First National Prevalence in Italian Horse Population and Phylogenesis Highlight a Fourth Sub-Type Candidate of Equine Hepacivirus.

Equine hepacivirus (EqHV, Flaviviridae, hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Hepatitis C virus infection reduces the lifespan of chimpanzees used in biomedical research.

Chimpanzees were used in hepatitis research for over three decades with the aim to identify and develop treatments for the virus, a leading cause of chronic liver disease in humans. We used a dataset of 120 chimpanzees housed at a single institution in Japan, 22 of whom became chronically infected with hepatitis C virus (HCV), to examine whether HCV infection results in a reduced lifespan as reported in humans. Survival analysis showed that HCV carriers experienced a higher mortality risk compared with non-carriers. Although no chimpanzee died from hepatic disease, carriers showed higher gamma-glutamyl transpeptidase (γGTP) levels compared with non-carriers suggesting that HCV infection negatively affected their liver condition. These results provide evidence that special attention is necessary to monitor the long-term condition of ex-biomedical primates.

Global Prevalence of RNA-Positive Horses for Hepacivirus (EqHV): Systematic Review and Meta-Analysis.

Equine hepacivirus (EqHV) belongs to the Flaviviridae family, genus Hepacivirus and has the greatest genomic identity with the hepatitis C virus (HCV), one of the main causes of chronic liver disease in humans. Due to the limited applicability of studies of HCV in animal hosts, the interest in studies of characterization of viral homologues has been growing. For this reason, we performed a systematic review of the literature with meta-analysis of the prevalence of EqHV and genetic sequencing studies. Twenty-three studies from four different continents were analyzed. The OR meta-analysis (0.98; 95% CI = 0.69-1.39) showed no influence of sex (female or male) on the risk of infection. Variables associated with EqHV infection were indirectly related to animal management such as transport, reproductive practices, among others. The combined prevalence of positive animals was 7.88% (95% CI = 5.23-11.69%), with the highest proportions in Asia (16.13%; 95% CI = 7.79-30.43%), followed by South America (12.03%; 95% CI = 9.58-15.01%), Africa (8.69%; 95% CI = 6.71-11.20%), and Europe (3.63%; 95% CI = 2.10-6.22%). However, these results represent the regional stratification of the epidemiological studies for EqHV published to date and, therefore, cannot extrapolate to determine the continental prevalence of EqHV. It is therefore important to update the systematic review as further research becomes available.

Seroreversion of IgG anti-HEV in HIV cirrhotic patients: A long-term multi-sampling longitudinal study.

The aim of our study was to evaluate HEV antibody kinetics in HIV/HCV-coinfected patients with cirrhosis. A longitudinal retrospective study was designed. Patients were followed up every 6 months; anti-HEV IgG and IgM antibodies levels and HEV-RNA by qPCR were analysed. The prevalence and incidence of every HEV infection marker were calculated. The kinetics of anti-HEV IgG and IgM during the follow-up were evaluated. Seventy-five patients comprised the study population. The seroprevalence observed was 17.3%. None showed IgM antibodies or HEV-RNA at baseline. None showed detectable HEV viral load during the study period. After a median follow-up of 5.1 years, two of 62 seronegative patients (3.2%) seroconverted to IgG antibody. The incidence for IgM was 2.7%. Of the 13 patients with IgG seropositivity at baseline, five (38.5%) seroreverted. Meanwhile, of the two patients who exhibited IgM positivity during the study, one (50%) showed intermittent positivity. We found that HEV seropositivity is common in HIV/HCV-coinfected cirrhotic patients. A remarkable rate of IgG seroreversions and IgM intermittence was found, limiting the use of antibodies for the diagnosis of HEV infection in this population.

The Bank Vole (Clethrionomys glareolus)-Small Animal Model for Hepacivirus Infection.

