Pathogens associated with hospitalization due to acute lower respiratory tract infections in children in rural Ghana: a case-control study.

Respiratory infections are one of the most common causes of death among children under the age of five years. Data on prevalence and relevance of specific organisms in African children are still lacking. This case-control-study investigated prevalence and relevance of specific organisms in Ghanaian children admitted to hospital with symptoms of lower respiratory tract infection (LRTI). Pharyngeal swabs were taken and tested by PCR for 19 respiratory isolates. Adjusted odds ratios (aORs) were calculated to estimate associations between isolates and admission with LRTI. Population attributable fractions (PAFs) were calculated to assess the proportion of LRTI cases due to a particular pathogen. The study included 327 cases and 562 controls. We found associations between detection and admission for LRTI for influenza (aOR 98.6; 95% confidence interval (CI) 20.0-1789.6), respiratory syncytial virus (aOR 40.2; 95% CI 7.2-758.6), H. influenzae (aOR 4.1; 95% CI 2.2-7.9) and S. pneumoniae (aOR 2.4; 95% CI 1.7-3.4). PAFs ≥ 10% were observed for S. pneumoniae (30%; 95% CI 26-42), H. influenzae (10%; 95% CI 2-19) and influenza (10%; 95% CI 2-18). This study highlights the need for heightened surveillance and development of effective vaccines for respiratory pathogens other than SARS-CoV-2 in the future.

Genetic characterization of varicella-zoster and HIV-1 viruses from the cerebrospinal fluid of a co-infected encephalitic patient, Ghana.

BACKGROUND: Encephalitis is a serious disease of the brain characterized by prodromal and specific neurological symptoms. HIV infections offer opportunistic viruses, such as Varicella-zoster virus (VZV), the chance to cause encephalitis in patients. There is a lack of information on the genetic diversity of VZV in Ghana and other parts of Africa which requires sequencing and characterization studies to address. The active evolution of HIV-1 in West Africa also requires continuous surveillance for the emergence of new genetic forms. CASE PRESENTATION: VZV was detected in the CSF sample of an 11-year-old patient presenting with symptoms of encephalitis by real-time PCR diagnostics. To identify possible unknown aetiological pathogens, next-generation sequencing was performed, and revealed an HIV-1 co-infection. Alignments of concatenated HIV-1 genome fragments in the gag, pol, vif, env and nef regions and a near-complete VZV genome were analyzed by Bayesian inference, and phylogenetic trees were generated. The VZV sequence belongs to clade 5 and the HIV-1 sequence is a member of the CRF02_AG predominant circulating recombinant form in Ghana. CONCLUSIONS: Diagnostic tests for CSF HIV would be useful where possible in patients presenting with encephalitis due to VZV and other opportunistic viruses in Kumasi to shed light on the role of HIV in encephalitis cases in Ghana. This report reaffirms the role of the CRF02_AG circulating recombinant form in HIV infections in Ghana and also gives a preliminary genetic characterization of VZV in Kumasi, Ghana.

Transmission of SARS-CoV-2 in northern Ghana: insights from whole-genome sequencing.

Following the detection of the first imported case of COVID-19 in the northern sector of Ghana, we molecularly characterized and phylogenetically analysed sequences, including three complete genome sequences, of severe acute respiratory syndrome coronavirus 2 obtained from nine patients in Ghana. We performed high-throughput sequencing on nine samples that were found to have a high concentration of viral RNA. We also assessed the potential impact that long-distance transport of samples to testing centres may have on sequencing results. Here, two samples that were similar in terms of viral RNA concentration but were transported from sites that are over 400 km apart were analyzed. All sequences were compared to previous sequences from Ghana and representative sequences from regions where our patients had previously travelled. Three complete genome sequences and another nearly complete genome sequence with 95.6% coverage were obtained. Sequences with coverage in excess of 80% were found to belong to three lineages, namely A, B.1 and B.2. Our sequences clustered in two different clades, with the majority falling within a clade composed of sequences from sub-Saharan Africa. Less RNA fragmentation was seen in sample KATH23, which was collected 9 km from the testing site, than in sample TTH6, which was collected and transported over a distance of 400 km to the testing site. The clustering of several sequences from sub-Saharan Africa suggests regional circulation of the viruses in the subregion. Importantly, there may be a need to decentralize testing sites and build more capacity across Africa to boost the sequencing output of the subregion.

At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions.

