Identification of Novel Rodent-Borne Orthohantaviruses in an Endemic Area of Chronic Kidney Disease of Unknown Etiology (CKDu) in Sri Lanka.

We reported the genetic evidence of circulating hantaviruses from small mammals captured in a chronic kidney disease of unknown etiology (CKDu) hotspot area of Sri Lanka. The high seroprevalence of anti-hantavirus antibodies against Thailand orthohantavirus (THAIV) has been reported among CKDu patients and rodents in Sri Lankan CKDu hotspots. We captured 116 small mammals from CKDu endemic regions in the Polonnaruwa District of Sri Lanka. Seven animals (five out of 11 Mus booduga and two out of 99 Rattus rattus) were PCR-positive for the hantavirus. A rat-borne sequence was grouped with a THAIV-like Anjozorobe virus. In contrast, Mus-borne sequences belonged to the THAIV lineage, suggesting a novel orthohantavirus species according to the phylogenetic analyses and whole-genome comparisons. Our genetic evidence indicates the presence of two THAIV-related viruses circulating in this CKDu endemic area, suggesting a basis for further investigations to identify the infectious virus in patients with CKDu and the CKDu induction mechanism of these viruses.

Orthohantavirus Isolated in Reservoir Host Cells Displays Minimal Genetic Changes and Retains Wild-Type Infection Properties.

: Orthohantaviruses are globally emerging zoonotic pathogens. While the reservoir host role of several rodent species is well-established, detailed research on the mechanisms of host-othohantavirus interactions has been constrained by the lack of an experimental system that is able to effectively replicate natural infections in controlled settings. Here we report the isolation, and genetic and phenotypic characterization of a novel Puumala orthohantavirus (PUUV) in cells derived from its reservoir host, the bank vole. The isolation process resulted in cell culture infection that evaded antiviral responses, persisted cell passaging, and had minor viral genome alterations. Critically, experimental infections of bank voles with the new isolate resembled natural infections in terms of viral load and host cell distribution. When compared to an attenuated Vero E6 cell-adapted PUUV Kazan strain, the novel isolate demonstrated delayed virus-specific humoral responses. A lack of virus-specific antibodies was also observed during experimental infections with wild-type PUUV, suggesting that delayed seroconversion could be a general phenomenon during orthohantavirus infection in reservoir hosts. Our results demonstrate that orthohantavirus isolation on cells derived from a vole reservoir host retains wild-type infection properties and should be considered the method of choice for experimental infection models to replicate natural processes.

Isolation and characterization of new Puumala orthohantavirus strains from Germany.

Orthohantaviruses are re-emerging rodent-borne pathogens distributed all over the world. Here, we report the isolation of a Puumala orthohantavirus (PUUV) strain from bank voles caught in a highly endemic region around the city Osnabrück, north-west Germany. Coding and non-coding sequences of all three segments (S, M, and L) were determined from original lung tissue, after isolation and after additional passaging in VeroE6 cells and a bank vole-derived kidney cell line. Different single amino acid substitutions were observed in the RNA-dependent RNA polymerase (RdRP) of the two stable PUUV isolates. The PUUV strain from VeroE6 cells showed a lower titer when propagated on bank vole cells compared to VeroE6 cells. Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. In conclusion, this is the first isolation of a PUUV strain from Central Europe and the generation of glycoprotein-specific monoclonal antibodies for this PUUV isolate. The obtained virus isolate and GPC-specific antibodies are instrumental tools for future reservoir host studies.

Orthohantavirus pulmonary syndrome in Santa Cruz and Tarija, Bolivia, 2018.

INTRODUCTION: Orthohantaviruses are still a significant public health threat in endemic countries, with high case fatality rates (CFR). In Bolivia, the reporting of small outbreaks occurred until 2012. The findings of 40 laboratory-confirmed cases diagnosed in two departments are reported herein. METHODS: This was an observational, retrospective and cross-sectional study. Data on laboratory-confirmed cases in 2018 were collected from the hospitals and departmental health services (SEDES) of Santa Cruz and Tarija. An ELISA was used for the detection of IgM antibody to hantavirus in the patient blood samples. RESULTS: Forty patients were IgM-positive. The median age of the patients was 24 years (interquartile range 19-41 years) and 72.5% were male. All patients were hospitalized; 57.5% were admitted to the intensive care unit and had cardiopulmonary compromise, with 83% of these presenting acute respiratory distress syndrome and 89.5% of these requiring mechanical ventilation. Six patients died (CFR 15%). Patients <15 or >60 years old were more prone to die (odds ratio 10.33, 95% confidence interval 1.411-75.694), as were those with comorbidities (odds ratio 16.5, 95% confidence interval 1.207-225.540). CONCLUSIONS: Orthohantavirus infections were associated with a high CFR. These cases occurred in areas with eco-epidemiological conditions facilitating viral transmission, including the presence of rodents, as well as the risk of spillover to humans due to social, environmental, and occupational factors.