Many people worldwide suffer from hepatitis C virus (HCV) infection, which is frequently persistent. The lack of efficient vaccines against HCV and the unavailability of or limited compliance with existing antiviral therapies is problematic for health care systems worldwide. Improved small animal models would support further hepacivirus research, including development of vaccines and novel antivirals. The recent discovery of several mammalian hepaciviruses may facilitate such research. In this study, we demonstrated that bank voles (Clethrionomys glareolus) were susceptible to bank vole-associated Hepacivirus F and Hepacivirus J strains, based on the detection of hepaciviral RNA in 52 of 55 experimentally inoculated voles. In contrast, interferon α/ß receptor deficient C57/Bl6 mice were resistant to infection with both bank vole hepaciviruses (BvHVs). The highest viral genome loads in infected voles were detected in the liver, and viral RNA was visualized by in situ hybridization in hepatocytes, confirming a marked hepatotropism. Furthermore, liver lesions in infected voles resembled those of HCV infection in humans. In conclusion, infection with both BvHVs in their natural hosts shares striking similarities to HCV infection in humans and may represent promising small animal models for this important human disease.

First identification and genomic characterization of equine hepacivirus subtype 2 in China.

Equine hepacivirus (EqHV) is a newly discovered hepatitis C virus-like virus that can infect equines. EqHV strains circulating worldwide have been classified into subtypes 1-3. In previous studies, we detected the presence of EqHV strains of subtype 1 and 3 in China. To determine whether EqHV strains of subtype 2 are prevalent in China, serum samples were collected from 133 racehorses in Guangdong province in 2021 and were tested for EqHV RNA by RT-PCR, and the positive rate was 9% (12/133). Sequencing of the NS3 gene revealed that one field strain (GD2021) had a high degree of genetic similarity to EqHV strains of subtype 2. Subsequent genome sequencing and analysis demonstrated that strain GD2021 belongs to subtype 2. The present study enriches our knowledge about the genetic diversity of EqHV in China.

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

Non-primate hepacivirus transmission and prevalence: Novel findings of virus circulation in horses and dogs in Morocco.

Non-primate hepacivirus (NPHV) is a homolog of hepatitis C virus and has been isolated from dogs and horses. Data on NPHV prevalence and distribution are not complete, and there is a particular lack of reports from the African continent. The present study represents the first investigation of NPHV prevalence in horses and dogs in North Africa. Blood was collected from 172 horses and 36 dogs at different locations in Morocco, and screened for NPHV RNA using nested PCR targeting 5'UTR and NS3 regions and analyzed for anti-NPHV NS3 antibody using a Gaussia luciferase immunoprecipitation system-to determine seroprevalence. Eight sequences of the NS3 region isolated from positive serum samples were targeted for phylogenetic analysis. Horses and dogs showed respective NPHV RNA positivity rates of 10.5% and 5.5%, and seroprevalences of 65.7% and 8.33%. Juvenile horses appeared more susceptible to infection, with a 23.5% NHPV RNA positivity rate. Seropositivity was more extensive in mares than stallions (77.14% vs. 46.27%, p < 0.0001). Phylogenetically, that NPHV NS3 genes isolated from horses and dog are clustered together. The NPHV strains we detected showed no correlation with geographic location within Morocco. In conclusion, Moroccan horses showed much evidence of previous and/or current NPHV infection, with young age and female sex as noted potential risk factors. Interestingly, NPHV is circulating in dogs as well as horses, suggesting that it has crossed species barriers and that horses and dogs are potential vectors by which an ancestor to hepatitis C virus was transmitted into human populations.

A hepatitis B virus causes chronic infections in equids worldwide.

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

First identification and genomic characterization of equine hepacivirus sub-type 3 strain in China.

