Molecular characterisation of fowl adenovirus associated with hydropericardium hepatitis syndrome in broiler and layer breeders in Azerbaijan.

BACKGROUND: Fowl adenovirus-4 is a causative agent of hydropericardium hepatitis syndrome (HHS) in chickens and has been frequently reported from many countries. Fowl adenoviruses cause severe disease and mortality in broiler and layer breeders in Azerbaijan. Therefore, in this study, pathological lesions and the dissemination of fowl adenovirus-4 into the visceral organs of infected birds were investigated as well as molecular characterisation of detected strains. For this, liver, heart and spleen from 20 necropsied chickens originated from a broiler breeder flock and a layer breeder flock were embeded on the FTA cards and the samples were analysed for adenovirus-DNA by PCR and sequencing. RESULTS: The findings of necropsy in both broiler and layer breeder chickens were similar, and the liver was severely effected showing hepatitis, and the heart with hydropericardium lesions. The kidneys were swollen with haemorrhages and small white foci on the surface of the spleens were noted. Intestinal congestion and ecchymotic hemorrhages were also observed in some birds. Fowl adenovirus-4-DNA was detected by PCR in all collected organs of 20 birds. The sequence analysis revealed that fowl adenovirus-4 present in Azerbaijan and close similarity of the hexon genes of the adenoviruses existing in the Middle East, North America, far east and Indian subcontinent were determined by phylogenetic analysis. However, sequence diversity was detected from the adenovirus strains circulating in Europe, North and South America. CONCLUSIONS: This study indicates the impact of fowl adenovirus-4 on the poultry health and production, and improved disease control and prevention strategies are necessary to reduce the HHS disease in chickens in Azerbaijan.

The diagnosis and molecular epidemiology investigation of avian hepatitis E in Shandong province, China.

BACKGROUND: Avian hepatitis E virus (HEV) is the pathogenic agent of big liver and spleen disease (BLS) and of hepatitis-splenomegaly syndrome (HSS) in chickens, which have caused economic losses to the poultry industry in China. In this study, 18 samples of BLS chickens were collected to reveal the molecular epidemiological characteristics of avian HEV in the province of Shandong, China. RESULTS: Gross and microscopic lesions of clinical samples were observed; then, virology detection and genetic analysis of avian HEV were performed. The results showed that there was significant swelling and rupture in the liver and that the spleen was enlarged. Microscopic lesions demonstrated obvious hemorrhage in the liver, with infiltration of heterophilic granulocytes, lymphocytes, and macrophages, as well as the reduction of lymphocytes in the spleen. Eleven of the 18 samples were positive for avian HEV, with a positive rate of 61.11%. More importantly, all avian HEV-positive samples were mixed infections: among these, the mixed infections of avian HEV and chicken infectious anemia virus (CIAV) and avian HEV and fowl adenovirus (FAdV) were the most common. Furthermore, the genetic evolution analysis showed that all avian HEV strains obtained here did not belong to the reported 4 genotypes, thus constituting a potential novel genotype. CONCLUSIONS: These results of this study further enrich the epidemiological data on avian HEV in Shandong, prove the genetic diversity of avian HEV in China, and uncover the complex mixed infections of avian HEV clinical samples.

Avian hepatitis E virus is widespread among chickens in Poland and belongs to genotype 2.

Big liver and spleen disease, caused by avian hepatitis E virus, has been reported in Poland, but the prevalence of the virus has not yet been investigated. In this study, 1034 serum samples from 57 breeder broiler and laying hen flocks were screened for the presence of anti-aHEV antibodies. In a random serology study, 56.1% of flocks were positive. Seroprevalence was higher in laying hen flocks than in broiler breeder flocks. Phylogenetic analysis of partial ORF1 and ORF2 sequences revealed that all Polish isolates belonged to genotype 2. This is the first time this genotype has been detected in Central Europe.

Meta-transcriptomic analysis reveals a new subtype of genotype 3 avian hepatitis E virus in chicken flocks with high mortality in Guangdong, China.