Bats host many viruses pathogenic to humans, and increasing evidence suggests that rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America, and Africa were PCR-positive for RVA, and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, which included evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite various levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses.IMPORTANCE The increased research on bat coronaviruses after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) allowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general, and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents sheds light on the vast genetic diversity of rotaviruses and also hints at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.

Epidemiological profile of SARS-CoV-2 among selected regions in Ghana: A cross-sectional retrospective study.

BACKGROUND: Global cases of COVID-19 continue to rise, causing havoc to several economies. So far, Ghana has recorded 48,643 confirmed cases with 320 associated deaths. Although summaries of data are usually provided by the Ministry of Health, detailed epidemiological profile of cases are limited. This study sought to describe the socio-demographic features, pattern of COVID-19 spread and the viral load dynamics among subjects residing in northern, middle and part of the southern belt of Ghana. METHODS: This was a cross-sectional retrospective study that reviewed records of samples collected from February to July, 2020. Respiratory specimens such as sputum, deep-cough saliva and nasopharyngeal swabs were collected from suspected COVID-19 subjects in 12 regions of Ghana for laboratory analysis and confirmation by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: A total of 72,434 samples were collected during the review period, with majority of the sampled individuals being females (37,464; 51.9%). The prevalence of SARS-CoV-2 identified in the study population was 13.2% [95%CI: 12.9, 13.4). Males were mostly infected (4,897; 51.5%) compared to females. Individuals between the ages 21-30 years recorded the highest number of infections (3,144, 33.4%). Symptomatic subjects had higher viral loads (1479.7 copies/µl; IQR = 40.6-178919) than asymptomatic subjects (49.9; IQR = 5.5-3641.6). There was significant association between gender or age and infection with SARS-CoV-2 (p<0.05). Among all the suspected clinical presentations, anosmia was the strongest predictor of SARS-CoV-2 infection (Adj. OR (95%CI): 24.39 (20.18, 29.49). We observed an average reproductive number of 1.36 with a minimum of 1.28 and maximum of 1.43. The virus trajectory shows a gradual reduction of the virus reproductive number. CONCLUSION: This study has described the epidemiological profile of COVID-19 cases in northern, middle and part of the southern belt of Ghana, with males and younger individuals at greater risk of contracting the disease. Health professionals should be conscious of individuals presenting with anosmia since this was seen as the strongest predictor of virus infection.

Diagnostics for COVID-19: A case for field-deployable, rapid molecular tests for community surveillance.

Across the globe, the outbreak of the COVID-19 pandemic is causing distress with governments doing everything in their power to contain the spread of the novel coronavirus (SARS-CoV-2) to prevent morbidity and mortality. Actions are being implemented to keep health care systems from being overstretched and to curb the outbreak. Any policy responses aimed at slowing down the spread of the virus and mitigating its immediate effects on health care systems require a firm basis of information about the absolute number of currently infected people, growth rates, and locations/hotspots of infections. The only way to obtain this base of information is by conducting numerous tests in a targeted way. Currently, in Ghana, there is a centralized testing approach, that takes 4-5 days for samples to be shipped and tested at central reference laboratories with results communicated to the district, regional and national stakeholders. This delay in diagnosis increases the risk of ongoing transmission in communities and vulnerable institutions. We have validated, evaluated and deployed an innovative diagnostic tool on a mobile laboratory platform to accelerate the COVID-19 testing. A preliminary result of 74 samples from COVID-19 suspected cases has a positivity rate of 12% with a turn-around time of fewer than 3 hours from sample taking to reporting of results, significantly reducing the waiting time from days to hours, enabling expedient response by the health system for contact tracing to reduce transmission and additionally improving case management. FUNDING: Test kits were provided by AngloGold Ashanti Obuasi Mine (AngloGold Ashanti Health Foundation). The American Leprosy Mission donated the PCR machine, and the mobile laboratory van was funded by the Embassy of the Kingdom of the Netherlands (EKN). AAS, YAA was supported by (PANDORA-ID-NET RIA2016E-1609) and ROP supported by EDCTP Senior Fellowship (TMA2016SF), both funded by the European and Developing Countries Clinical Trials Partnership (EDCTP2) programme which is supported under Horizon 2020, the European Union.

