Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Cardiovascular disease in central bearded dragons (Pogona vitticeps): 54 cases (2007-2022).

OBJECTIVE: To describe the clinical features, histopathologic lesions, and outcome of cardiovascular disease in central bearded dragons. ANIMALS: 54 bearded dragons. METHODS: Retrospective evaluation of captive bearded dragons with antemortem imaging or postmortem diagnosis of cardiovascular disease from 2007 to 2022 from 6 hospitals. RESULTS: The total prevalence of cardiovascular disease was 3.3% (54/1,655). Physical examination findings were available in 46 cases with change in mentation being the most common finding (n = 28/46 [60.9%]), followed by dehydration (17/46 [37%]), palpable coelomic mass (13/46 [28.3%]), dyspnea (10/46 [21.7%]), and sunken eyes (10/46 [21.7%)]. Doppler auscultation revealed an arrhythmia in 5/34 (14.7%) animals. Diagnostic imaging was only performed on 21 animals, and 10 (47.6%) had cardiovascular abnormalities described. In total, 84 cardiovascular diagnoses were found in 54 animals. The most common diagnosis was myocarditis (n = 14) followed by aneurysms (11), pericardial effusion (9), atherosclerosis (7), epicarditis (7), and myocardial degeneration/necrosis (7). Overall, 62 causes of death were identified in 52 cases, with cardiovascular disease being the most common (n = 18/52 [34.5%]). Only 3/54 animals were diagnosed with congestive heart failure. Animals with aneurysms were more likely to die to due cardiovascular disease compared to other types of cardiovascular diagnoses (OR, 43.75; 95% CI, 4.88 to 392.65; P < .001). CLINICAL RELEVANCE: Diagnosis of cardiovascular disease in bearded dragons is challenging given the inconsistent clinical presentation; however, it should remain a differential in animals with nonspecific signs of illness. Antemortem diagnostics are recommended in suspected cases, including diagnostic imaging. Of the cardiovascular diseases described, aneurysms most often contributed to clinical demise.

CLINICAL AND HISTOPATHOLOGIC OCULAR FINDINGS IN AQUARIUM-HOUSED COWNOSE RAYS (RHINOPTERA BONASUS).

Cownose rays (Rhinoptera bonasus) are susceptible to ocular disease with their prominent globes, but despite being popular animals housed in aquaria, there is little published information about their normal ocular anatomy and common pathologic ocular findings. A total of 63 live cownose rays (CNR) from three unrelated, separately housed groups had ocular examinations, and 5 adult rays were selected for ocular ultrasound. All examinations were performed out of the water, and most without anesthesia. Clinical findings were described, categorized, and scored by severity. Sixty-two of 63 rays (123 eyes) had clinical abnormalities, including 110 eyes with corneal pathology (mild = 76, moderate/severe = 34) and 74 eyes with intraocular pathology (mild = 44, moderate/severe = 30). Grey-to-white corneal opacities were the most common pathology (n = 58 rays/100 eyes) followed by cataracts (n = 41 rays/58 eyes), then persistent (or dysplastic) pupillary membranes (n = 14 rays/15 eyes). Most pathologic findings appeared inactive, but one aquarium had several CNR with active ocular pathology. There was a significant association between the diagnosis of moderate/severe corneal and intraocular pathology with age (P = 0.008 and P = 0.014, respectively) and weight (P = 0.001 and P = 0.039, respectively), as well as moderate/severe corneal pathology and group sampled (P = 0.03). There were no other significant variables identified. Additionally, histopathology of 14 eyes (11 rays) from two different facilities were examined, with keratitis (n = 8) and uveitis (n = 2) as the most common lesions. This study shows a high prevalence of pathologic ocular findings in cownose ray eyes with heavier adults more likely to be affected than lighter juveniles. Comprehensive ocular evaluation is important in this species and serial ocular exams and future studies should be pursued to monitor ocular disease progression and better understand possible etiologies.

INTRAOCULAR PRESSURES OF AQUARIUM-HOUSED COWNOSE RAYS (RHINOPTERA BONASUS) WITH NORMAL AND ABNORMAL OPHTHALMIC EXAMS.