Equine Hepacivirus (EqHV) is a newly discovered equine virus that is classified under the Hepacivirus genus of the Flaviviridae family. There are three sub-types of EqHV worldwide namely; sub-types 1-3. The majority of EqHV sub-type 1 strains were found in China. While different sub-types have been found in Japan and USA, therefore, to investigate whether the other sub-types of EqHV strains were present in China, a total of 60 horse serum samples were collected and screened for EqHV RNA through RT-PCR. The results revealed that 19 serum samples were RNA-positive (19/60) and the EqHV detection rate was 31.67%. One EqHV strain named GD23 was obtained and its near-complete genome sequence was acquired. Analysis of nucleotide p-distance with reference to the entire polyprotein gene revealed that GD23 was classified into sub-type 3. In addition, the phylogenetic analysis demonstrated that GD23 was clustered together with EqHV strains of sub-type 3 in other countries. The present study is the first to identify an EqHV sub-type 3 strain in China.

Tupaia MAVS Is a Dual Target during Hepatitis C Virus Infection for Innate Immune Evasion and Viral Replication via NF-κB.

Hepatitis C virus (HCV) infection is the cause of severe liver disease in many people. The restricted species tropism of HCV hinders the research and development of drugs and vaccines. The Chinese tree shrew (Tupaia belangeri chinensis) is a close relative of primates and can be infected by HCV, but the underlying mechanisms are unknown. In this study, we have characterized the functions of tree shrew MAVS (tMAVS) in response to HCV infection and defined the capacity of HCV replication. HCV was shown to be colocalized with tMAVS in primary tree shrew hepatocytes and cleaved tMAVS at site Cys508 via its NS3/4A protease, with a modulating effect by site Glu506 of tMAVS. The tMAVS cleavage by HCV NS3/4A impaired the IRF3-mediated induction of IFN-ß but maintained the activated NF-κB signaling in the tree shrew primary cells. Activation of the tMAVS-dependent NF-κB signaling inversely inhibited HCV replication and might limit the establishment of persistent infection. Overall, our study has revealed an elegant example of the balance between the host defenses and HCV infection via the MAVS-mediated antiviral signaling and has provided an insight into the mechanisms underpinning HCV infection in the Chinese tree shrew.

Genetic heterogeneity of bovine hepacivirus in Italy.

Viruses similar to human hepatitis C virus (HCV) in the Hepacivirus genus have been identified in several animal hosts, including cattle. Since its first discovery in Germany, bovine hepacivirus (BovHepV) has been described in several countries globally. However, limited data are available on BovHepV epidemiology and genetic variability. The aim of this study was to investigate the prevalence and genetic diversity of BovHepV in Italy. Viral RNA was identified in 37 (0.15%) of 24,820 bovine sera, with titres ranging from 1.09 × 103 to 8.27 × 106 RNA copies/ml. Upon sequencing and phylogenetic analysis of the 5'UTR and NS3 genomic portions, the Italian BovHepV strains segregated into at least four distinct subtypes (A, B, C and F) that are also co-circulating globally.

Prevalence of equine hepacivirus infection in Mongolia.

Equine hepacivirus (EHV) belongs to the hepacivirus A and is related to hepatitis C virus (HCV). This virus shows hepatic tropism and is known to chronically infect horses. EHV has been reported from various countries, but the prevalence in Mongolia, where large horse populations are pastured, remains unknown. This study collected serum samples from horses in six areas across Mongolia, in order to investigate the status of infection. The possibility of human infection was also examined. The results showed an infection rate among horses of about 40 % in all regions. However, no evidence of EHV viremia was found in human serum. A mutation characteristic of Mongolian EHV was found in the 5'-untranslated region of the viral sequence. Molecular phylogenetic trees for core, NS3, and NS5B sequences showed the formation of two clusters depending on the area from which samples were taken. The same results were obtained from molecular phylogenetic analyses using the full genome. From detailed calculations of genetic diversity calculated using the full genome, EHV appears divisible into two subgenotypes. Blood samples were collected again after a 7-month interval to examine infection persistence. Seventeen of 19 horses retested showed positive results for EHV after 7 months, suggesting a high rate of persistent infection. These results indicate a relatively higher frequency of EHV infection in Mongolia than in Europe or North America, with virus strains divided into at least two subgenotypes.