Discovery of a highly divergent hepadnavirus in shrews from China.

Limited sampling means that relatively little is known about the diversity and evolutionary history of mammalian members of the Hepadnaviridae (genus Orthohepadnavirus). An important case in point are shrews, the fourth largest group of mammals, but for which there is limited knowledge on the role they play in viral evolution and emergence. Here, we report the discovery of a novel shrew hepadnavirus. The newly discovered virus, denoted shrew hepatitis B virus (SHBV), is divergent to be considered a new species of Orthohepadnavirus. Phylogenetic analysis revealed that these viruses were usually most closely related to TBHBV (tent-making bat hepatitis B virus), known to be able to infect human hepatocytes, and had a similar genome structure, although SHBV fell in a more basal position in the surface protein phylogeny. In sum, these data suggest that shrews are natural hosts for hepadnaviruses and may have played an important role in their long-term evolution.

A Novel Orthohepadnavirus Identified in a Dead Maxwell's Duiker (Philantomba maxwellii) in Taï National Park, Côte d'Ivoire.

New technologies enable viral discovery in a diversity of hosts, providing insights into viral evolution. We used one such approach, the virome capture sequencing for vertebrate viruses (VirCapSeq-VERT) platform, on 21 samples originating from six dead Maxwell's duikers (Philantomba maxwellii) from Taï National Park, Côte d'Ivoire. We detected the presence of an orthohepadnavirus in one animal and characterized its 3128 bp genome. The highest viral copy numbers were detected in the spleen, followed by the lung, blood, and liver, with the lowest copy numbers in the kidney and heart; the virus was not detected in the jejunum. Viral copy numbers in the blood were in the range known from humans with active chronic infections leading to liver histolytic damage, suggesting this virus could be pathogenic in duikers, though many orthohepadnaviruses appear to be apathogenic in other hosts, precluding a formal test of this hypothesis. The virus was not detected in 29 other dead duiker samples from the Côte d'Ivoire and Central African Republic, suggesting either a spillover event or a low prevalence in these populations. Phylogenetic analysis placed the virus as a divergent member of the mammalian clade of orthohepadnaviruses, though its relationship to other orthohepadnaviruses remains uncertain. This represents the first orthohepadnavirus described in an artiodactyl. We have tentatively named this new member of the genus Orthohepadnavirus (family Hepadnaviridae), Taï Forest hepadnavirus. Further studies are needed to determine whether it, or some close relatives, are present in a broader range of artiodactyls, including livestock.

Development of a serosurveillance assay for detection of Necoclí virus exposure.

Hantavirus cardiopulmonary syndrome (HPS) has gained importance in Latin America as an emerging disease, with reports of about 4000 HPS cases; however, this is probably an underestimate because of limited surveillance programs and diagnostic tools to confirm HPS. In order to address this issue and develop better serosurveillance capability, we evaluated three recombinant peptides from the Necoclí virus (NECV) nucleocapsid in antibody-capture ELISA. We cloned and expressed antigens representing the whole NECV nucleocapsid protein (NECV-rN), the immunodominant domain (NECV-rN100), and a serospecific domain (NECV-rN428), and then we compared these antigens in ELISA to detect IgG antibodies to NECV in human sera. We evaluated human sera collected during two epidemiological studies from the area where NECV was discovered. The first group included 609 sera from healthy individuals, and the second one included 89 samples from patients with undifferentiated febrile illness. In these two groups, hantavirus infection had previously been determined by the presence of IgG to Maciel virus (MCLV), a hantavirus closely related to NECV. The number of IgG-positive sera was higher using the Necoclí ELISA with the rN100 protein, which detected antibodies in a higher percentage of healthy individuals, 129/609 (21.2%), as well as in febrile patients, 11/89 (12.3%). In contrast, using MCLV ELISA, 8 of 609 (1.3%) and 4 of 89 (4.5%) samples from healthy and febrile patients, respectively, were seropositive. The agreement between the NECV and MCLV ELISA assays was ≥ 82.3%; however, the kappa indices were weak but statistically significant for rN (0.251 CI; 0.138-0.365) and rN100rN (0.153 CI; 0.084-0.223). The weak kappa indices were attributed to decreased MCLV ELISA assay sensitivity. These results suggest that NECV rN and rN100 have increased specificity and could be further validated for improved diagnosis of hantavirus infections.