Cownose rays (Rhinoptera bonasus) are common elasmobranchs in zoos and aquaria; however, there is a lack of published information regarding ocular findings in this species. Intraocular pressure (IOP) was measured in a total of 52 cownose rays (Rhinoptera bonasus) from two unrelated aquaria (n = 22 from A1, n = 30 from A2) using a TonoVet rebound tonometer on two settings (dog = D, and unidentified species = P) as part of a full ophthalmologic examination. Adult (n = 38) and juvenile (n = 14) rays were sampled out of water briefly in sternal recumbency. Intraocular pressure (mean ± SD [range]) in the D setting (9.10 ± 2.57 [4-18] mmHg) was higher than the P setting (5.21 ± 2.32 [0-12] mmHg) (P<0.001). Statistical analysis revealed no difference in IOP between right and left eyes, and no correlation between body weight and IOP. No differences in IOP between sex, age group, and location were identified in either setting. However, a significant difference was observed between levels of severity of corneal disease in IOP D setting (P=0.006) and P setting (P=0.024), and levels of severity of intraocular disease in IOP D setting (P=0.034) only. This study provides baseline IOP values using rebound tonometry in aquarium-housed cownose rays with apparent corneal and intraocular lesions and reveals that the D setting may be more sensitive in identifying IOP changes in eyes with intraocular disease.

HEMATOLOGY AND PLASMA CHEMISTRY COMPARISONS AMONG JUVENILE AMERICAN BLACK BEARS (URSUS AMERICANUS) UNDERGOING REHABILITATION.

The American black bear (Ursus americanus) is an opportunistic and adaptable species with high rehabilitation success rates. Injured, ill, and orphaned bears across the southeastern United States are examined and treated at the University of Tennessee College of Veterinary Medicine followed by rehabilitation at Appalachian Bear Rescue (ABR). Hematology and biochemistry reference ranges exist for healthy adult black bears; however, most bears presenting to ABR are young and of variable health status. Thus, further investigation into the difference of blood values at varying ages and presentations is warranted. ABR records from 1996 to 2022 included 106 bears with completed hematology and plasma biochemistry panels (22 paired samples at intake and release, 84 at intake only). Intake-only samples consisted of 12 neonates (<3 mon old), 64 cubs (3-12 mon), and 30 yearlings (1-2 yr). Bears presented as orphaned neonates (22%), orphaned cubs (45%), malnourished yearlings (24%), and injured/ill (9%) during fall (16%), winter (13%), spring (32%), and summer (39%). Changes in hematology and plasma biochemistry results between intake and release included an increase in hematocrit and glucose. Injured/ill bears presented with higher total leukocyte count (WBC), absolute neutrophils (ABS segs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (P < 0.05). Positive correlation between ALT, AST, proteins, and blood urea nitrogen and negative correlation between absolute lymphocytes and alkaline phosphatase were noted with age. Both WBC and ABS segs were lower during winter (P < 0.05). Understanding what factors affect juvenile black bear blood values improves clinical expectations and evaluation upon intake, clinical evaluation, and treatment.

Butorphanol-Azaperone-Medetomidine Is as Safe and Effective as Nalbuphine-Azaperone-Medetomidine for Immobilization of Juvenile American Black Bears (Ursus americanus).

Immobilization kits including butorphanol-azaperone-medetomidine (BAM) and nalbuphine-azaperone-medetomidine can provide effective, safe, and easy-to-use protocols in bears. Nalbuphine-azaperone-medetomidine is not commercially available but may be useful for wildlife agencies because it does not contain controlled substances. This study directly compared BAM to nalbuphine-azaperone-medetomidine immobilization in 10 juvenile healthy black bears (10 mo old; four females, six males) undergoing prerelease examinations after rehabilitation. Bears were immobilized via remote delivery of 1 mL of BAM (n=5) or nalbuphine-azaperone-medetomidine (n=5) intramuscularly in the shoulder during December (randomized, blinded trial). Bears were intubated, monitored with an electrocardiogram, pulse oximeter, capnograph, noninvasive blood pressure cuff, and rectal thermometer, and underwent physical examination, sample collection, morphometrics, and ear-tag placement. Induction, physiologic, and recovery parameters were recorded, including arterial blood gas analysis. The anesthetic agents were antagonized with atipamezole and naltrexone. There were no differences between protocols in induction or recovery times. There were no differences between protocols in heart rate, respiratory rate, temperature, oxygen saturation, end-tidal CO2, mean arterial pressure, or blood gas analysis or any differences between male and female bears in any parameters. Bears were hypertensive and normoxemic with low oxygen saturation via pulse oximeter, but all recovered smoothly and were released within 2 h of recovery. This study supports that nalbuphine-azaperone-medetomidine is clini-cally as safe and effective as BAM in American black bears.

