Virological evidence of the impact of non-pharmaceutical interventions against COVID-19 in Ecuador, a resource-limited setting.

Ecuador had substantial COVID-19-mortality during 2020 despite early implementation of non-pharmaceutical interventions (NPIs). Resource-limited settings like Ecuador have high proportions of informal labour which entail high human mobility, questioning efficacy of NPIs. We performed a retrospective observational study in Ecuador's national reference laboratory for viral respiratory infections during March 2020-February 2021 using stored respiratory specimens from 1950 patients, corresponding to 2.3% of all samples analysed within the Ecuadorian national surveillance system per week. During 2020, detection of SARS-CoV-2 (Pearson correlation; r = -0.74; p = 0.01) and other respiratory viruses (Pearson correlation; r = -0.68; p = 0.02) by real-time RT-PCR correlated negatively with NPIs stringency. Among respiratory viruses, adenoviruses (Fisher's exact-test; p = 0.026), parainfluenzaviruses (p = 0.04), enteroviruses (p < 0.0001) and metapneumoviruses (p < 0.0001) occurred significantly more frequently during months of absent or non-stringent NPIs (characterized by <55% stringency according to the Oxford stringency index data for Ecuador). Phylogenomic analyses of 632 newly characterized SARS-CoV-2 genomes revealed 100 near-parallel SARS-CoV-2 introductions during early 2020 in the absence of NPIs. NPI stringency correlated negatively with the number of circulating SARS-CoV-2 lineages during 2020 (r = -0.69; p = 0.02). Phylogeographic reconstructions showed differential SARS-CoV-2 dispersion patterns during 2020, with more short-distance transitions potentially associated with recreational activity during non-stringent NPIs. There were also fewer geographic transitions during strict NPIs (n = 450) than during non-stringent or absent NPIs (n = 580). Virological evidence supports that NPIs had an effect on virus spread and distribution in Ecuador, providing a template for future epidemics in resource-limited settings and contributing to a balanced assessment of societal costs entailed by strict NPIs.

Epidemiology of carpal fractures: is it only about the scaphoid?

Because of their low incidence, studies about carpal fractures are rare. The aim of the present study was to analyze epidemiology and treatment of fractured carpal bones. We retrospectively analyzed data of 178 patients admitted to our emergency room with carpal fractures over 6 years. More males than woman were injured. In 91%, a CT scan was performed. The most commonly affected bone was the triquetrum followed by the scaphoid. Almost all triquetral fractures were treated conservatively as opposed to perilunate dislocations that were all operated on. Half of all patients with scaphoid fractures were operated. Young men had the highest risk to sustain a carpal fracture. The triquetrum and the scaphoid are most frequently affected. Usually a CT scan is needed. Treatment of scaphoid and perilunate luxation fractures is rather operative whereas the other fractures mostly allow conservative casting. Nevertheless, correct indication for treatment is important to avoid sequelae.

Genomic determinants of Furin cleavage in diverse European SARS-related bat coronaviruses.

The furin cleavage site (FCS) in SARS-CoV-2 is unique within the Severe acute respiratory syndrome-related coronavirus (SrC) species. We re-assessed diverse SrC from European horseshoe bats and analyzed the spike-encoding genomic region harboring the FCS in SARS-CoV-2. We reveal molecular features in SrC such as purine richness and RNA secondary structures that resemble those required for FCS acquisition in avian influenza viruses. We discuss the potential acquisition of FCS through molecular mechanisms such as nucleotide substitution, insertion, or recombination, and show that a single nucleotide exchange in two European bat-associated SrC may suffice to enable furin cleavage. Furthermore, we show that FCS occurrence is variable in bat- and rodent-borne counterparts of human coronaviruses. Our results suggest that furin cleavage sites can be acquired in SrC via conserved molecular mechanisms known in other reservoir-bound RNA viruses and thus support a natural origin of SARS-CoV-2.

Epidemiology and treatment of acute elbow dislocations: current concept based on primary surgical ligament repair of unstable simple elbow dislocations.

