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Tibroviruses are novel rhabdoviruses detected in humans, cattle, and arthropods. Four tibroviruses are known to infect humans: Bas-Congo virus (BASV), Ekpoma virus 1 (EKV-1), Ekpoma virus 2, and Mundri virus. However, since none of them has been isolated, their biological properties are largely unknown. We aimed to characterize the human tibrovirus glycoprotein (G), which likely plays a pivotal role in viral tropism and pathogenicity. Human tibrovirus Gs were found to share some primary structures and display 14 conserved cysteine residues, although their overall amino acid homology was low (29%-48%). Multiple potential glycosylation sites were found on the G molecules, and endoglycosidase H- and peptide-N-glycosidase F-sensitive glycosylation was confirmed. AlphaFold-predicted three-dimensional (3D) structures of human tibrovirus Gs were overall similar. Membrane fusion mediated by these tibrovirus Gs was induced by acidic pH. The low pH-induced conformational change that triggers fusion was reversible. Virus-like particles (VLPs) were produced by transient expression of Gs in cultured cells and used to produce mouse antisera. Using vesicular stomatitis Indiana virus pseudotyped with Gs, we found that the antisera to the respective tibrovirus VLPs showed limited cross-neutralizing activity. It was also found that human C-type lectins and T-cell immunoglobulin mucin 1 acted as attachment factors for G-mediated entry into cells. Interestingly, BASV-G showed the highest ability to utilize these molecules. The viruses infected a wide range of cell lines with preferential tropism for human-derived cells whereas the preference of EKV-1 was unique compared with the other human tibroviruses. These findings provide fundamental information to understand the biological properties of the human tibroviruses. IMPORTANCE: Human tibroviruses are poorly characterized emerging rhabdoviruses associated with either asymptomatic infection or severe disease with a case fatality rate as high as 60% in humans. However, the extent and burden of human infection as well as factors behind differences in infection outcomes are largely unknown. In this study, we characterized human tibrovirus glycoproteins, which play a key role in virus-host interactions, mainly focusing on their structural and antigenic differences and cellular tropism. Our results provide critical information for understanding the biological properties of these novel viruses and for developing appropriate preparedness interventions such as diagnostic tools, vaccines, and effective therapies.
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Proteínas do Envelope Viral , Humanos , Animais , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Camundongos , Glicosilação , Internalização do Vírus , Tropismo Viral , Linhagem Celular , Mucina-1/metabolismo , Células HEK293 , Anticorpos Antivirais/imunologia , Sequência de AminoácidosRESUMO
BACKGROUND: Bas-Congo virus (BASV), an emerging tibrovirus, was associated with an outbreak of acute haemorrhagic fever in Mangala, Democratic Republic of the Congo, in 2009. In 2012, neutralising antibodies to BASV were detected in the lone survivor and one of his close contacts. However, subsequent serological and molecular surveys were unsuccessful as neither BASV antibodies nor its RNA were detected. In this study, we determined the seroprevalence of BASV infection in Mangala 13 years after the initial outbreak. METHODS: We conducted a population-based serological survey from Jan 17 to Jan 23, 2022. Consenting individuals at least 5 years of age, living in Mangala for at least 4 weeks, and who had no contraindication to venepuncture were enrolled. Participants were interviewed using a pre-tested questionnaire for sociodemographic and clinical characteristics. We supplemented the collected serum samples with 284 archived samples from Matadi and Kinshasa. All samples were tested for antibodies to BASV and other tibroviruses using a pseudovirus-based neutralisation test. FINDINGS: Among the 267 individuals from Mangala, the prevalence of BASV antibodies was 55% (95% CI 49-61; n=147). BASV seropositivity odds significantly increased with age (5·2 [95% CI 2·1-12·9] to 83·9 [20·8-337·7] times higher in participants aged 20 years or older than participants aged 5-19 years). Some occupational categories (eg, farmer or public servant) were associated with seropositivity. Only nine (6%) of 160 samples from Matadi and one (<1%) of 124 samples from Kinshasa had neutralising antibodies to BASV. Moreover, we also detected neutralising antibodies to other tibroviruses-Ekpoma virus 1, Ekpoma virus 2, and Mundri virus-in 84 (31%), 251 (94%), and 219 (82%) of 267 Mangala samples; 14 (9%), 62 (39%), and 120 (75%) of 160 Matadi samples; and six (5%), five (4%), and 33 (27%) of 124 Kinshasa samples, respectively. INTERPRETATION: Human infection with BASV and other tibroviruses seems common in Mangala, although no deadly outbreak has been reported since 2009. Exposure to BASV might be highly restricted to Mangala and the increasing prevalence of neutralising antibodies with age suggests regular contact with the virus in this city. Altogether, our findings suggest that human infection with tibroviruses could be common in the study areas and not associated with deadly haemorrhagic or debilitating syndromes. FUNDING: Japan Agency for Medical Research and Development (AMED) and Japan International Cooperation Agency (JICA) under the Science and Technology Research Partnership for Sustainable Development (SATREPS) and Japan Program for Infectious Diseases Research and Infrastructure from AMED.