Detection and genomic characterization of hepatitis E virus genotype 3 from pigs in Ghana, Africa.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of human hepatitis worldwide. Zoonotic genotypes of the virus have been found in diverse animal species with pigs playing a major role. Putative risk of zoonotic infection from livestock particularly swine in Sub-Saharan Africa including Ghana is poorly understood due to scarcity of available data, especially HEV sequence information. METHODS: Serum samples were collected from cattle, sheep, goats and pigs from Kumasi in the Ashanti region of Ghana. Samples were subjected to nested RT-PCR screening and quantification of HEV RNA-positive samples using real-time RT-PCR and the World Health Organization International Standard for HEV. Testing of all pig samples for antibodies was done by ELISA. Sanger sequencing and genotyping was performed and one representative complete genome was generated to facilitate genome-wide comparison to other available African HEV sequences by phylogenetic analysis. RESULTS: A total of 420 samples were available from cattle (n = 105), goats (n = 124), pigs (n = 89) and sheep (n = 102). HEV Viral RNA was detected only in pig samples (10.1%). The antibody detection rate in pigs was 77.5%, with positive samples from all sampling sites. Average viral load was 1 × 105 (range 1.02 × 103 to 3.17 × 105) International Units per mL of serum with no statistically significant differences between age groups (≤ 6 month, > 6 months) by a T-test comparison of means (t = 1.4272, df = 7, p = 0.1966). Sequences obtained in this study form a monophyletic group within HEV genotype 3. Sequences from Cameroon, Ghana, Burkina Faso and Madagascar were found to share a most recent common ancestor; however this was not the case for other African HEV sequences. CONCLUSION: HEV genotype 3 is highly endemic in pigs in Ghana and likely poses a zoonotic risk to people exposed to pigs. HEV genotype 3 in Ghana shares a common origin with other virus strains from Sub-Saharan Africa.

Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission.

A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (-29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23-fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS.

Burden of influenza among hospitalized febrile children in Ghana.

BACKGROUND: Influenza surveillance data from Africa indicate a substantial disease burden with high mortality. However, local influenza data from district hospitals with limited laboratory facilities are still scarce. OBJECTIVES: To identify the frequency and seasonal distribution of influenza among hospitalized febrile children in a rural hospital in Ghana and to describe differential diagnoses to other severe febrile infections. METHODS: Between January 2014 and April 2015, all children with a temperature of ≥38°C admitted to a district hospital in Ghana were screened for influenza A and B by RT-PCR and differentiated to subtypes A(H1N1)pdm09 and A(H3N2). Malaria microscopy and blood cultures were performed for each patient. RESULTS: A total of 1063 children with a median age of 2 years (IQR: 1-4 years) were recruited. Of those, 271 (21%) were classified as severe acute respiratory infection (SARI) and 47 (4%) were positive for influenza, namely 26 (55%) influenza B, 15 (32%) A(H1N1)pdm09, and 6 (13%) A(H3N2) cases. Influenza predominantly occurred in children aged 3-5 years and was more frequently detected in the major rainy season (OR = 2.9; 95% CI: 1.47-6.19) during the first half of the year. Two (4%) and seven (15%) influenza-positive children were co-diagnosed with an invasive bloodstream infection or malaria, respectively. CONCLUSION: Influenza contributes substantially to the burden of hospitalized febrile children in Ghana being strongly dependent on age and corresponds with the major rainy season during the first half-year.

Evidence for widespread infection of African bats with Crimean-Congo hemorrhagic fever-like viruses.

Crimean Congo hemorrhagic fever virus (CCHFV) is a highly virulent tick-borne pathogen that causes hemorrhagic fever in humans. The geographic range of human CCHF cases largely reflects the presence of ticks. However, highly similar CCHFV lineages occur in geographically distant regions. Tick-infested migratory birds have been suggested, but not confirmed, to contribute to the dispersal. Bats have recently been shown to carry nairoviruses distinct from CCHFV. In order to assess the presence of CCHFV in a wide range of bat species over a wide geographic range, we analyzed 1,135 sera from 16 different bat species collected in Congo, Gabon, Ghana, Germany, and Panama. Using a CCHFV glycoprotein-based indirect immunofluorescence test (IIFT), we identified reactive antibodies in 10.0% (114/1,135) of tested bats, pertaining to 12/16 tested species. Depending on the species, 3.6%-42.9% of cave-dwelling bats and 0.6%-7.1% of foliage-living bats were seropositive (two-tailed t-test, p = 0.0447 cave versus foliage). 11/30 IIFT-reactive sera from 10 different African bat species had neutralizing activity in a virus-like particle assay. Neutralization of full CCHFV was confirmed in 5 of 7 sera. Widespread infection of cave-dwelling bats may indicate a role for bats in the life cycle and geographic dispersal of CCHFV.

