results show that simulated microgravity affects colony growth in a strain-dependent manner, leading to thicker biofilms (vegetative mycelium) and increased spore production. We suggest that the Rho GTPase RacA might play a role in A. niger's adaptation to simulated microgravity, as deletion of ΔracA leads to changes in biofilm thickness, spore production and total biomass. We also propose that FwnA-mediated melanin production plays a role in A. niger's microgravity response, as ΔfwnA mutant colonies grown under SMG conditions showed increased colony area and spore production. Taken together, our study shows that simulated microgravity does not inhibit A. niger growth, but rather indicates a potential increase in surface-colonization. Further studies addressing fungal growth and surface contaminations in spaceflight should be conducted, not only to reduce the risk of negatively impacting human health and spacecraft material safety, but also to positively utilize fungal-based biotechnology to acquire needed resources in situ.

Assessing and improving the validity of COVID-19 autopsy studies - A multicentre approach to establish essential standards for immunohistochemical and ultrastructural analyses.

BACKGROUND: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. METHODS: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FINDINGS: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and provide recommendations for optimized sampling and analysis. 135 of 144 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM sections as a reference and for training purposes. INTERPRETATION: Since detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. FUNDING: German Federal Ministry of Health, German Federal Ministry of Education and Research, Berlin University Alliance, German Research Foundation, German Center for Infectious Research.

The Sensilla-Specific Expression and Subcellular Localization of SNMP1 and SNMP2 Reveal Novel Insights into Their Roles in the Antenna of the Desert Locust Schistocerca gregaria.

Insect olfactory sensilla house olfactory sensory neurons (OSNs) and supports cells (SCs). The olfactory sensory processes require, besides the odorant receptors (ORs), insect-specific members of the CD36 family, named sensory neuron membrane proteins (SNMPs). While SNMP1 is considered to act as a coreceptor in the OR-mediated detection of pheromones, SNMP2 was found to be expressed in SCs; however, its function is unknown. For the desert locust, Schistocerca gregaria, we previously visualized mRNA for SNMP1 in OSNs and SNMP2 mRNA in cells associated with OSN clusters. Towards an understanding of their functional implication, it is imperative to explore the cellular and the subcellular localization the SNMP proteins. Therefore, we have generated polyclonal antibodies against SNMP1 and SNMP2 and used fluorescence immunohistochemistry (FIHC) to visualize the SNMP proteins. We found SNMP1 in the somata and respective dendrites of all OSNs in trichoid sensilla and in subsets of OSNs in basiconic sensilla. Notably, SNMP1 was also detected in SCs of these sensilla types. In contrast, SNMP2 protein was only visualized in SCs of basiconic and coeloconic sensilla, but not of trichoid sensilla. Exploring the subcellular localization by electron microscopy using anti-SNMP1-ab and anti-SNMP2-ab revealed an immunogold labelling of SC microvilli bordering the sensillum lymph. Together our findings suggest a dual role of SNMP1 in the antenna of S. gregaria, in some OSN subpopulations in odor detection as well as in functions of some SCs, whereas the role of SNMP2 is limited to the functions of support cells.

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

Ultrastructural examination of lung "cryobiopsies" from a series of fatal COVID-19 cases hardly revealed infected cells.

Ultrastructural analysis of autopsy samples from COVID-19 patients usually suffers from significant structural impairment possibly caused by the rather long latency between death of the patient and an appropriate sample fixation. To improve structural preservation of the tissue, we obtained samples from ventilated patients using a trans-bronchial "cryobiopsy" within 30 min after their death and fixed them immediately for electron microscopy. Samples of six COVID-19 patients with a documented histopathology were systematically investigated by thin section electron microscopy. The different samples and areas inspected revealed the ultrastructural correlates of the different phases of diffuse alveolar damage, including detachment of the alveolar epithelium, hyperplasia of type 2 cells, exudates, and accumulation of extracellular material, such as the hyaline membranes and fibrin. Macrophages and neutrophilic granulocytes were regularly detected. Structural integrity of endothelium was intact in regions where the alveolar epithelium was already detached. Aggregates of erythrocytes, leukocytes with fibrin, and thrombocytes were not observed. Coronavirus particles were only found in and around very few cells in one of the six patient samples. The type and origin of these cells could not be assessed although the overall structural preservation of the samples allowed the identification of pulmonary cell types. Hence, the observed alveolar damage is not associated with virus presence or structural impairment due to ongoing replication at later stages of the disease in fatal cases, which implies that the lung damage in these patients is at least propagated by alternative mechanisms, perhaps, an inappropriate immune or stress response.