Silent Orthohantavirus Circulation Among Humans and Small Mammals from Central Minas Gerais, Brazil.

New World orthohantaviruses are emerging RNA viruses that cause hantavirus cardiopulmonary syndrome (HCPS). These viruses are a burden to public health around the world with a lethality rate of around 60%. In South America, rodents of Sigmodontinae subfamily are the main reservoirs of orthohantaviruses. We described a serosurvey for orthohantaviruses circulation in an apparently healthy human population and small mammals from rural areas in Central Minas Gerais State, Brazil. A total of 240 individuals and 50 small mammals (26 rodents belonging to 10 different species and 24 marsupials from 4 different species) were sampled during 2012-2013. The seroprevalence rates of IgG/IgM antibodies in humans were 7.1 and 1.6%, respectively. Only one rodent, an Oligoryzomys nigripes captured in peridomestic area, tested positive for IgG antibodies and viral RNA. Our findings suggest a silent circulation of orthohantaviruses in a region of intensive agriculture production. The detection of seropositive humans in an area with a lack of previous HCPS reports highlights potential oligosymptomatic cases and the need for surveillance strategies that could reduce the risk of future outbreaks.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses.

BACKGROUND & AIMS: All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. METHODS: We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. RESULTS: We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. CONCLUSIONS: Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. LAY SUMMARY: The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

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

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

Hepatitis virus in long-fingered bats, Myanmar.

During an analysis of the virome of bats from Myanmar, a large number of reads were annotated to orthohepadnaviruses. We present the full genome sequence and a morphological analysis of an orthohepadnavirus circulating in bats. This virus is substantially different from currently known members of the genus Orthohepadnavirus and represents a new species.

Prevalence of a virus similar to human hepatitis B virus in swine.

BACKGROUND: The objective of this study is to established evidence of the existence of a novel member of the hepadnavirus family endemic in swine. Temporarily this virus was designated as swine hepatitis B virus (SHBV). This SHBV can be detected by using human hepatitis B virus diagnostic kits including ELISA, immunohistochemical staining, and transmission electron microscopy (TEM). Also seroprevalence of pig farms in Beijing, China, and pathological features of SHBV infection was determined. RESULTS: Screened result shows that overall prevalence of HBsAg was 24.8%, closed to that of anti-HBsAg, whereas HBeAg and anti-HBe were barely detectable. The distribution of HBsAg and HBcAg was examined by immunohistochemistry of liver samples. Typical hepatitis pathological change, such as spotty parenchymal cell degeneration, necrosis of hepatocytes and proliferation of fibrous connective tissue were observed during histopathological analysis. Analysis of HBsAg-positive serum with TEM revealed two morphologic forms, 20 nm and 40 nm sized particles, similar to small spherical and Danes particles of HBV. Observation of the ultrastructure of the liver also found HBV-like particles in the nucleus of hepatocytes. CONCLUSION: Our research result implies that SHBV could be a causative agent of swine. The discovery of SHBV will unveil novel evolutionary aspects of hepatitis and provides new information for further hepadnavirus research.

Acute hepatitis E virus infection in Taiwan 2002-2006 revisited: PCR shows frequent co-infection with multiple hepatitis viruses.

Sporadic cases of acute hepatitis E virus (HEV) infection with production of anti-HEV IgM have been reported occasionally in Taiwan despite no reported outbreaks in the past. This study was undertaken to determine whether serological markers correlated with virus detection. From 2002 to 2006, 72 reported cases of acute hepatitis E seropositive for anti-HEV IgM in Taiwan were enrolled for investigation. Acute phase serum samples were collected for detection of HEV RNA, HBV DNA, HCV RNA, and GBV-C RNA by PCR. The results showed that viral sequences of HEV, HBV, HCV and GBV-C were detected in 54 (75%), 21 (29.2%), 9 (12.5%), and 22 (30.6%) of cases, respectively. Acute hepatitis A co-infection was excluded in all patients because none were seropositive for anti-HAV IgM and, nine patients (12.5%) did not seroconvert to anti-HEV IgG. These results suggest that serum markers did not correlate completely with viremia in the diagnosis of acute HEV infection. Multiple viruses may co-infect with acute hepatitis E virus in Taiwan. Detection of hepatitis E viremia together with seropositivity for anti-HEV IgM and followed by seroconversion to anti-HEV IgG should be included in the diagnostic criteria for HEV infection.