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establishing persistence.

The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity.

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

Mouse Liver-Expressed Shiftless Is an Evolutionarily Conserved Antiviral Effector Restricting Human and Murine Hepaciviruses.

Mice are refractory to infection with human-tropic hepatitis C virus (HCV), although distantly related rodent hepaciviruses (RHV) circulate in wild rodents. To investigate whether liver intrinsic host factors can exhibit broad restriction against these distantly related hepaciviruses, we focused on Shiftless (Shfl), an interferon (IFN)-regulated gene (IRG) which restricts HCV in humans. Unusually, and in contrast to selected classical IRGs, human and mouse SHFL orthologues (hSHFL and mSHFL, respectively) were highly expressed in hepatocytes in the absence of viral infection, weakly induced by IFN, and highly conserved at the amino acid level (>95%). Replication of both HCV and RHV subgenomic replicons was suppressed by ectopic expression of mSHFL in human or rodent hepatoma cell lines. Gene editing of endogenous mShfl in mouse liver tumor cells increased HCV replication and virion production. Colocalization of mSHFL protein with viral double-stranded RNA (dsRNA) intermediates was confirmed and could be ablated by mutational disruption of the SHFL zinc finger domain, concomitant with a loss of antiviral activity. In summary, these data point to an evolutionarily conserved function for this gene in humans and rodents: SHFL is an ancient antiviral effector which targets distantly related hepaciviruses via restriction of viral RNA replication. IMPORTANCE Viruses have evolved ways to evade or blunt innate cellular antiviral mechanisms within their cognate host species. However, these adaptations may fail when viruses infect new species and can therefore limit cross-species transmission. This may also prevent development of animal models for human-pathogenic viruses. HCV shows a narrow species tropism likely due to distinct human host factor usage and innate antiviral defenses limiting infection of nonhuman liver cells. Interferon (IFN)-regulated genes (IRGs) partially inhibit HCV infection of human cells by diverse mechanisms. Here, we show that mouse Shiftless (mSHFL), a protein that interferes with HCV replication factories, inhibits HCV replication and infection in human and mouse liver cells. We further report that the zinc finger domain of SHFL is important for viral restriction. These findings implicate mSHFL as a host factor that impairs HCV infection of mice and provide guidance for development of HCV animal models needed for vaccine development.

Retrospective evaluation of wobbly hedgehog syndrome in 49 African pygmy hedgehogs (Atelerix albiventris): 2000-2020.

OBJECTIVE: To retrospectively evaluate the prevalence and clinical progression of wobbly hedgehog syndrome (WHS) and concurrent incidence of neoplasia in a cohort of African pygmy hedgehogs (Atelerix albiventris). ANIMALS: 49 hedgehogs. CLINICAL PRESENTATION AND PROCEDURES: Medical records of hedgehogs from 7 institutions across the US over a 20-year period (2000 to 2020) were retrospectively reviewed. Inclusion criteria were hedgehogs of any sex or age with postmortem CNS histopathology consistent with WHS. Collected data included sex, age at onset and euthanasia, major histopathologic findings, reported neurologic clinical signs, and treatments administered. RESULTS: 24 males and 25 females were included. Fifteen of 49 (31%) individuals had subclinical WHS with no reported antemortem neurologic clinical signs. In neurologically affected (clinical) hedgehogs (n = 34), the mean ± SD age at onset was 3.3 ± 1.5 years with a median (range) time from onset to euthanasia of 51 days (1 to 319 days). In neurologically affected hedgehogs, the most commonly reported clinical signs were ataxia (n = 21) and pelvic limb paresis (16) and the most commonly administered treatment was meloxicam (13). Overall, 31 of 49 (63%) hedgehogs had a concurrent histopathologic diagnosis of neoplasia outside of the CNS. CLINICAL RELEVANCE: The prognosis for hedgehogs with WHS is poor. No treatment had a significant effect on survival time, and neoplasia was a common comorbidity in the current cohort. A small but clinically relevant subset of neurologically normal hedgehogs had a histopathologic diagnosis of WHS.

UTILITY OF URINE ANTIGEN TESTING FOR DIAGNOSIS OF BLASTOMYCOSIS IN NONDOMESTIC FELIDS.

