Rabbit hemorrhagic disease virus 2, 2010-2023: a review of global detections and affected species.

Rabbit hemorrhagic disease virus 2/genotype GI.2 (RHDV2/GI.2; Caliciviridae, Lagovirus) causes a highly contagious disease with hepatic necrosis and disseminated intravascular coagulation in several Leporidae species. RHDV2 was first detected in European rabbits (Oryctolagus cuniculus) in France in 2010 and has since spread widely. We gather here data on viral detections reported in various countries and affected species, and discuss pathology, genetic differences, and novel diagnostic aspects. RHDV2 has been detected almost globally, with cases reported in Europe, Africa, Oceania, Asia, and North America as of 2023. Since 2020, large scale outbreaks have occurred in the United States and Mexico and, at the same time, cases have been reported for the first time in previously unaffected countries, such as China, Japan, Singapore, and South Africa, among others. Detections have been notified in domestic and wild European rabbits, hares and jackrabbits (Lepus spp.), several species of cottontail and brush rabbits (Sylvilagus spp.), pygmy rabbits (Brachylagus idahoensis), and red rock rabbits (Pronolagus spp.). RHDV2 has also been detected in a few non-lagomorph species. Detection of RHDV2 causing RHD in Sylvilagus spp. and Leporidae species other than those in the genera Oryctolagus and Lepus is very novel. The global spread of this fast-evolving RNA virus into previously unexploited geographic areas increases the likelihood of host range expansion as new species are exposed; animals may also be infected by nonpathogenic caliciviruses that are disseminated by almost all species, and with which genetic recombination may occur.

First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

The cytosolic hyperoxidation-sensitive and -robust Leishmania peroxiredoxins cPRX1 and cPRX2 are both dispensable for parasite infectivity.

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.

Snapshot of the Phylogenetic Relationships among Avian Poxviruses Circulating in Portugal between 2017 and 2023.

Avipoxvirus (APV), a linear dsDNA virus belonging to the subfamily Chordopoxvirinae of the family Poxviridae, infects more than 278 species of domestic and wild birds. It is responsible for causing avian pox disease, characterized by its cutaneous and diphtheric forms. With a high transmission capacity, it can cause high economic losses and damage to the ecosystem. Several diagnostic methods are available, and bird vaccination can be an effective preventive measure. Ten APV-positive samples were analyzed to update the molecular characterization and phylogenetic analysis of viruses isolated in Portugal between 2017 and 2023. A P4b gene fragment was amplified using a PCR, and the nucleotide sequence of the amplicons was determined using Sanger sequencing. The sequences obtained were aligned using ClustalW, and a maximum likelihood phylogenetic tree was constructed. With this study, it was possible to verify that the analyzed sequences are distributed in subclades A1, A2, B1, and B3. Since some of them are quite similar to others from different countries and obtained in different years, it is possible to conclude that there have been several viral introductions in Portugal. Finally, it was possible to successfully update the data on Avipoxviruses in Portugal.

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.

Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis.

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994-1999, none MYXV-DNA positive, and n = 28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Co-infection by classic MYXV and ha-MYXV in Iberian hare (Lepus granatensis) and European wild rabbit (Oryctolagus cuniculus algirus).