PURPOSE: Acute elbow dislocations are complex injuries that predispose to chronic instability and pain. The ideal treatment strategy is part of controversial discussion and evidence-based recommendations for the treatment could not be concluded from the literature. The purpose of the present study was to assess current epidemiological data, injury pattern, and the changing trend for treatment. METHODS: This study presents a retrospective review of 72 patients ≥ 18 years of age who were treated in our level I trauma centre with acute elbow dislocations from 2014 to 2018. The data were acquired by analysis of the institution's database, and radiological examinations. RESULTS: The average age of the patients was 48.5 years (range 18-86). The ratio of male to female patients was 1.9:1. A fall onto the outstretched arm (42%) was the most common injury mechanism. By classification, 42% of the elbow dislocations were simple, and 58% complex. A total of 85% of patients underwent surgery including 73% of the simple elbow dislocations due to remaining instability or non-congruency of the reduced elbow. The indication for surgical treatment correlated merely with the grade of instability and displacement, but not with age. CONCLUSION: Acute elbow dislocations need identification of the precise injury pattern and instability after reduction of the elbow joint. To achieve a congruent and stable joint, we recommend primary surgical repair as first-line treatment for patients with unstable simple and complex elbow dislocation independent of age.

Assessment of muscle volume using magnetic resonance imaging (MRI) in football players after hamstring injuries.

Muscle injuries of the hamstrings are among the most frequent in football and a main cause for significant time away from training and competition. The purpose of this study was to prospectively evaluate the loss of muscle volume in recreational football players three and six weeks after initial trauma. We hypothesized that significant muscle volume loss occurs within 6 weeks after the initial injury event. Twenty recreational football players (mean-age=25 ± 4years; mean-height=181 ± 8cm; mean-weight=81 ± 10kg) with type3a (minor partial muscle tear) and type3b (moderate partial muscle tear) injuries were included. Muscle volume was determined using established methods for the hamstrings and the quadriceps femoris muscle within 3 days and after 3 and 6 weeks following the initial injury. The injured hamstrings lost 6.5% (mean=64 cm3(95%CI=31-98 cm3), p<0.001), the healthy hamstrings lost 2.1% (mean=21 cm3(3-44 cm3),p=0.096) of muscle volume after six weeks. The quadriceps in the injured leg lost 3.8% (mean=78 cm3(51-104 cm3), p<0.001) and 4.5% (83 cm3 (45-121 cm3), p<0.001) in the healthy leg. Muscle volume loss inversely correlated with activity levels in the healthy leg for the quadriceps (r=0.96 (0.90-0.98); R2=0.92; p<0.001) and the hamstrings (r=0.72 (0.40-0.88); R2=0.51; p<0.001), as well as the quadriceps in the injured leg (r=0.70 (0.37-0.87); R2=0.49; p<0.001), but not the injured hamstrings. Muscle volume ratio of hamstrings to quadriceps in the control limb was 0.52 ± 0.06 and 0.53 ± 0.06 in the injured leg. The rehabilitation period of six weeks did not have a relevant negative or a positive effect on ratios. Significant muscle volume loss in the upper thigh occurs in recreational soccer players within three, and within six weeks after a hamstring injury and lies between 2% and 7%.

Epidemiology of complete knee dislocations: an updated classification system.