Further Evidence for in Utero Transmission of Equine Hepacivirus to Foals.

(1) Background: Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses-and dogs in some instances-and is closely related to hepatitis C virus (HCV) that has infected up to 3% of the world's human population, causing an epidemic of liver cirrhosis and cancer. EqHV also chronically infects the liver of horses, but does not appear to cause serious liver damages. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. (2) Methods: In this retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. (3) Results: Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. (4) Conclusions: As seemingly benign viruses could turn deadly (e.g., Zika flavivirus) and EqHV happens to have infected a significant proportion of the world's horse herds, EqHV infectious cycle should be further clarified.

No Evidence of Mosquito Involvement in the Transmission of Equine Hepacivirus (Flaviviridae) in an Epidemiological Survey of Austrian Horses.

Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Concurrently, 386 serum samples from horses in eastern Austria were analyzed for EqHV-specific antibodies by luciferase immunoprecipitation system (LIPS) and for EqHV RNA by RT-qPCR. Additionally, liver-specific biochemistry parameters were compared between EqHV RNA-positive horses and EqHV RNA-negative horses. Phylogenetic analysis was conducted in comparison to previously published sequences from various origins. No EqHV RNA was detected in mosquito pools. Serum samples yielded an EqHV antibody prevalence of 45.9% (177/386) and RNA prevalence of 4.15% (16/386). EqHV RNA-positive horses had significantly higher glutamate dehydrogenase (GLDH) levels (p = 0.013) than control horses. Phylogenetic analysis showed high similarity between nucleotide sequences of EqHV in Austrian horses and EqHV circulating in other regions. Despite frequently detected evidence of EqHV infection in Austrian horses, no viral RNA was found in mosquitoes. It is therefore unlikely that mosquitoes are vectors of this flavivirus.

Evaluation of testicular cytotoxicity and genotoxicity of sofosbuvir and sofosbuvir - ribavirin in the adult male albino rats

Hepatitis C is a widely distributed problem all over the world, especially Egypt. Chronically infected people develop serious liver disease and now it is the most common cause for liver transplantation. Recently, a new regimen, sofosbuvir (sovaldi), alone or with combinations as sovaldi-ribavirin, was approved for treating this disease. There are limited studies that explore the effects of these drugs on the reproductive organs, and hence affection of male fertility while using these drugs. This study aims to throw more light on whether sovaldi or sovaldi-ribavirin causes testicular damaging effects in the adult male albino rats. We investigated the effect of this regimen in a dose equivalent to that used in the human (41 mg/kg once daily orally for sovaldi and 41 mg/kg twice daily orally for ribavirin) for consecutive 5 and 10 days. There was highly significant decrease in testosterone hormone level and marked degenerative changes in the seminiferous tubules and the testicular interstitium, with increase in collagen deposits in sovaldi treated rats, and in a more extensive manner in sovaldi-ribavirin treated rats. There was a significant increase of deoxyribonucleic acid (DNA) fragmentation in the treated groups after 10 days. However, there was a non-significant difference in DNA fragmentation in the treated groups after 5 days when compared with control. Immuno-histochemistry detection of caspase-3 showed significant increase in its expression in the treated groups after either 5 or 10 days. This denoted the specificity of caspase-3 immunohistochemistry technique in the detection of early apoptotic changes. It was concluded that sovaldi and sovaldi ribavirin induced gonado toxic effects through induction of DNA fragmentation via up regulation of caspase-3, and that the resulting damaging effects increased with longer duration of drug in take

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Highly divergent cattle hepacivirus N in Southern Brazil.