BACKGROUND: Hepatitis E virus (HEV) is one of most important zoonotic viruses, and it can infect a wide range of host species. Avian HEV has been identified as the aetiological agent of big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens. HEV infection is common among chicken flocks in China, and there are currently no practical measures for preventing the spread of the disease. The predominant avian HEV genotype circulating in China have been identified as genotype 3 strains, although some novel genotypes have also been identified from chicken flocks in China. RESULTS: In this study, we used a meta-transcriptomics approach to identify a new subtype of genotype 3 avian HEV in broiler chickens at a poultry farm located in Shenzhen, Guangdong Province, China. The complete genome sequence of the avian HEV, designated CaHEV-GDSZ01, is 6655-nt long, including a 5' UTR of 24 nt and a 3' UTR of 125 nt (excluding the poly(A) tail), and contains three open reading frames (ORFs). Sequence analysis indicated that the complete ORF1 (4599 nt/1532 aa), ORF2 (1821 nt/606 aa) and ORF3 (264 nt/87 aa) of CaHEV-GDSZ01 share the highest nucleotide sequence identity (85.8, 86.7 and 95.8%, respectively) with the corresponding ORFs of genotype 3 avian HEV. Phylogenetic analyses further demonstrated that the avian HEV identified in this study is a new subtype of genotype 3 avian HEV. CONCLUSIONS: Our results demonstrate that a new subtype of genotype 3 avian HEV is endemic in Guangdong, China, and could cause high mortality in infected chickens. This study also provides full genomic data for better understanding the evolutionary relationships of avian HEV circulating in China. Altogether, the results presented in this study suggest that more attention should be paid to avian HEV and its potential disease manifestation.

Molecular characterization of fowl aviadenoviruses species D and E associated with inclusion body hepatitis in chickens and falcons indicates possible cross-species transmission.

During the period from 2015 to 2017, frequent outbreaks of inclusion body hepatitis (IBH) were observed in broiler chickens and falcons in Saudi Arabia. Fifty samples were collected from both species. The histopathological examination and polymerase chain reaction confirmed the IBH infection in eight samples (five samples from chickens and three samples from falcons). The genomic sequence and phylogenetic analysis based on nucleotide and amino acid sequences of Saudi strains, reference fowl aviadenoviruses (FAdVs) and field viruses available in Genbank revealed that all investigated FAdVs clustered into FAdV-2 (species D) and FAdV-6 (species E). The host-dependent characterization revealed that falcon origin strains showed low identity (∼35%) with falcon adenoviruses isolated from USA, which clustered into a separate group. The identification of FAdV-D and FAdV-E in diseased falcons and chickens indicates cross-species transmission although falcons and chickens are phylogenetically different. The control of IBH infection in falcons and chickens should be based on the separation of carriers and susceptible chickens as well as falcons to prevent cross-species contact. Vaccination is an important method for prevention of IBH. The characterization of newly emerging FAdV strains provides valuable information for the development of an efficacious control strategy based on the molecular structure of current circulating FAdV strains in different species of birds.

Production-level risk factors for syncytial hepatitis in farmed tilapia (Oreochromis niloticus L).

Syncytial hepatitis (SHT) is an emerging viral disease of tilapia characterized by significant morbidity and mortality. This study aimed to establish the production-level risk factors associated with presence and severity of SHT. Production factors were analysed during multiple outbreaks of SHT that occurred between 2011 and 2013 on a single tilapia farm in Ecuador and compared with the year 2010 before the SHT outbreaks. Relative risks, t tests, modified Poisson and forward stepwise linear regression analyses were performed using EPIINFO™. Compared to other strains, Chitralada had an elevated risk of SHT [RR = 2.1 (95%CI 1.8-2.4)]. Excessive mortality associated with the presence (and severity) of SHT increased by 611 (365), 6,814 (5,768) and 388 (340) deaths per 100,000 fry when stocking density, dissolved oxygen and pond production cycles were raised by 1 fish/m2 , 1 mg/L and 1 cycle, respectively. Excessive mortality associated with the presence (and severity) of SHT decreased by 337 (258) and 1,354 (1,025) deaths per 100,000 when stocking weight and water temperature increased by 1 g and 1°C, respectively. Time (season and stocking year) was not significantly associated with SHT. This study shows that some production factors increase the risk incidence and severity of SHTon a farm.

New Avian Hepadnavirus in Palaeognathous Bird, Germany.

In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous (Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.