Rare isolation of human-tropic recombinant porcine endogenous retroviruses PERV-A/C from Göttingen minipigs.

BACKGROUND: Porcine endogenous retroviruses (PERVs) can infect human cells and pose a risk for xenotransplantation when pig cells, tissues or organs are transplanted to human recipients. Xenotransplantation holds great promise to overcome the shortage of human donor organs after solving the problems of rejection, functionality and virus safety. We recently described the transmission of a human-tropic recombinant PERV-A/C, designated PERV-F, from peripheral blood mononuclear cells (PBMCs) of a Göttingen Minipig (GöMP) to human 293 cells (Krüger et al., in Viruses 12(1):38, 2019). The goal of this study was to characterize PERV-F in more detail and to analyze the probability of virus isolation from other animals. METHODS: The recombination site in the envelope (env) gene, the long terminal repeats (LTR), the proteins and the morphology of the recombinant PERV-F were characterized by polymerase chain reaction (PCR), sequencing, Western blot analysis, immunofluorescence, and transmissible electron microscopy. Mitogen-stimulated PBMCs from 47 additional pigs, including 17 new GöMP, were co-cultured with highly susceptible human 293 T cells, and the PERV-A/C prevalence and PERV transmission was analyzed by PCR. RESULTS: PERV-F, isolated from a GöMP, is an infectious human-tropic PERV-A/C virus with a novel type of recombination in the env gene. The length of the LTR of PERV-F increased after passaging on human cells. In a few minipigs, but not in German landrace pigs, PERV-A/C were found. There was no transmission of human-tropic PERV-A/C from additional 47 pigs, including 17 GöMP, to human cells. CONCLUSION: These data show that human-tropic recombinant PERV-A/C proviruses can only be found in a very small number of minipigs, but not in other pigs, and that their isolation as infectious virus able to replicate on human cells is an extremely rare event, even when using highly susceptible 293 cells.

Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway.

Apicomplexan parasites, such as Toxoplasma gondii, are unusual in that each cell contains a single apicoplast, a plastid-like organelle that compartmentalizes enzymes involved in the essential 2C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis. The last two enzymatic steps in this organellar pathway require electrons from a redox carrier. However, the small iron-sulfur cluster-containing protein ferredoxin, a likely candidate for this function, has not been investigated in this context. We show here that inducible knockdown of T. gondii ferredoxin results in progressive inhibition of growth and eventual parasite death. Surprisingly, this phenotype is not accompanied by ultrastructural changes in the apicoplast or overall cell morphology. The knockdown of ferredoxin was instead associated with a dramatic decrease in cellular levels of the last two metabolites in isoprenoid biosynthesis, 1-hydroxy-2-methyl-2-(E)- butenyl-4-pyrophosphate, and isomeric dimethylallyl pyrophosphate/isopentenyl pyrophosphate. Ferredoxin depletion was also observed to impair gliding motility, consistent with isoprenoid metabolites being important for dolichol biosynthesis, protein prenylation, and modification of other proteins involved in motility. Significantly, pharmacological inhibition of isoprenoid synthesis of the host cell exacerbated the impact of ferredoxin depletion on parasite replication, suggesting that the slow onset of parasite death after ferredoxin depletion is because of isoprenoid scavenging from the host cell and leading to partial compensation of the depleted parasite metabolites upon ferredoxin knockdown. Overall, these findings show that ferredoxin has an essential physiological function as an electron donor for the 2C-methyl-D-erythritol 4-phosphate pathway and is a potential drug target for apicomplexan parasites.