Viral Shedding and Antibody Response in 37 Patients With Middle East Respiratory Syndrome Coronavirus Infection.

BACKGROUND: The Middle East respiratory syndrome (MERS) coronavirus causes isolated cases and outbreaks of severe respiratory disease. Essential features of the natural history of disease are poorly understood. METHODS: We studied 37 adult patients infected with MERS coronavirus for viral load in the lower and upper respiratory tracts (LRT and URT, respectively), blood, stool, and urine. Antibodies and serum neutralizing activities were determined over the course of disease. RESULTS: One hundred ninety-nine LRT samples collected during the 3 weeks following diagnosis yielded virus RNA in 93% of tests. Average (maximum) viral loads were 5 × 10(6) (6 × 10(10)) copies/mL. Viral loads (positive detection frequencies) in 84 URT samples were 1.9 × 10(4) copies/mL (47.6%). Thirty-three percent of all 108 serum samples tested yielded viral RNA. Only 14.6% of stool and 2.4% of urine samples yielded viral RNA. All seroconversions occurred during the first 2 weeks after diagnosis, which corresponds to the second and third week after symptom onset. Immunoglobulin M detection provided no advantage in sensitivity over immunoglobulin G (IgG) detection. All surviving patients, but only slightly more than half of all fatal cases, produced IgG and neutralizing antibodies. The levels of IgG and neutralizing antibodies were weakly and inversely correlated with LRT viral loads. Presence of antibodies did not lead to the elimination of virus from LRT. CONCLUSIONS: The timing and intensity of respiratory viral shedding in patients with MERS closely matches that of those with severe acute respiratory syndrome. Blood viral RNA does not seem to be infectious. Extrapulmonary loci of virus replication seem possible. Neutralizing antibodies do not suffice to clear the infection.

Filovirus receptor NPC1 contributes to species-specific patterns of ebolavirus susceptibility in bats.

Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature.

Lagos bat virus transmission in an Eidolon helvum bat colony, Ghana.

A brain sample of a straw-coloured fruit bat (Eidolon helvum) from Ghana without evident signs of disease tested positive by generic Lyssavirus RT-PCR and direct antigen staining. Sequence analysis confirmed the presence of a Lagos bat virus belonging to phylogenetic lineage A. Virus neutralization tests using the isolate with sera from the same group of bats yielded neutralizing antibodies in 74% of 567 animals. No cross-neutralization was observed against a different Lagos bat virus (lineage B).

Serological evidence of influenza A viruses in frugivorous bats from Africa.

Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona- and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes--H17N10 and H18N11--in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5 µl of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.

Highly divergent hepaciviruses from African cattle.

UNLABELLED: The hepatitis C virus (HCV; genus Hepacivirus) is a highly relevant human pathogen. Unique hepaciviruses (HV) were discovered recently in animal hosts. The direct ancestor of HCV has not been found, but the genetically most closely related animal HVs exist in horses. To investigate whether other peridomestic animals also carry HVs, we analyzed sera from Ghanaian cattle for HVs by reverse transcription-PCR (RT-PCR). Nine of 106 specimens from different sampling sites contained HV RNA (8.5%) at median viral loads of 1.6 × 10(5) copies/ml. Infection seemed unrelated to cattle age and gender. Near-full-genome sequencing of five representative viruses confirmed taxonomic classifications. Cattle HVs formed two distinct phylogenetic lineages that differed by up to 17.7% on the nucleotide level in the polyprotein-encoding region, suggesting cocirculation of different virus subtypes. A conserved microRNA122-binding site in the 5' internal ribosomal entry site suggested liver tropism of cattle HVs. Phylogenetic analyses suggested the circulation of HVs in cattle for several centuries. Cattle HVs were genetically highly divergent from all other HVs, including HCV. HVs from genetically related equine and bovine hosts were not monophyletic, corroborating host shifts during the evolution of the genus Hepacivirus. Similar to equine HVs, the genetic diversity of cattle HVs was low compared to that of HCV genotypes. This suggests an influence of the human-modified ecology of peridomestic animals on virus diversity. Further studies should investigate the occurrence of cattle HVs in other geographic areas and breeds, virus pathogenicity in cattle, and the potential exposure of human risk groups, such as farmers, butchers, and abattoir workers. IMPORTANCE: HCV (genus Hepacivirus) is a major human pathogen, causing liver failure and cancer. Unique hepaciviruses (HVs) were discovered over the last few years in animals, but the direct ancestor of HCV has not been found. The animal HV most closely related to HCV so far originated from horses, suggesting that other livestock animals also harbor HVs. Therefore, we investigated African cattle and discovered previously unknown HVs at high prevalence and viral loads. Because of the agricultural importance of cattle, it may be relevant to investigate HV pathogenicity. The frequent exposure of humans to cattle also may warrant investigations of the zoonotic potential of these viruses. Evolutionary analyses suggested that cattle HVs have existed for centuries. Despite the genetic relatedness of their animal hosts, HVs from cattle and horses were not phylogenetically related, corroborating frequent host shifts during the evolution of the genus Hepacivirus.