Blastomycosis caused by the fungus Blastomyces dermatitidis has been reported to cause disease in numerous species of nondomestic felids. Diagnosis of blastomycosis in domestic species often relies on the combination of clinical signs, radiographic findings, and commercial urinary antigen testing. In this report, the sensitivity, specificity, and positive and negative predictive values for urine blastomyces antigen testing for use in nondomestic felids were examined and compared with findings on postmortem examination. The study showed a sensitivity of 100%, specificity of 91.86%, positive predictive value of 50%, and negative predictive value of 100% for urine antigen testing. Furthermore, radiographic and hematologic findings were compared with those of animals diagnosed with blastomycosis. Radiographic evidence consistent with blastomycosis was found in those animals diagnosed via urine antigen testing, but no significant differences in plasma biochemistry parameters between diseased and nondiseased animals were found. This study provides evidence that a positive blastomycosis antigenuria test result should be combined with other diagnostic methods to confirm the presence of infection with B. dermatitidis, whereas a negative antigenuria test result is 100% effective in predicting the absence of disease.

RABIES DIAGNOSIS AND RESPONSE TO VACCINATION IN SOUTHERN TAMANDUA (TAMANDUA TETRADACTYLA).

Rabies has rarely been described in Xenarthra, and rabies vaccine response has not been documented. A southern tamandua (Tamandua tetradactyla) presented with nonspecific clinical signs and was euthanatized. Subsequently, immunohistochemistry and RT-PCR confirmed a rabies diagnosis. Following these tests, a group of eight captive tamanduas were vaccinated with a killed rabies vaccine, and titers were measured at the time of vaccination and 23 d later. One animal had day 0 titers suggestive of previous vaccination or exposure. All animals had detectable neutralizing rabies virus antibody titers after vaccination, but one animal failed to meet the World Organization for Animal Health's definition for adequate vaccination (≥0.5 IU/ml), and two other animals had low antibody titers (0.56 and 0.6 IU/ml). Rabies should be considered as a possible cause of illness in tamanduas, and rabies vaccination may be a useful preventative measure when anthropic interaction through medical care or ambassador roles is occurring.

Failure of Passive Transfer in Camel Calves: 4 Cases (2010-2019).

Failure of passive transfer is a management concern for all ruminant species, but is not well described in the literature for camel calves. This case series presents four camel calves (Camelus dromedarius and Camelus bactrianus) referred to a North American veterinary teaching hospital for diagnosis and management of failure of passive transfer. Diagnostics utilized included hematology, serum biochemistry, and immunologic methods as described for crias. Management included antimicrobial, anti-inflammatory, and plasma transfusion therapies. Three of the four calves survived to discharge, and common diagnostic practices such as evaluation of total solids, total protein, immunoglobulin G, and sodium sulfite appear to be correlate to passive transfer status in these four calves. Xenotransfusion with llama plasma was well tolerated by two calves, and xenotransfusion with bovine plasma was well tolerated by an additional calf in this study. An additional work is necessary to develop validated breakpoints for diagnosis of passive transfer status in camel calves.

MORBIDITY AND MORTALITY OF FREE-RANGING AMERICAN BLACK BEARS (URSUS AMERICANUS) UNDERGOING REHABILITATION IN EASTERN TENNESSEE, USA, 1996-2021.

Free-ranging American black bears (Ursus americanus) often share habitat with humans and domestic animals, predisposing them to anthropogenic conflicts. Rehabilitation under professional care is a management option for orphaned, injured, and/or ill bears. Across several southeastern states, rescued bears are assessed and treated at the University of Tennessee and rehabilitated at Appalachian Bear Rescue (ABR). Records from 1996-2021 showed 337 bears (170 males, 166 females, 1 unknown) from nine states were admitted to ABR. Three bears were admitted twice, resulting in 340 admissions (42 neonates <3 mo old, 206 cubs 3-12 mo, 87 yearlings 1-2 yr, and 5 adults >2 yr). Bears presented as orphans (58%), malnourished (24%), injured or ill (12%), or confiscated/other (6%). Individuals were returned to the wild (85%); died or were euthanized (12%); or were placed into professional care (3%). Of released bears, 195 had complete medical records available for evaluation; 31% were healthy upon intake while the remaining were treated successfully for malnutrition and internal parasites (49%), orthopedic (9%) and soft tissue injuries (5%), or other diseases (5%). Causes of death determined during necropsies performed (n=30) were classified as trauma (50%), developmental (13%), undetermined (13%), malnutrition (13%), infectious or inflammatory (7%), and toxicosis (3%). Despite the lack of maternal care and high prevalence of malnutrition and trauma, most bears recovered to release with appropriate husbandry and medical care. This study provides a foundation for research to further improve care of rehabilitating black bears.