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Anticorpos Antivirais , Humanos , República Democrática do Congo/epidemiologia , Masculino , Estudos Soroepidemiológicos , Adulto , Estudos Transversais , Adolescente , Feminino , Pessoa de Meia-Idade , Criança , Adulto Jovem , Anticorpos Antivirais/sangue , Pré-Escolar , Anticorpos Neutralizantes/sangue , Idoso , Surtos de DoençasRESUMO
Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.
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The recent outbreaks of Marburg virus disease (MVD) in Guinea, Ghana, Equatorial Guinea, and Tanzania, none of which had reported previous outbreaks, imply increasing risks of spillover of the causative viruses, Marburg virus (MARV) and Ravn virus (RAVV), from their natural host animals. These outbreaks have emphasized the need for the development of rapid diagnostic tests for this disease. Using monoclonal antibodies specific to the viral nucleoprotein, we developed an immunochromatography (IC) assay for the rapid diagnosis of MVD. The IC assay was found to be capable of detecting approximately 102-4 50% tissue culture infectious dose (TCID50)/test of MARV and RAVV in the infected culture supernatants. We further confirmed that the IC assay could detect the MARV and RAVV antigens in the serum samples from experimentally infected nonhuman primates. These results indicate that the IC assay to detect MARV can be a useful tool for the rapid point-of-care diagnosis of MVD.
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Doença do Vírus de Marburg , Marburgvirus , Animais , Anticorpos Monoclonais , Nucleoproteínas , Cromatografia de AfinidadeRESUMO
Mosquitoes interact with various organisms in the environment, and female mosquitoes in particular serve as vectors that directly transmit a number of microorganisms to humans and animals by blood-sucking. Comprehensive analysis of mosquito-borne viruses has led to the understanding of the existence of diverse viral species and to the identification of zoonotic arboviruses responsible for significant outbreaks and epidemics. In the present study on mosquito-borne bunyaviruses we employed a broad-spectrum RT-PCR approach and identified eighteen different additional species in the Phenuiviridae family and also a number of related but unclassified bunyaviruses in mosquitoes collected in Zambia. The entire RNA genome segments of the newly identified viruses were further analyzed by RNA sequencing with a ribonuclease R (RNase R) treatment to reduce host-derived RNAs and enrich viral RNAs, taking advantage of the dsRNA panhandle structure of the bunyavirus genome. All three or four genome segments were identified in eight bunyavirus species. Furthermore, L segments of three different novel viruses related to the Leishbunyaviridae were found in mosquitoes together with genes from the suspected host, the Crithidia parasite. In summary, our virus detection approach using a combination of broad-spectrum RT-PCR and RNA sequencing analysis with a simple virus enrichment method allowed the discovery of novel bunyaviruses. The diversity of bunyaviruses is still expanding and studies on this will allow a better understanding of the ecology of hematophagous mosquitoes.
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Arbovírus , Culicidae , Orthobunyavirus , Vírus de RNA , Animais , Humanos , Feminino , Mosquitos Vetores , Orthobunyavirus/genética , Vírus de RNA/genética , Arbovírus/genéticaRESUMO
Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission.