Myxomatosis is an emergent disease in the Iberian hare (Lepus granatensis). In this species, the disease is caused by a natural recombinant virus (ha-myxoma virus [MYXV]) identified for the first time in 2018 and has since been responsible for a large number of outbreaks in Spain and Portugal. The ha-MYXV, which harbours a 2.8 Kb insert-disrupting gene M009L, can also infect and cause disease in wild and domestic rabbits, despite being less frequently identified in rabbits. During the laboratory investigations of wild leporids found dead in Portugal carried out within the scope of a Nacional Surveillance Plan (Dispatch 4757/17, MAFDR), co-infection events by classic (MYXV) and naturally recombinant (ha-MYXV) strains were detected in both one Iberian hare and one European wild rabbit (Oryctolagus cuniculus algirus). These two cases were initially detected by a multiplex qPCR detection of MYXV and ha-MYXV and subsequently confirmed by conventional PCR and sequencing of the M009L gene, which contains an ha-MYXV-specific insertion. To our knowledge, this is the first documented report of co-infection by classic MYXV and ha-MYXV strains either in Iberian hare or in European wild rabbit. It is also the first report of infection of an Iberian hare by a classic MYXV strain. These findings highlight the continuous evolution of the MYXV and the frequent host range changes that justify the nonstop monitoring of the sanitary condition of wild Leporidae populations in the Iberian Peninsula.

Evaluation of Commercial Myxomatosis Vaccines against Recombinant Myxoma Virus (ha-MYXV) in Iberian Hare and Wild Rabbit.

The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with the Mixohipra-H vaccine or the Nobivac Myxo-RHD PLUS (MSD Animal Health) using the recommended doses for domestic rabbits. After the vaccination trials, the animals were challenged with a wild type strain of ha-MYXV. The results showed that no protection to ha-MYXV challenge was afforded when a commercial dose of Mixohipra-FSA or Mixohipra-H vaccine was used in hares. However, the application of a higher dose of Mixohipra-FSA vaccine may induce protection and could possibly be used to counteract the accelerated decrease of wild hare populations due to ha-MYXV emergence. The two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) tested in wild rabbits were fully protective against ha-MYXV infection. This knowledge gives more insights into ha-MYXV management in hares and rabbits and emphasises the importance of developing a vaccine capable of protecting wild populations of Iberian hare and wild rabbit towards MYXV and ha-MYXV strains.

Does cultural background influence the dissemination and severity of the COVID-19 pandemic?

The COVID-19 pandemic has spread throughout the globe affecting countries worldwide. However, several differences have been observed in the number of daily new cases, the COVID-19 reproduction rate, and the severity of the disease in different countries. Previous studies have mostly highlighted government restriction policies to mitigate the pandemic effects as reasons for such differences. This study focuses on 101 countries and proposes that each country's cultural background is also accountable for such differences. We considered the six Hofstede's cultural dimensions (power distance, individualism, masculinity, uncertainty avoidance, long term orientation, and indulgence) and statistically analyzed their correlation with several COVID-19 impact metrics in comparison to several restriction policies. Our results support our claim that national culture influences both acceptance and subsequent adoption of restriction policies and the implementation by each government of those policies. We highlight that the attitudes towards and trust in political institutions, policies and governance is influenced by the cultural background, which is reflected in the pandemic numbers. As a main takeaway from this study, we conclude that data-driven models which aim at predicting the pandemic impact evolution at a global scale should also include variables that reflect the cultural background of each nation.

West Nile virus transmission potential in Portugal.

It is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country's adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966-2020), explored mosquito (2016-2019) and land type distributions (1992-2019), and used climate data (1981-2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.

Spillover events of rabbit haemorrhagic disease virus 2 (recombinant GI.4P-GI.2) from Lagomorpha to Eurasian badger.

Rabbit haemorrhagic disease (RHD) is a major threat to domestic and wild European rabbits. Presently, in Europe, the disease is caused mainly by Rabbit haemorrhagic disease virus 2 (RHDV2/b or Lagovirus europaeus GI.2), the origin of which is still unclear, as no RHDV2 reservoir hosts were identified. After the RHDV2 emergence in 2010, viral RNA was detected in a few rodent species. Furthermore, RHDV2 was found to cause disease in some hare species resembling the disease in rabbits, evidencing the ability of the virus to cross the species barrier. In this study, through molecular, histopathologic, antigenic and morphological evidences, we demonstrate the presence and replication of RHDV2 in Eurasian badgers (Meles meles) found dead in the district of Santarém, Portugal, between March 2017 and January 2020. In these animals, we further classify the RHDV2 as a Lagovirus europaeus recombinant GI.4P-GI.2. Our results indicate that Meles meles is susceptible to RHDV2, developing systemic infection, and excreting the virus in the faeces. Given the high viral loads seen in several organs and matrices, we believe that transmission to the wild rabbit is likely. Furthermore, transmission electron microscopy data show the presence of calicivirus compatible virions in the nucleus of hepatocytes, which constitutes a paradigm shift for caliciviruses' replication cycle.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I.