Clinical disease and treatment of Leptospira kirschneri sv Grippotyphosa in a Sumatran tiger (Panthera tigris sumatrae).

CASE DESCRIPTION: A 12-year-old sexually intact male zoo-managed Sumatran tiger (Panthera tigris sumatrae) was evaluated for a 3-day history of vomiting, hyporexia, and lethargy. Radiographs were supportive of gastrointestinal obstruction, and an exploratory laparotomy was performed. CLINICAL FINDINGS: Diffuse tan foci were present on the liver parenchyma, and the tiger became icteric throughout the procedure. Hepatic histopathology and immunohistochemistry resulted in a diagnosis of leptospirosis. Serum microagglutination testing for Leptospira spp antibody titers were positive for L kirschneri serovar Grippotyphosa, rising from 1:400 to 1:3,200 in 2 days. TREATMENT AND OUTCOME: The tiger was treated with antimicrobials, ursodiol, and mirtazapine, and increased biosecurity measures were instituted. Free-ranging wildlife on grounds were trapped, euthanized, and submitted for necropsy to screen for disease vectors. The tiger's urine was intermittently opportunistically collected from the enclosure and remained PCR assay negative for Leptospira spp until being positive once again on day 595. Although the tiger was without clinical signs at that time, antimicrobial therapy and increased biosecurity protocols were instituted a second time until urinary Leptospira shedding was confirmed to have stopped. By 1,071 days after initial presentation, the tiger remained nonclinical, with no additional urinary shedding episodes. CLINICAL RELEVANCE: While domestic and nondomestic free-ranging felids have been reported as subclinical Leptospira spp carriers, this report indicates the clinical importance of leptospirosis when a tiger presents with generalized gastrointestinal signs and icterus. Due to the zoonotic potential, biosecurity measures are necessary. This patient had a clinically successful outcome with antimicrobial therapy and supportive care.

Disseminated Mycobacterium genavense infection in a guinea pig (Cavia porcellus): a case report.

BACKGROUND: Mycobacteria are found in many environmental conditions and infect a variety of species, including rodents and rabbits. Guinea pigs are used experimentally as a model for Mycobacterium tuberculosis, but natural mycobacteriosis in guinea pigs has not been reported. CASE PRESENTATION: A 1.5-year-old female guinea pig was found acutely deceased with no premonitory illness. On gross post-mortem examination, multifocal to coalescing, raised, firm, pale tan nodules with discrete, irregular margins were noted over the surfaces of all lung lobes. Histopathology revealed nodules composed of clustered foamy macrophages and multinucleated giant cells containing numerous bacterial rods. Similar bacteria-laden macrophages were noted within sections of the liver, heart, palpebral conjunctiva, duodenum, and cecum. Polymerase chain reaction was performed on tissues collected during post-mortem examination. The 16S rRNA gene product was sequenced and was identical to the Mycobacterium genavense type strain. CONCLUSIONS: To the best of the author's knowledge, this report details the first documented case of Mycobacterium genvaense infection in a guinea pig and a follow up investigation of close-contact animals. Given their experimental susceptibility and this clinical case report, mycobacteriosis should be considered as a differential in guinea pigs exhibiting weight loss in the absence of other clinical signs. With the potential for zoonotic transmission in immunosuppressed individuals, precautions should be taken to safeguard human health in cases of guinea pigs with suspected M. genavense infection.

Interdependent Impact of Lipoprotein Receptors and Lipid-Lowering Drugs on HCV Infectivity.