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Quirópteros , Murinae , Infecções por Rotavirus , Rotavirus , Animais , Quirópteros/virologia , Diarreia/veterinária , Diarreia/virologia , Genoma Viral , Genótipo , Quênia , Filogenia , Rotavirus/genética , Rotavirus/isolamento & purificação , Infecções por Rotavirus/veterinária , Murinae/virologiaRESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV), a nairovirus, is a tick-borne zoonotic virus that causes hemorrhagic fever in humans. The CCHFV nucleoprotein (NP) is the antigen most used for serological screening of CCHFV infection in animals and humans. To gain insights into antibody epitopes on the NP molecule, we produced recombinant chimeric NPs between CCHFV and Nairobi sheep disease virus (NSDV), which is another nairovirus, and tested rabbit and mouse antisera/immune ascites, anti-NP monoclonal antibodies, and CCHFV-infected animal/human sera for their reactivities to the NP antigens. We found that the amino acids at positions 161-320 might include dominant epitopes recognized by anti-CCHFV IgG antibodies, whereas cross-reactivity between anti-CCHFV and anti-NSDV antibodies was limited. Their binding capacities were further tested using a series of synthetic peptides whose sequences were derived from CCHFV NP. IgG antibodies in CCHFV-infected monkeys and patients were reactive to some of the synthetic peptide antigens (e.g., amino acid residues at positions 131-150 and 211-230). Only a few peptides were recognized by IgG antibodies in the anti-NSDV serum. These results provide useful information to improve NP-based antibody detection assays as well as antigen detection tests relying on anti-NP monoclonal antibodies.
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Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Epitopos , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/diagnóstico , Humanos , Imunoglobulina G , Camundongos , Nucleoproteínas , Coelhos , OvinosRESUMO
Influenza A viruses (IAVs) cause highly contagious respiratory diseases in humans and animals. In 2009, a swine-origin pandemic H1N1 IAV, designated A(H1N1)pdm09 virus, spread worldwide, and has since frequently been introduced into pig populations. Since novel reassortant IAVs with pandemic potential may emerge in pigs, surveillance for IAV in pigs is therefore necessary not only for the pig industry but also for public health. However, epidemiological information on IAV infection of pigs in Africa remains sparse. In this study, we collected 246 serum and 605 nasal swab samples from pigs in Zambia during the years 2011-2018. Serological analyses revealed that 49% and 32% of the sera collected in 2011 were positive for hemagglutination-inhibition (HI) and neutralizing antibodies against A(H1N1)pdm09 virus, respectively, whereas less than 5.3% of sera collected during the following period (2012-2018) were positive in both serological tests. The positive rate and the neutralization titres to A(H1N1)pdm09 virus were higher than those to classical swine H1N1 and H1N2 IAVs. On the other hand, the positive rate for swine H3N2 IAV was very low in the pig population in Zambia in 2011-2018 (5.3% and 0% in HI and neutralization tests, respectively). From nasal swab samples, we isolated one H3N2 and eight H1N1 IAV strains with an isolation rate of 1.5%. Phylogenetic analyses of all eight gene segments revealed that the isolated IAVs were closely related to human IAV strains belonging to A(H1N1)pdm09 and seasonal H3N2 lineages. Our findings indicate that reverse zoonotic transmission from humans to pigs occurred during the study period in Zambia and highlight the need for continued surveillance to monitor the status of IAVs circulating in swine populations in Africa.
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Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Doenças dos Suínos , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A/genética , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Filogenia , Suínos , Zâmbia/epidemiologiaRESUMO
BACKGROUND: Pteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia. METHODS: Egyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017-2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively. RESULTS: An PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient. CONCLUSIONS: A high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans.
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Quirópteros/virologia , Orthoreovirus/isolamento & purificação , Infecções por Reoviridae/veterinária , Animais , Chlorocebus aethiops , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/virologia , Células Vero , Zâmbia/epidemiologiaRESUMO
Ticks are obligate ectoparasites as they require to feed on their host blood during some or all stages of their life cycle. In addition to the pathogens that ticks harbor and transmit to vertebrate hosts, they also harbor other seemingly nonpathogenic microorganisms including nutritional mutualistic symbionts. Tick nutritional mutualistic symbionts play important roles in the physiology of the host ticks as they are involved in tick reproduction and growth through the supply of B vitamins as well as in pathogen maintenance and propagation. Coxiella-like endosymbionts (CLEs) are the most widespread endosymbionts exclusively reported in ticks. Although CLEs have been investigated in ticks in other parts of the world, there is no report of their investigation in ticks in Zambia. To investigate the occurrence of CLEs, their maintenance, and association with host ticks in Zambia, 175 ticks belonging to six genera, namely Amblyomma, Argas, Haemaphysalis, Hyalomma, Ornithodoros, and Rhipicephalus, were screened for CLEs, followed by characterization of CLEs by multi-locus sequence typing of the five Coxiella housekeeping genes (dnaK, groEL, rpoB, 16S rRNA, and 23S rRNA). The results showed that 45.7% (n = 80) were positive for CLEs. The comparison of the tick 16S rDNA phylogenetic tree with that of the CLEs concatenated sequences showed that there was a strong correlation between the topology of the trees. The results suggest that most of the CLEs have evolved within tick species, supporting the vertical transmission phenomenon. However, the negative results for CLE in some ticks warrants further investigations of other endosymbionts that the ticks in Zambia may also harbor.