Performance evaluation of the QIAGEN EZ1 DSP Virus Kit with Abbott RealTime HIV-1, HBV and HCV assays.

BACKGROUND: Automated sample preparation systems must meet the demands of routine diagnostics laboratories with regard to performance characteristics and compatibility with downstream assays. OBJECTIVES: In this study, the performance of QIAGEN EZ1 DSP Virus Kit on the BioRobot EZ1 DSP was evaluated in combination with the Abbott RealTime HIV-1, HCV, and HBV assays, followed by thermalcycling and detection on the Abbott m2000rt platform. STUDY DESIGN: The following performance characteristics were evaluated: linear range and precision, sensitivity, cross-contamination, effects of interfering substances and correlation. RESULTS: Linearity was observed within the tested ranges (for HIV-1: 2.0-6.0 log copies/ml, HCV: 1.3-6.9 log IU/ml, HBV: 1.6-7.6 log copies/ml). Excellent precision was obtained (inter-assay standard deviation for HIV-1: 0.06-0.17 log copies/ml (>2.17 log copies/ml), HCV: 0.05-0.11 log IU/ml (>2.09 log IU/ml), HBV: 0.03-0.07 log copies/ml (>2.55 log copies/ml)), with good sensitivity (95% hit rates for HIV-1: 50 copies/ml, HCV: 12.5 IU/ml, HBV: 10 IU/ml). No cross-contamination was observed, as well as no negative impact of elevated levels of various interfering substances. In addition, HCV and HBV viral load measurements after BioRobot EZ1 DSP extraction correlated well with those obtained after Abbott m2000sp extraction. CONCLUSIONS: This evaluation demonstrates that the QIAGEN EZ1 DSP Virus Kit provides an attractive solution for fully automated, low throughput sample preparation for use with the Abbott RealTime HIV-1, HCV, and HBV assays.

Liver cirrhosis in HIV-infected patients: prevalence, aetiology and clinical outcome.

Liver disease is frequently seen in HIV+ patients as a result of coinfection with hepatitis B (HBV) or C (HCV) viruses, alcohol abuse and/or exposure to hepatotoxic drugs. The aim of this study was to assess the prevalence of liver cirrhosis, its main causes and clinical presentation in HIV+ patients. Observational, cross-sectional, retrospective study of all HIV+ individuals followed at one reference HIV outpatient clinic in Madrid. Liver fibrosis was measured in all cases using transient elastometry (FibroScan). All 2168 HIV+ patients on regular follow-up (76% males, 46% injecting drug users) were successfully examined by FibroScan) between October 2004 and August 2006. Liver cirrhosis was recognized in 181 (overall prevalence, 8.3%), and the main aetiologies were HCV, 82.3%; HBV, 1.6%; dual HBV/HCV, 2.8%; and triple HBV/HCV/ hepatitis delta virus (HDV) infection, 6.6%. The prevalence of cirrhosis differed among patients with distinct chronic viral hepatitis: HCV, 19.2%; HBV, 6.1%; HBV/HCV, 41.7%; and HBV/HCV/HDV, 66.7%. In 12 patients with cirrhosis (6.7%), no definite aetiology was recognized. Overall, cirrhotics had lower mean CD4 counts than noncirrhotics (408 vs 528 cells/microL respectively; P = 0.02), despite similar proportion of subjects with undetectable viraemia on highly active antiretroviral therapy. Clinical manifestations of liver cirrhosis were: splenomegaly, 61.5%; oesophageal varices, 59.8%; ascites, 22.6%; encephalopathy, 12.1%; and variceal bleeding, 6.1%. Liver cirrhosis and hepatic decompensation events are relatively frequent in HIV+ individuals. Chronic HCV and alcohol abuse, but not chronic HBV, play a major role. Transient elastometry may allow the identification of a significant number of HIV+ individuals with asymptomatic liver cirrhosis.

Analysis of integrated ground squirrel hepatitis virus and flanking host DNA in two hepatocellular carcinomas.

We cloned the integrated ground squirrel hepatitis B virus (GSHV) sequences from two hepatomas showing a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other hepadnaviruses. Insertional activation of a cellular gene appears unlikely: the integrated GSHV sequences lacked the known viral enhancers and were not expressed in the tumors, and we found no evidence for the presence of a gene at the integration site. Our results, together with those earlier studies, suggest that GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitis virus. GSHV may thus cause carcinogenesis by more indirect mechanisms, as does the human hepatitis B virus.