INTRODUCTION: Current classifications of complete knee dislocations do not capture the extent of the complex concomitant ligamentous and bony injuries, which may have an impact on future outcomes. The purpose of this retrospective study was to evaluate the epidemiology of complete knee dislocations as well as to present an updated classification system based on the author's experience at a Level-I trauma center. MATERIALS AND METHODS: Only patients with complete loss of contact of the articulating bones and ≥ 18 years of age who admitted in our level-I trauma center between 2002 and 2019 were included. Patients were identified using a retrospective systematical query in the Hospital Information System (HIS) using the International Statistical Classification of Diseases and Related Health Problems Version10 (ICD-10) codes of the German Diagnosis Related Groups (G-DRG). RESULTS: Final data included 80 patients, with the majority of patients being male (n = 64; 80.0%). Mean age was 34.9 years (range: 18-70 years). External protective fixation was applied in 32 patients (40.0%). Reconstruction of the posterior cruciate ligament and the anterior cruciate ligament were performed in 56.3% (n = 45) and 55.0% (n = 44) of cases, respectively. The lateral collateral ligament complex was surgically addressed in 47.5% (n = 38), while the medial collateral ligament complex was reconstructed in 40% (n = 32). Surgery of the lateral meniscus and the medial meniscus was needed in 31.1% (n = 25) and 30.0% (n = 24). Neurovascular surgery occurred in 13.8% (n = 11). From the characteristic injury-patterns the authors of this study present a new classification system that ranks the injuries from Grade-A to Grade-D according to their severity. CONCLUSION: This retrospective study demonstrates that the historically used classification systems for dislocations of the knee are insufficient for these severe injuries. Concomitant ligamentous, neurovascular, bony, and meniscal injuries were frequent, and required several staged procedures. Consequently, an updated classification system is proposed.

Simplified abductor pollicis longus suspension interposition arthroplasty for thumb carpometacarpal joint osteoarthritis.

PURPOSE: The primary treatment goals for advanced-stage thumb carpometacarpal (CMC) joint osteoarthritis are complete pain relief and restoration of thumb strength. The purpose of the present study was to introduce a variation of the abductor pollicis longus (APL) suspension arthroplasty using a single looping of a radial slip from the APL tendon around the flexor carpi radialis (FCR) tendon combined with RegJoint™ interposition and to determine its efficacy in the treatment of thumb CMC joint osteoarthritis. METHODS: Between 2015 and 2017, 21 patients were included. The average age was 60.8 years (range 48-79). The mean follow-up was 27.7 months (range 8-50). Evaluation included pain, radial and palmar abduction, tip pinch and grip strength, and Disabilities of the Arm, Shoulder, and Hand (DASH) score. RESULTS: Pain averaged 0.3 (range 0-4) at rest and 1.4 (range 0-4) on exertion. The radial and palmar abduction were 97% and 99% compared to the contralateral side. The tip pinch and grip strength were 4.1 kg (range 3-6.5) and 22 kg (range 13.3-40), respectively. The DASH score accounted for 18.5 (range 0.8-41.7). CONCLUSION: The modified APL suspension interposition arthroplasty was an efficient and simplified option for the treatment of thumb CMC joint osteoarthritis, with results comparable or better than other published procedures. The APL suspension technique was easy to perform avoiding difficult bone tunneling and incision of the FCR tendon. The RegJoint™ interposition as spacer prevented impingement of the first metacarpal base on the second metacarpal base or the trapezoid bone.

Emergence of SARS-CoV-2 Delta Variant, Benin, May-July 2021.

Severe acute respiratory syndrome coronavirus 2 Delta variant epidemiology in Africa is unknown. We found Delta variant was introduced in Benin during April-May 2021 and became predominant within 2 months, after which a steep increase in reported coronavirus disease incidence occurred. Benin might require increased nonpharmaceutical interventions and vaccination coverage.

Epidemiology and treatment of phalangeal fractures: conservative treatment is the predominant therapeutic concept.

PURPOSE: Despite the high number of patients with phalangeal fractures, evidence-based recommendations for the treatment of specific phalangeal fractures could not be concluded from the literature. The purpose of the present study was to assess current epidemiological data, classification of the fracture type, and mode of treatment. METHODS: This study presents a retrospective review of 261 patients with 283 phalangeal fractures ≥ 18 years of age who were treated in our level I trauma centre between 2017 and 2018. The data were obtained by the analysis of the institution's database, and radiological examinations. RESULTS: The average age of the patients was 40.4 years (range 18-98). The ratio of male to female patients was 2.7:1. The two most typical injury mechanisms were crush injuries (33%) and falls (23%). Most phalangeal fractures occurred in the distal phalanx (P3 43%). The 4th ray (D4 29%) was most frequently affected. The P3 tuft fractures, and the middle phalanx (P2) base fractures each accounted for 25% of fracture types. A total of 74% of fractures were treated conservatively, and 26% required surgery, with Kirschner wire(s) (37%) as the preferred surgical treatment. The decision for surgical treatment correlated with the degree of angular and/or rotational deformity, intraarticular step, and sub-/luxation of specific phalangeal fractures, but not with age and gender. CONCLUSIONS: Our findings demonstrated the popularity of conservative treatment of phalangeal fractures, while surgery was only required in properly selected cases. The correct definition of precise fracture pattern in addition to topography is essential to facilitate treatment decision-making.