Epidemiological and pathological investigation of fowl aviadenovirus serotypes 8b and 11 isolated from chickens with inclusion body hepatitis in Spain (2011-2013).

Inclusion body hepatitis caused by different fowl aviadenovirus (FAdV) serotypes has been described in several countries in recent years. In Spain, from the spring of 2011 to 2013, an increased number of outbreaks in broiler and broiler breeder flocks from different regions occurred. The objectives of the present work were to carry out the molecular characterization of FAdV strains from Spanish inclusion body hepatitis cases and to study the pathogenicity and viral dynamics of these strains in specific pathogen-free (SPF) chickens. A total of 52 inclusion body hepatitis clinical cases, including 45 from broiler farms and seven from broiler breeder farms, were analysed by conventional polymerase chain reaction and sequencing targeting the FAdV hexon gene. From these, 37 strains were classified as FAdV type 8b, while the remaining 15 were classified as FAdV types 11 (n = 10), 2 (n = 4) and 8a (n = 1). In addition, two different FAdVs belonging to the genotypes 8b and 11 were used for experimental infection. Specific pathogen-free five-day-old birds were inoculated intramuscularly with a high (106.5 tissue culture infective dose (TCID)50/ml) or low (104 TCID50/ml) dose of the above-mentioned FAdVs. No mortality was observed in any of the experimental groups, and only one bird showed evident clinical signs. However, macroscopic and microscopic hepatic lesions, as well as viral DNA, were detected in birds from all infection groups. Inclusion bodies and viral DNA were also detected in the pancreas and in the small and the large intestine in some birds. Long-lasting shedding and transmission to contact birds were confirmed in all infected groups.

Molecular epidemiology and genetic diversity of duck hepatitis A virus type 3 in Shandong province of China, 2012-2014.

 The infections with duck hepatitis A virus type 3 (DHAV-3) become common in eastern Asia. To better understand the molecular evolution and genetic variation of DHAV-3, a total of 482 dead Cherry Valley duckling liver samples collected from Shandong province of China during 2012-2014 were tested, and the complete P1 coding sequences of 18 DHAV-3 strains were analyzed. The detection rate of DHAV-3 was 64.5% (311/482) in clinical liver samples and 73.0% (92/126) in duckling flocks. The P1 genes of the 18 DHAV-3 isolates shared 91.9%-99.0% nucleotide similarity and 95.2%-100% amino acid similarity with those of the other 26 reference strains. Based on the P1 and VP1 gene sequences, phylogenetic analysis results indicated that the genotyping of DHAV-3 strains presented a distinct geographical distribution. Except B63 strain, all Chinese strains isolated from different host species (duck or goose) at different time were classed into the CH genotype. All Korean and Vietnamese strains belonged to the KV genotype, and all the Korean strains were clustered into KV1 subgenotype, while B63 strain and the Vietnamese strains from different host species (duck or goose) were clustered into KV2 subgenotype. Ten variable amino acid residues were highly conserved within genotypes or subgenotypes in the VP0, VP3 and VP1, respectively, which were possibly the geographic molecular markers of DHAV-3. To the best of our knowledge, this is the first study about the genetic variation of the P1 gene of different DHAV-3 strains, which will be helpful for understanding of the molecular epidemiology of DHAV-3.

Clinical course of infection and viral tissue tropism of hepatitis C virus-like nonprimate hepaciviruses in horses.

UNLABELLED: Hepatitis C virus (HCV) has a very narrow species and tissue tropism and efficiently replicates only in humans and the chimpanzee. Recently, several studies identified close relatives to HCV in different animal species. Among these novel viruses, the nonprimate hepaciviruses (NPHV) that infect horses are the closest relatives of HCV described to date. In this study, we analyzed the NPHV prevalence in northern Germany and characterized the clinical course of infection and viral tissue tropism to explore the relevance of HCV-related horse viruses as a model for HCV infection. We found that approximately 31.4% of 433 horses were seropositive for antibodies (Abs) against NPHV and approximately 2.5% carried viral RNA. Liver function analyses revealed no indication for hepatic impairment in 7 of 11 horses. However, serum gamma-glutamyl transferase (GGT) concentrations were mildly elevated in 3 horses, and 1 horse displayed even highly elevated GGT levels. Furthermore, we observed that NPHV infection could be cleared in individual horses with a simultaneous emergence of nonstructural (NS)3-specific Abs and transient elevation of serum levels of liver-specific enzymes indicative for a hepatic inflammation. In other individual horses, chronic infections could be observed with the copresence of viral RNA and NS3-specific Abs for over 6 months. For the determination of viral tissue tropism, we analyzed different organs and tissues of 1 NPHV-positive horse using quantitative real-time polymerase chain reaction and fluorescent in situ hydridization and detected NPHV RNA mainly in the liver and at lower amounts in other organs. CONCLUSION: Similar to HCV infections in humans, this work demonstrates acute and chronic stages of NPHV infection in horses with viral RNA detectable predominantly within the liver.