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Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district's owned dog population was estimated at 29.0% (95% confidence interval: 22.4-35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.
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Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.
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Doenças dos Bovinos/parasitologia , Vírus da Febre Hemorrágica da Crimeia-Congo/isolamento & purificação , Febre Hemorrágica da Crimeia/veterinária , Carrapatos/virologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/epidemiologia , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Febre Hemorrágica da Crimeia/sangue , Febre Hemorrágica da Crimeia/epidemiologia , Febre Hemorrágica da Crimeia/virologia , Humanos , Imunoglobulina G/sangue , Filogenia , Testes Sorológicos , Zâmbia/epidemiologiaRESUMO
BACKGROUND: An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01-0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8-51.6%. This low coverage was principally attributed to the owners' lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8-76.2%. CONCLUSIONS/SIGNIFICANCE: This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.
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Doenças do Cão/prevenção & controle , Vacina Antirrábica/imunologia , Raiva/veterinária , Cobertura Vacinal/estatística & dados numéricos , Animais , Teorema de Bayes , Estudos Transversais , Cães , Feminino , Masculino , Vacinação em Massa/veterinária , Propriedade , Raiva/prevenção & controle , Vacina Antirrábica/administração & dosagem , População Rural , ZâmbiaRESUMO
Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.
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Modelos Animais de Doenças , Ebolavirus/genética , Marburgvirus/genética , Estomatite Vesicular/virologia , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Antivirais/administração & dosagem , Cricetinae , Suscetibilidade a Doenças , Avaliação Pré-Clínica de Medicamentos , Ebolavirus/imunologia , Mesocricetus , Camundongos , Ratos , Vacinas Sintéticas , Estomatite Vesicular/patologia , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/terapia , Vesiculovirus/patogenicidade , Proteínas do Envelope Viral/imunologia , Carga ViralRESUMO
The influenza A virus (IAV) matrix-2 (M2) protein is an antigenically conserved viral envelope protein that plays an important role in virus budding together with another envelope protein, hemagglutinin (HA). An M2-specific mouse monoclonal IgG antibody, rM2ss23, which binds to the ectodomain of the M2 protein, has been shown to be a non-neutralizing antibody, but inhibits plaque formation of IAV strains. In this study, we generated chimeric rM2ss23 (ch-rM2ss23) IgG and IgA antibodies with the same variable region and compared their antiviral activities. Using gel chromatography, ch-rM2ss23 IgA were divided into three antibody subsets: monomeric IgA (m-IgA), dimeric IgA (d-IgA), and trimeric and tetrameric IgA (t/q-IgA). We found that t/q-IgA had a significantly higher capacity to reduce the plaque size of IAVs than IgG and m-IgA, most likely due to the decreased number of progeny virus particles produced from infected cells. Interestingly, HA-M2 colocalization was remarkably reduced on the infected cell surface in the presence of ch-rM2ss23 antibodies. These results indicate that anti-M2 polymeric IgA restricts IAV budding more efficiently than IgG and suggest a role of anti-M2 IgA in cross-protective immunity to IAVs.