11,12 Epoxyeicosatrienoic Acid Rescues Deteriorated Wound Healing in Diabetes.

Epoxyeicosatrienoic acids (EET) facilitate regeneration in different tissues, and their benefit in dermal wound healing has been proven under normal conditions. In this study, we investigated the effect of 11,12 EET on dermal wound healing in diabetes. We induced diabetes by i.p. injection of streptozotocin 2 weeks prior to wound creation on the dorsal side of the mouse ear. 11,12 EET was applied every second day on the wound, whereas the control groups received only solvent. Epithelialization was monitored every second day intravitally up to wound closure. Wounds were stained for VEGF, CD31, TGF-ß, TNF-α, SDF-1α, NF-κB, and Ki-67, and fibroblasts were counted after hematoxylin-eosin stain on days 3, 6, 9, and 16 after wounding. After induction of diabetes, wounds closed on day 13.00 ± 2.20 standard deviation (SD). Local 11,12 ETT application improved wound closure significantly to day 8.40 ± 1.39 SD. EET treatment enhanced VEGF and CD31 expression in wounds on day 3. It also seemed to raise TNF-α level on all days investigated as well as TGF-ß level on days 3 and 6. A decrease in NF-κB could be observed on days 9 and 16 after EET application. The latter findings were not significant. SDF-1α expression was not influenced by EET application, and Ki-67 was significantly less in the EET group on day 9 after EET application. The number of fibroblasts was significantly increased on day 9 after the 11,12 EET application. 11,12 EET improve deteriorated wound healing in diabetes by enhancing neoangiogenesis, especially in the early phase of wound healing. Furthermore, they contribute to the dissolution of the initial inflammatory reaction, allowing the crucial transition from the inflammatory to proliferative phase in wound healing.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic diversity through space and time can reveal trends in virus importation and domestic circulation and permit the exploration of questions regarding the early transmission dynamics. Here, we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the coronavirus-19 pandemic. We generated and analysed 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylogeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions, with differential degrees of persistence and national dissemination.

Mutations Associated with SARS-CoV-2 Variants of Concern, Benin, Early 2021.

Intense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa might promote emergence of variants. We describe 10 SARS-CoV-2 lineages in Benin during early 2021 that harbored mutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients, warranting accelerated vaccination in Africa.

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador.

Characterisation of SARS-CoV-2 genetic diversity through space and time can reveal trends in virus importation and domestic circulation, and permit the exploration of questions regarding the early transmission dynamics. Here we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the COVID-19 pandemic. We generate and analyse 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylgeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions (NPIs), with differential degrees of persistence and national dissemination.

An Observational Laboratory-Based Assessment of SARS-CoV-2 Molecular Diagnostics in Benin, Western Africa.