Circulation and in vivo distribution of duck hepatitis A virus types 1 and 3 in infected ducklings.

The circulation of duck hepatitis A virus types 1 (DHAV-1) and 3 (DHAV-3) in Southeast Asia has resulted in a continuously changing epidemiological scenario. In this study, a duplex real-time PCR assay for simultaneous quantitative detection of DHAV-1 and DHAV-3 was established, and 200 liver samples from dead ducklings collected from 31 different flocks in Shandong province, China, were tested. Fifty-eight (29.0 %) samples from 13 flocks were positive for DHAV-1 single infection, 113 (56.5 %) samples from 13 other flocks were positive for DHAV-3 single infection, and 24 samples (12.0 %) from four flocks were positive for both viruses. DHAV-1 and DHAV-3 were detected with high viral loads in all of the organs tested (liver, spleen, pancreas, kidney, heart, thymus, bursa of Fabricius and brain). No significant difference in DHAV-1 and DHAV-3 viral loads was found between singly infected and coinfected samples, and there was no correlation between the viral loads of the two viruses and the age of dead ducklings. To the best of our knowledge, this is the first report about the in vivo distribution of DHAV-1 and DHAV-3 in clinically infected ducklings.

Prevalence of inclusion body hepatitis (IBH) in Poland from 2010-2014.

This study investigated the prevalence of inclusion body hepatitis (IBH) infections during the past five years (2010-2014) in broiler chicken flocks in Poland. The diagnosis of IBH was based on anatomopathological investigations and histopathological analyzes. IBH was the most prevalent disease in broiler flocks in Poland (10.4%) during this time period, and occurred as a primary infection. The fewest cases of IBH occurred in the summer, suggesting that the temperature may have been a possible stressor that increased susceptibility to IBH. However, over the last 5 years the occurrence of clinical IBH cases in Poland has systematically decreased.

Identification of a novel Orthohepadnavirus in pomona roundleaf bats in China.

Bats in Myanmar, Gabon, and Panama have been found to harbor diverse hepadnaviruses. Here, we report a novel hepadnavirus in 4 of 20 pomona roundleaf bats from Yunnan province, China. This virus contains 3,278 nucleotides (nt) in the full circularized genome, with four predicted open frames (ORFs) reading in the same direction. Full genomic sequence comparisons and evolutionary analysis indicate that this virus is a member of a new species within the genus Orthohepadnavirus.

The prevalence of duck hepatitis A virus types 1 and 3 on Korean duck farms.

This study reports the prevalence of duck hepatitis A virus (DHAV) types 1 and 3 on Korean duck farms. By RT-nested PCR assays specific for DHAV-1 or DHAV-3, DHAV-1 was detected in 9 of 157 liver samples (5.7 %) from 2 of 30 farms (6.7 %), and DHAV-3 was positive in 104 of 157 liver samples (66.2 %) from 23 of 30 farms (76.7 %). Dual infections with DHAV-1 and DHAV-3 were detected in 23 of 157 samples (14.6 %) from 5 of 30 farms (16.7 %). The data indicate that DHAV-3 infections are prevalent and that DHAV-1 reemerged in Korea, resulting in dual infections on several farms. Our data will help to establish a vaccination policy against DHAV-1 and DHAV-3 in Korea.

Avian hepatitis E virus in chickens, Taiwan, 2013.

A previously unidentified strain of avian hepatitis E virus (aHEV) is now endemic among chickens in Taiwan. Analysis showed that the virus is 81.5%-86.5% similar to other aHEVs. In Taiwan, aHEV infection has been reported in chickens without aHEV exposure, suggesting transmission from asymptomatic cases or repeated introduction through an unknown common source(s).