The genus Hepacivirus includes 14 species (Hepacivirus A-N). In this study, we determined a partial genome sequence of a highly divergent bovine hepacivirus (hepacivirus N, HNV) isolate from cattle in Southern Brazil. Previously described HNV isolates have shared 80-99.7% nucleotide sequence identity in the NS3 coding region. However, the sequence determined in this study had 72.6% to 73.8% nucleotide sequence identity to known HNV NS3 sequences. This high divergence could be seen in a phylogenetic tree, suggesting that it represents a new genotype of HNV. These data expand our knowledge concerning the genetic variability and evolution of hepaciviruses.

Presence of a Novel Subtype of Bovine Hepacivirus in China and Expanded Classification of Bovine Hepacivirus Strains Worldwide into 7 Subtypes.

The newest member of the Hepacivirus genus, bovine hepacivirus (BovHepV), was first identified in cattle in 2015 and is a novel hepacivirus C virus (HCV)-like virus. This virus has been detected in five countries so far and is classified into four subtypes. Bovine serum is commonly used for cell cultures and is considered the major source of viral contamination of pharmaceutical products. In this study, bovine serum samples were collected from seven countries located in Asia, America, Oceania, and Europe and were tested for BovHepV RNA using nested PCR, in order to: (i) obtain more knowledge on the geographical distribution and subtypes of BovHepV; and (ii) detect the potential contamination of BovHepV in commercial bovine serum samples used for cell culture propagation. The results demonstrated that bovine serum samples from individual donor cattle in China contained BovHepV RNA. After PCR, sequencing, and assembly, the genomes of the Chinese BovHepV strains were obtained. Genetic analysis of the polyprotein gene revealed a protein identity of <77% and a nucleotide identity of <85% between the Chinese BovHepV strains and all other previously reported BovHepV strains. Using cut-off values for determination of HCV genotypes and subtypes, BovHepV strains worldwide were classified into one unique genotype and seven subtypes. The BovHepV strains identified in the present study were classified into a novel subtype, which was provisionally designated subtype G. The genetic relationships among the different BovHepV subtypes were further confirmed through phylogenetic analysis. The present study provides critical insights into BovHepV's geographical distribution and genetic variability.

Chronic equine hepacivirus infection in an adult gelding with severe hepatopathy.

BACKGROUND: Equine hepacivirus (EqHV) in equids represents the closest homologue to hepatitis C virus (HCV) infecting humans. A majority of HCV infected patients develop a chronic course of infection leading to liver fibrosis, cirrhosis and liver failure. However, in horses mostly transient mild subclinical infections are reported for EqHV to date. OBJECTIVES: EqHV can be involved in chronic liver diseases of horses. METHODS: Biochemical parameters in serum samples were measured. Viral load was determined using qPCR. Next generation sequencing (NGS) of serum was performed. Liver tissue was stained with haematoxylin and eosin and analysed for viral RNA with fluorescent in situ-hybridization. RESULTS: The horse showed symptoms of severe hepatopathy and was chronically infected with EqHV. Viral RNA was detectable in the liver during disease. To rule out other infectious agents NGS was performed and showed the highest abundance for EqHV. The identified virus sequence was similar to other circulating equine hepaciviruses. CONCLUSIONS: EqHV can be associated with liver disease in horses. Whether it causes the disease or contributes in a multifactorial manner needs further investigation.

Natural recombination of equine hepacivirus subtype 1 within the NS5A and NS5B genes.

Equine hepacivirus (EqHV) was first reported in 2012 and is the closest known homolog of hepatitis C virus (HCV). A number of studies have reported HCV recombination events. The aim of this study was to determine whether recombination events occur in EqHV strains. Considering that no information on the Chinese EqHV genome sequence is available, we first sequenced the near-complete genomes of three field EqHV strains. Through systemic analysis, we obtained strong evidence supporting a recombination event within the NS5A and NS5B genes in the American EqHV strains, but not in the strains from China or other countries. Finally, using cut-off values for determination of HCV genotypes and subtypes, we classified the EqHV strains from around the world into one unique genotype and three subtypes. The recombination event occurred in subtype 1 EqHV strains. This study provides critical insights into the genetic variability and evolution of EqHV.