Information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread in Africa is limited by insufficient diagnostic capacity. Here, we assessed the coronavirus disease (COVID-19)-related diagnostic workload during the onset of the pandemic in the central laboratory of Benin, Western Africa; characterized 12 SARS-CoV-2 genomes from returning travelers; and validated the Da An RT-PCR-based diagnostic kit that is widely used across Africa. We found a 15-fold increase in the monthly laboratory workload due to COVID-19, dealt with at the cost of routine activities. Genomic surveillance showed near-simultaneous introduction of distinct SARS-CoV-2 lineages termed A.4 and B.1, including the D614G spike protein variant potentially associated with higher transmissibility from travelers from six different European and African countries during March-April 2020. We decoded the target regions within the ORF1ab and N genes of the Da An dual-target kit by MinION-based amplicon sequencing. Despite relatively high similarity between SARS-CoV-2 and endemic human coronaviruses (HCoVs) within the ORF1ab target domain, no cross-detection of high-titered cell culture supernatants of HCoVs was observed, suggesting high analytical specificity. The Da An kit was highly sensitive, detecting 3.2 to 9.0 copies of target-specific in vitro transcripts/reaction. Although discrepant test results were observed in low-titered clinical samples, clinical sensitivity of the Da An kit was at least comparable to that of commercial kits from affluent settings. In sum, virologic diagnostics are achievable in a resource-limited setting, but unprecedented pressure resulting from COVID-19-related diagnostics requires rapid and sustainable support of national and supranational stakeholders addressing limited laboratory capacity.IMPORTANCE Months after the start of the COVID-19 pandemic, case numbers from Africa are surprisingly low, potentially because the number of SARS-CoV-2 tests performed in Africa is lower than in other regions. Here, we show an overload of COVID-19-related diagnostics in the central laboratory of Benin, Western Africa, with a stagnating average number of positive samples irrespective of daily sample counts. SARS-CoV-2 genomic surveillance confirmed a high genomic diversity in Benin introduced by travelers returning from Europe and other African countries, including early circulation of the D614G spike mutation associated with potentially higher transmissibility. We validated a widely used RT-PCR kit donated by the Chinese Jack Ma Foundation and confirmed high analytical specificity and clinical sensitivity equivalent to tests used in affluent settings. Our assessment shows that although achievable in an African setting, the burden from COVID-19-related diagnostics on national reference laboratories is very high.

Outcome of conservative treatment for triangular fibrocartilage complex lesions with stable distal radioulnar joint.

PURPOSE: Triangular fibrocartilage complex (TFCC) lesions have high clinical relevance. Although multiple studies have been done in the past, there is a lack of data after conservative treatment and controversy remains regarding management. The purpose of this study was to compare the outcome of symptomatic TFCC lesions after conservative treatment and arthroscopic debridement. METHODS: Between 2012 and 2017, 33 patients were included. 16 patients were treated conservatively and 17 patients with arthroscopic debridement. The average age was 41 years (range 18-63). The mean follow-up was 22.2 months (range 6-74). Evaluation included pain, range of motion (ROM), grip strength, Disabilities of the Arm, Shoulder, and Hand (DASH) score, Modified Mayo Wrist Score (MMWS), and Purdue Pegboard test. RESULTS: Pain averaged 0.1 (range 0-1) in the conservative group compared to 1.3 (range 0-6) in the arthroscopic group. The mean ROM was 99% for wrist extension, and 100% for flexion and pro-/supination in the conservative group, and 96% for extension and flexion, and 100% for pro-/supination in the arthoscopic group compared to the contralateral side. Grip strength was 88% (range 63-100) in the conservative group versus 89% (range 33-100) in the arthroscopic group. The conservative group reached a DASH score of 16.8 and MMWS of 94.3 compared to 22.1 and 87.2 in the arthroscopic group. The differences were not statistically significant. CONCLUSIONS: Our study demonstrated similar results of conservative compared to arthroscopic treatment. Because conservative treatment was a sufficient and reliable option, we propose it as first-line treatment for TFCC lesions with stable distal radioulnar joint.

Limited Specificity of Serologic Tests for SARS-CoV-2 Antibody Detection, Benin.

We used commercially available ELISAs to test 68 samples from coronavirus disease cases and prepandemic controls from Benin. We noted <25% false-positive results among controls, likely due to unspecific immune responses elicited by acute malaria. Serologic tests must be carefully evaluated to assess coronavirus disease spread and immunity in tropical regions.

Cross-order host switches of hepatitis C-related viruses illustrated by a novel hepacivirus from sloths.