Identification and characterization of avian hepatitis E virus in 2013 outbreaks of hepatitis-splenomegaly syndrome in two US layer operations.

Two commercial Midwestern egg-type chicken flocks experienced significant increases in mortality rates in April 2013 with clinical signs appearing in 17-week-old pullets on Farm A and in 46-week-old hens on Farm B. Average weekly mortality was 0.44% over a 4-week period on Farm A and 0.17% over an 8-week period on Farm B. On Farm A, flocks in the affected house had a 45% decrease in daily egg production from weeks 19 to 27 when compared with standard egg production curves (P < 0.01) while no decrease in egg production was noticed on Farm B. Post-mortem examination revealed changes consistent with hepatitis-splenomegaly syndrome, including hepatomegaly with serosanguineous fluid in the coelomic cavity and hepatic subcapsular haemorrhages. Microscopic lesions were characterized by multifocal necrotizing hepatitis and intrahepatic haemorrhage. No significant bacteria were recovered from liver samples, but 72 to 100% of the liver samples from affected chickens on Farm A (8/11) and Farm B (7/7) contained detectable amounts of avian hepatitis E virus (aHEV) RNA as determined by polymerase chain reaction. Sequencing and phylogenetic analysis of a 361-base-pair fragment of the helicase gene demonstrated 98.6 to 100% nucleotide identity between the aHEV genomes from Farm A and Farm B, whereas identities ranged from 74.6 to 90.5% when compared with other representative sequences. Sequences from this study clustered within aHEV genotype 2 previously recognized in the USA. In contrast to other reported aHEV outbreaks that occurred in 30-week-old to 80-week-old chickens, in the present investigation clinical aHEV was identified in 17-week-old chickens on one of the farms.

Retrospective study of turkey viral hepatitis in California turkey flocks, 2000-2012.

Turkey viral hepatitis (TVH) is a disease characterized by an inflammation of the liver, and occasionally of the pancreas, of turkeys. Little is known about the occurrence of TVH in turkey flocks; thus, the aim of the present article is to summarize retrospectively 76 cases of TVH diagnosed at the California Animal Health and Food Safety System (CAHFS), University of California, Davis, in the years 2000 through 2012. Flocks diagnosed with TVH were between 7 and 61 days old, with an average age of 29.4 days and a median age of 28 days. In the majority of cases, increased mortality was reported. In 55 cases, gross lesions were present in the liver; the most common lesions were a few to numerous pale white foci in 35 cases. In livers of 74 cases, histologic lesions were observed. Multifocal necrosis with inflammation was observed in livers of 42 cases, inflammation but no necrosis in 22 cases, and necrosis without inflammation in 8 cases. In 17 cases, pale white foci were found in the pancreas; in 4 cases, larger areas of the organ were pale. In 33 cases, histologic changes were observed in the pancreas. Necrosis with inflammation was observed in the pancreas of 17 cases, necrosis without inflammation in 7 cases, and inflammation without necrosis in 9 cases. No Salmonella was isolated from any of the livers cultured. Transmission electron microscopy of liver and pancreas demonstrated viral particles between 23 and 25 nm in two cases. Concurrent diseases were mostly poult enteritis (65 cases), but also respiratory diseases and colibacillosis. TVH occurred more often in turkeys during winter months. In conclusion, TVH was observedon a regular basis in California turkey flocks during the last 12 yr.

First report of Equine Parvovirus-Hepatitis (EqPV-H) in Argentina.