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I-expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I-overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2-an enzyme without a counterpart in mammals-as a candidate target for leishmanicidal drugs.

Variation and Selection in the Putative Sperm-Binding Region of ZP3 in Muroid Rodents: A Comparison between Cricetids and Murines.

In mammals, the zona pellucida glycoprotein 3 (ZP3) is considered a primary sperm receptor of the oocyte and is hypothesized to be involved in reproductive isolation. We investigated patterns of diversity and selection in the putative sperm-binding region (pSBR) of mouse ZP3 across Cricetidae and Murinae, two hyperdiverse taxonomic groups within muroid rodents. In murines, the pSBR is fairly conserved, in particular the serine-rich stretch containing the glycosylation sites proposed as essential for sperm binding. In contrast, cricetid amino acid sequences of the pSBR were much more variable and the serine-rich motif, typical of murines, was generally substantially modified. Overall, our results suggest a general lack of species specificity of the pSBR across the two muroid families. We document statistical evidence of positive selection acting on exons 6 and 7 of ZP3 and identified several amino acid sites that are likely targets of selection, with most positively selected sites falling within or adjacent to the pSBR.

Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures.

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.

Fibropapillomatosis and the Chelonid Alphaherpesvirus 5 in Green Turtles from West Africa.

Fibropapillomatosis (FP) is a tumorigenic panzootic disease of sea turtles, most common in green turtles (Chelonia mydas). FP is linked to the chelonid alphaherpesvirus 5 (ChAHV5) and to degraded habitats and, though benign, large tumours can hinder vital functions, causing death. We analyse 108 green turtles, captured in 2018 and 2019, at key foraging grounds in Guinea-Bissau and Mauritania, West Africa, for the presence of FP, and use real-time PCR to detect ChAHV5 DNA, in 76 individuals. The prevalence of FP was moderate; 33% in Guinea-Bissau (n = 36) and 28% in Mauritania (n = 72), and most turtles were mildly affected, possibly due to low human impact at study locations. Juveniles had higher FP prevalence (35%, n = 82) compared to subadults (5%, n = 21), probably because individuals acquire resistance over time. ChAHV5 DNA was detected in 83% (n = 24) of the tumour biopsies, consistent with its role as aetiological agent of FP and in 26% (n = 27) of the 'normal' skin (not showing lesions) from FP turtles. Notably, 45% of the asymptomatic turtles were positive for ChAHV5, supporting multifactorial disease expression. We report the first baselines of FP and ChAHV5 prevalence for West Africa green turtles, essential to assess evolution of disease and future impacts of anthropogenic activities.

Harmless or Threatening? Interpreting the Results of Molecular Diagnosis in the Context of Virus-Host Relationships.

Molecular methods, established in the 1980s, expanded and delivered tools for the detection of vestigial quantities of nucleic acids in biological samples. Nucleotide sequencing of these molecules reveals the identity of the organism it belongs to. However, the implications of such detection are often misinterpreted as pathogenic, even in the absence of corroborating clinical evidence. This is particularly significant in the field of virology where the concepts of commensalism, and other benign or neutral relationships, are still very new. In this manuscript, we review some fundamental microbiological concepts including commensalism, mutualism, pathogenicity, and infection, giving special emphasis to their application in virology, in order to clarify the difference between detection and infection. We also propose a system for the correct attribution of terminology in this context.

A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies.

In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.