Fusion activity of African henipavirus F proteins with a naturally occurring start codon directly upstream of the signal peptide.

Compared to the fusion proteins of pathogenic Nipah and Hendra viruses, the F protein of prototype African henipavirus GH-M74a displays a drastically reduced surface expression and fusion activity. A probable reason for limited F expression is the unusually long sequence located between the gene start and the signal peptide (SP) not present in other henipaviruses. Such a long pre-SP extension can prevent efficient ER translocation or protein maturation and processing. As its truncation can therefore enhance surface expression, the recent identification of a second in-frame start codon directly upstream of the SP in another African henipavirus F gene (GH-UP28) raised the question if such a naturally occurring minor sequence variation can lead to the synthesis of a pre-SP truncated translation product, thereby increasing the production of mature F proteins. To test this, we analyzed surface expression and biological activity of F genes carrying the second SP-proximal start codon of GH-UP28. Though we observed minor differences in the expression levels, introduction of the additional start codon did not result in an increased fusion activity, even if combined with further mutations in the pre-SP region. Thus, limited bioactivity of African henipavirus F protein is maintained even after sequence changes that alter the gene start allowing the production of F proteins without an unusually long pre-SP.

A novel rhabdovirus isolated from the straw-colored fruit bat Eidolon helvum, with signs of antibodies in swine and humans.

UNLABELLED: Bats have been implicated as reservoirs of emerging viruses. Bat species forming large social groups and roosting in proximity to human communities are of particular interest. In this study, we sampled a colony of ca. 350,000 individuals of the straw-colored fruit bat Eidolon helvum in Kumasi, the second largest city of Ghana. A novel rhabdovirus (Kumasi rhabdovirus [KRV]) was isolated in E. helvum cell cultures and passaged to Vero cells as well as interferon-competent human and primate cells (A549 and MA104). Genome composition was typical for a rhabdovirus. KRV was detected in 5.1% of 487 animals, showing association with the spleen but not the brain. Antibody prevalence was 11.5% by immunofluorescence and 6.4% by plaque reduction virus neutralization test (PRNT). Detection throughout 3 sampling years was pronounced in both annual wet seasons, of which only one overlaps the postparturition season. Juvenile bats showed increased viral prevalence. No evidence of infection was obtained in 1,240 female mosquitos (6 different genera) trapped in proximity to the colony to investigate potential vector association. Antibodies were found in 28.9% (5.4% by PRNT) of 107 swine sera but not in similarly large collections of sheep, goat, or cattle sera. The antibody detection rate in human subjects with occupational exposure to the bat colony was 11% (5/45 persons), which was significantly higher than in unexposed adults (0.8% [1/118]; chi square, P < 0.001). KRV is a novel bat-associated rhabdovirus potentially transmitted to humans and swine. Disease associations should be investigated. IMPORTANCE: Bats are thought to carry a huge number of as-yet-undiscovered viruses that may pose epidemic threats to humans and livestock. Here we describe a novel dimarhabdovirus which we isolated from a large colony of the straw-colored fruit bat Eidolon helvum in Ghana. As these animals are exposed to humans and several livestock species, we looked for antibodies indicating infection in humans, cattle, swine, sheep, and goats. Signs of infection were found in swine and humans, with increased antibody findings in humans who are occupationally exposed to the bat colony. Our data suggest that it is worthwhile to look for diseases caused by the novel virus in humans and livestock.

Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.

Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes.

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Evidence for novel hepaciviruses in rodents.

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.