The HCV replication cycle is tightly associated with host lipid metabolism: Lipoprotein receptors SR-B1 and LDLr promote entry of HCV, replication is associated with the formation of lipid-rich membranous organelles and infectious particle assembly highjacks the very­low-density lipoprotein (VLDL) secretory pathway. Hence, medications that interfere with the lipid metabolism of the cell, such as statins, may affect HCV infection. Here, we study the interplay between lipoprotein receptors, lipid homeostasis, and HCV infection by genetic and pharmacological interventions. We found that individual ablation of the lipoprotein receptors SR­B1 and LDLr did not drastically affect HCV entry, replication, or infection, but double lipoprotein receptor knock-outs significantly reduced HCV infection. Furthermore, we could show that this effect was neither due to altered expression of additional HCV entry factors nor caused by changes in cellular cholesterol content. Strikingly, whereas lipid­lowering drugs such as simvastatin or fenofibrate did not affect HCV entry or infection of immortalized hepatoma cells expressing SR-B1 and/or LDLr or primary human hepatocytes, ablation of these receptors rendered cells more susceptible to these drugs. Finally, we observed no significant differences between statin users and control groups with regards to HCV viral load in a cohort of HCV infected patients before and during HCV antiviral treatment. Interestingly, statin treatment, which blocks the mevalonate pathway leading to decreased cholesterol levels, was associated with mild but appreciable lower levels of liver damage markers before HCV therapy. Overall, our findings confirm the role of lipid homeostasis in HCV infection and highlight the importance of the mevalonate pathway in the HCV replication cycle.

MEASUREMENT OF INTRAOCULAR PRESSURE USING REBOUND TONOMETRY IN ANESTHETIZED PERUVIAN FUR SEALS (ARCTOCEPHALUS AUSTRALIS) FROM PUNTA SAN JUAN, PERU.

Ocular disease in pinnipeds under human care is well described, and intraocular pressure (IOP) can be impacted by a variety of ophthalmic conditions. Species-specific reference parameters from clinically normal animals are instrumental for understanding how ophthalmic diseases may impact ocular pressures. IOP measurements were obtained using rebound tonometry from free-ranging Peruvian fur seals (Arctocephalus australis unnamed subspecies) at Punta San Juan, Peru, over a 6-yr period (2010-2016). Retrospective data obtained from 108 (81 adults and 27 neonates comprising 69 females and 39 males) anesthetized fur seals with normal anterior segment ophthalmic examinations was included in the analysis. Differences in IOP from each eye were compared to categorical variables (age, year, sex, restraint) using an independent-samples t test. All univariate results with a significance of P < 0.05 were included in multivariate analysis. Of the 13 general linear models evaluated, the top two for both the right and the left eye included age class when all variables were evaluated simultaneously. Neonates had significantly lower IOP values than adults in both the right eye (17.5 mm Hg; 95% confidence interval [CI]: 14.0-21.1 mm Hg compared to 33.5 mm Hg; 95% CI: 31.0-36.1 mm Hg, respectively) and the left eye (18.4 mm Hg; 95% CI: 14.4-22.5 mm Hg compared to 32.3 mm Hg; 95% CI: 29.3-35.3 mm Hg, respectively). Anesthesia method was not statistically significant (P > 0.05). This is the first report of normal IOP measurements for any fur seal species. Described data can be used to improve diagnosis and management of ocular alterations in pinnipeds.

Liver-expressed Cd302 and Cr1l limit hepatitis C virus cross-species transmission to mice.

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.

Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus.

OBJECTIVE AND DESIGN: Human stem cell-derived hepatocyte-like cells (HLCs) have shown high potential as authentic model for dissection of the HCV life cycle and virus-induced pathogenesis. However, modest HCV replication, possibly due to robust innate immune responses, limits their broader use. To overcome these limitations and to dissect the mechanisms responsible for control of HCV, we analysed expression of key components of the interferon (IFN) system in HLCs, assessed permissiveness for different HCV strains and blocked innate immune signalling by pharmacological intervention. RESULTS: Transcriptional profiling revealed that HLCs constitutively express messenger RNA of RLRs, and members of the IFN pathway. Moreover, HLCs upregulated IFNs and canonical interferon-regulated genes (IRGs) upon transfection with the double-stranded RNA mimic poly(I:C). Infection of HLCs with Jc1-HCVcc produced only limited viral progeny. In contrast, infection with p100, a Jc1-derived virus population with enhanced replication fitness and partial resistance to IFN, resulted in robust yet transient viraemia. Viral titres declined concomitant with a peak of IRG induction. Addition of ruxolitinib, a JAK/STAT inhibitor, permitted chronic infection and raised p100 infectious virus titres to 1×105 FFU/mL. IRGs expression profiling in infected HLCs revealed a landscape of HCV-dependent transcriptional changes similar to HCV-infected primary human hepatocytes, but distinct from Huh-7.5 cells. Withdrawal of ruxolitinib restored innate immune responses and resulted in HCV clearance. CONCLUSION: This authentic human cell model is well suited to examine acute and chronic host-HCV interactions, particularly IFN-triggered antiviral effector functions and mechanisms of innate immune control of HCV infection.