The genealogy of the hepatitis C virus (HCV) and the genus Hepacivirus remains elusive despite numerous recently discovered animal hepaciviruses (HVs). Viruses from evolutionarily ancient mammals might elucidate the HV macro-evolutionary patterns. Here, we investigated sixty-seven two-toed and nine three-toed sloths from Costa Rica for HVs using molecular and serological tools. A novel sloth HV was detected by reverse transcription polymerase chain reaction (RT-PCR) in three-toed sloths (2/9, 22.2%; 95% confidence interval (CI), 5.3-55.7). Genomic characterization revealed typical HV features including overall polyprotein gene structure, a type 4 internal ribosomal entry site in the viral 5'-genome terminus, an A-U-rich region and X-tail structure in the viral 3'-genome terminus. Different from other animal HVs, HV seropositivity in two-toed sloths was low at 4.5 per cent (3/67; CI, 1.0-12.9), whereas the RT-PCR-positive three-toed sloths were seronegative. Limited cross-reactivity of the serological assay implied exposure of seropositive two-toed sloths to HVs of unknown origin and recent infections in RT-PCR-positive animals preceding seroconversion. Recent infections were consistent with only 9 nucleotide exchanges between the two sloth HVs, located predominantly within the E1/E2 encoding regions. Translated sequence distances of NS3 and NS5 proteins and host comparisons suggested that the sloth HV represents a novel HV species. Event- and sequence distance-based reconciliations of phylogenies of HVs and of their hosts revealed complex macro-evolutionary patterns, including both long-term evolutionary associations and host switches, most strikingly from rodents into sloths. Ancestral state reconstructions corroborated rodents as predominant sources of HV host switches during the genealogy of extant HVs. Sequence distance comparisons, partial conservation of critical amino acid residues associated with HV entry and selection pressure signatures of host genes encoding entry and antiviral protein orthologs were consistent with HV host switches between genetically divergent mammals, including the projected host switch from rodents into sloths. Structural comparison of HCV and sloth HV E2 proteins suggested conserved modes of hepaciviral entry. Our data corroborate complex macro-evolutionary patterns shaping the genus Hepacivirus, highlight that host switches are possible across highly diverse host taxa, and elucidate a prominent role of rodent hosts during the Hepacivirus genealogy.

Epidemiology and treatment of distal radius fractures: current concept based on fracture severity and not on age.

PURPOSE: A shift towards surgical treatment of distal radius fractures seems to be apparent. The purpose of the present study was to assess current epidemiological data, the fracture severity according to the AO classification and the changing trend in the treatment. METHODS: This study presents a retrospective review of 268 patients ≥ 18 years of age admitted to our level I trauma center with distal radius fractures between 2013 and 2015. The data were obtained from the hospital inpatient enquiry system and review of the radiological examinations. RESULTS: The ratio between males and females was 87:181. The average age of the patients was 56.9 (18-99) years. A fall from standing position was the most common mechanism of injury. According to the AO classification, 43.3% were type A fractures, 13.1% type B, and 43.7% type C. As the intensity of the injury mechanism increased, the percentage of type C fractures also increased. A total of 61.9% of patients underwent surgery with open reduction and internal fixation with volar locking plates used as the preferred surgical treatment. Fracture severity correlated well with surgical decision. Elderly patients were just as likely to get surgical treatment as adult patients. CONCLUSIONS: Our findings confirmed the increasing popularity of surgical intervention with open reduction and internal fixation. Contrary to previous studies, the fracture type profile showed an equal proportion of type A and C fractures and the indication for surgical treatment correlated with fracture severity and not age.

Sloths host Anhanga virus-related phleboviruses across large distances in time and space.

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod-borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two-toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two-toed (Choloepus hoffmanni, n = 65) and three-toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT-PCR. A clinically healthy adult Hoffmann's two-toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 107  RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non-synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro-evolutionary processes associated with immune evasion during the genealogy of sloth-associated phleboviruses. However, gene-wide mean dN/dS ratios were low at 0.02-0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth-associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely-related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV-related phleboviruses with the two extant species of two-toed sloths.

Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses.

Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.