The novel Equine Parvovirus-Hepatitis (EqPV-H) was first identified in the serum and liver of a horse that died of equine serum hepatitis, also known as Theiler's disease. Several reports in recent years strongly suggest that EqPV-H is the etiologic agent of Theiler's disease. Brazil is the only South American country where infection with this virus has been reported. This study investigated the presence of EqPV-H DNA in horse serum pools (n=51), commercial horse serum batches (n=5) and individual serum samples from donor horses (n=175) from Argentina. All serum samples were analyzed by quantitative polymerase chain reaction (qPCR) and samples with positive or indeterminate results were further analyzed by NS1 nested-PCR for phylogenetic studies. None of the serum pools was positive by qPCR but 9/51 pools were indeterminate (one or both test sample's Ct values were higher than the limit of detection). The NS1 nested-PCR detected the EqPV-H DNA in 8 of these indeterminate samples (15.7 % of serum pools). Three of the commercial horse serum batches (60 %) contained EqPV-H DNA, detected either by qPCR and/or nested-PCR. From the 175 individual horse serum samples, three (1.71 %) were positive for EqPV-H by both techniques. The genetic analysis of the 12 partial NS1 sequences obtained showed that the local isolates were similar to EqPV-H sequences from Germany and China. This study provides the first evidence of the presence of EqPV-H in horses and in horse sera commercially available in Argentina and emphasizes the importance of controlling the biosecurity of commercial equine sera as well as any other blood-derived biological products of equine origin. DATA AVAILABILITY: Viral sequences generated in this study were uploaded to the NCBI nucleotide database and are available with the accession numbers PP408676-PP408687.

The biological characteristics of DAstV molecular epidemiology and pathogenicity of duck astrovirus causing hepatitis in ducks and chickens in Southeast China.

A suspected outbreak of duck astrovirus (DAstV) disease occurred in a large Muscovy duck farm in Guangdong Province, China, in June 2022, which severely affected the production performance and health of Muscovy ducks. This study aimed to investigate the prevalence of DAstV disease in Southeast China. Herein, we employed semi-nested PCR ethodto screen 5203 swab and liver samples from 11 Muscovy duck farms in 5 provinces of China for the presence of DAstV. Among them, 1356 samples (26.06%, 1356/5203) tested positive for DAstV, out of which 11 DAstV strains were isolated after 10 generations of blind transmission through Leghorn male hepatoma (LMH) cells and performed their whole-genome sequencing. The alignment results showed that the 11 DAstV isolates exhibited relatively low homology (15.4%-75%) with the astrovirus isolates from other species published in GenBank, whereas their homology (nucleotide: 90.4%-99.99%; amino acid: 94%-99.8%) with the DAstV type 1 (DAstV-1) reference strain was higher, indicating considerable homology. The results indicated that DAstV-1 was the main pathogenic factor. Herein, we successfully recreated the clinical symptoms of natural infection in 28-day-old specific-pathogen-free (SPF) ducks using the DAstV-1-GDB-2022 strain. The primary clinical manifestations included liver enlargement, hemorrhaging, and disruptions in liver function. Additionally, we confirmed the cross-species transmission potential of DAstV-1, marking the first occurrence of clinical symptoms of DAstV in 28-day-old SPF chickens. Our findings provide new perspectives on the epidemiology and pathogenicity of DAstV-1 and may help in advancing the development of DAstV vaccines.

Subclinical infection and potential shedding routes of equine parvovirus-hepatitis among hospitalized horses in Austria.

BACKGROUND: Equine parvovirus hepatitis (EqPV-H) can cause Theiler's disease and subclinical hepatitis in horses. OBJECTIVES: Assess the frequency of subclinical EqPV-H infection in hospitalized horses and to study viral transmission by investigating potential shedding routes. ANIMALS: One hundred sixteen equids, that presented to the University Equine Hospital of the University of Veterinary Medicine Vienna between February 2021 and March 2022, for causes other than hepatopathy. METHODS: In this cross-sectional study, samples (serum, feces, nasal, and buccal swabs) of hospitalized horses were collected. Sera were screened for the presence of anti-EqPV-H antibodies by a luciferase immunoprecipitation system assay. Quantitative PCR was used for the detection of EqPV-H DNA in the samples and a nested PCR was used for further validation. RESULTS: Seroprevalence was 10.3% (12/116) and viremia occurred in 12.9% (15/116) of the serologically positive horses. The detected viral load in serum varied from non-quantifiable amount to 1.3 × 106 genome equivalents per milliliter of serum. A low viral load of EqPV-H DNA was detected in 2 nasal swabs and 1 fecal sample. CONCLUSION AND CLINICAL IMPORTANCE: EqPV-H DNA was detected in nasal secretions and feces of viremic horses, which could pose a risk to naive hospitalized horses. It is advisable to screen hospitalized horses that are potential donors of blood or plasma to reduce the risk of iatrogenic EqPV-H transmission.