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Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Vírus da Influenza A/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Western Blotting , Reações Cruzadas/imunologia , Cães , Ensaio de Imunoadsorção Enzimática , Humanos , Vírus da Influenza A/ultraestrutura , Células Madin Darby de Rim Canino/virologia , Camundongos , Microscopia Eletrônica de Transmissão , Testes de Neutralização , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Ensaio de Placa ViralRESUMO
IgA antibodies on mucosal surfaces are known to play an important role in protection from influenza A virus (IAV) infection and are believed to be more potent than IgG for cross-protective immunity against IAVs of multiple hemagglutinin (HA) subtypes. However, in general, neutralizing antibodies specific to HA are principally HA subtype specific. Here, we focus on nonneutralizing but broadly cross-reactive HA-specific IgA antibodies. Recombinant IgG, monomeric IgA (mIgA), and polymeric secretory IgA (pSIgA) antibodies were generated based on the sequence of a mouse anti-HA monoclonal antibody (MAb) 5A5 that had no neutralizing activity but showed broad binding capacity to multiple HA subtypes. While confirming that there was no neutralizing activity of the recombinant MAbs against IAV strains A/Puerto Rico/8/1934 (H1N1), A/Adachi/2/1957 (H2N2), A/Hong Kong/483/1997 (H5N1), A/shearwater/South Australia/1/1972 (H6N5), A/duck/England/1/1956 (H11N6), and A/duck/Alberta/60/1976 (H12N5), we found that pSIgA, but not mIgA and IgG, significantly reduced budding and release of most of the viruses from infected cells. Electron microscopy demonstrated that pSIgA deposited newly produced virus particles on the surfaces of infected cells, most likely due to tethering of virus particles. Furthermore, we found that pSIgA showed significantly higher activity to reduce plaque sizes of the viruses than IgG and mIgA. These results suggest that nonneutralizing pSIgA reactive to multiple HA subtypes may play a role in intersubtype cross-protective immunity against IAVs.IMPORTANCE Mucosal immunity represented by pSIgA plays important roles in protection from IAV infection. Furthermore, IAV HA-specific pSIgA antibodies are thought to contribute to cross-protective immunity against multiple IAV subtypes. However, the mechanisms by which pSIgA exerts such versatile antiviral activity are not fully understood. In this study, we generated broadly cross-reactive recombinant IgG and pSIgA having the same antigen-recognition site and compared their antiviral activities in vitro These recombinant antibodies did not show "classical" neutralizing activity, whereas pSIgA, but not IgG, significantly inhibited the production of progeny virus particles from infected cells. Plaque formation was also significantly reduced by pSIgA, but not IgG. These effects were seen in infection with IAVs of several different HA subtypes. Based on our findings, we propose an antibody-mediated host defense mechanism by which mucosal immunity may contribute to broad cross-protection from IAVs of multiple HA subtypes, including viruses with pandemic potential.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina A/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Proteção Cruzada , Reações Cruzadas , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/classificação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunidade nas Mucosas , Imunoglobulina A/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Liberação de VírusRESUMO
Orthoreoviruses have been indentified in several mammals, however, there is no information about orthoreoviruses in shrews. In this study, we screened wild animals in Zambia, including shrews, rodents, and bats for the detection of orthoreoviruses. Two orthoreovirus RNA genomes were detected from a shrew intestinal-contents (1/24) and a bat colon (1/96) sample by reverse-transcription (RT)-PCR targeting the RNA-dependent RNA polymerase gene of orthoreoviruses. Phylogenetic analyses revealed that each of the identified orthoreoviruses formed a distinct branch among members of the Orthoreovirus genus. This is the first report that shrews are susceptible to orthoreovirus infection. Our results suggest the existence of undiscovered orthoreoviruses in shrews and provide important information about the genetic diversity of orthoreoviruses.
Assuntos
Quirópteros/virologia , Orthoreovirus/classificação , Orthoreovirus/genética , Musaranhos/virologia , Animais , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Filogenia , RNA Viral/isolamento & purificação , Infecções por Reoviridae/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zâmbia/epidemiologiaRESUMO
Rabies is endemic in Zambia and Zimbabwe. The previously investigated strains of rabies virus in central Zambia belong to the Africa 1b lineage, with similar circulating virus strains found in the various tested hosts and regions. However, prior work assessed only limited regions and host species. Thus, this study aimed to more comprehensively determine the genetic diversity of rabies virus across regions of Zambia and Zimbabwe. RNA (n = 76) was extracted from positive direct fluorescent antibody test brain tissues from dog, cow, goat, cat, pig, human, and jackal collected from Zambia and Zimbabwe. The amplicons of the nucleoprotein and glycoprotein genes were obtained from all examined samples by nested RT-PCR and subsequently sequenced. A phylogenetic analysis of the N gene confirmed that all the endemic strains of rabies virus in Zambia and Zimbabwe belong to the Africa 1b lineage. The obtained viral gene sequences were phylogenetically divided into two clusters. Cluster II comprised only Zambian strains. In contrast, cluster I comprised both Zambia and Zimbabwe strains, with strains from Zimbabwe forming a distinct lineage from Zambian strains, implying viral genetic divergence due to geographical barriers. However, no evidence of clustering based on host or region was observed, implying the circulation of similar virus strains occurs in different hosts and regions of Zambia and Zimbabwe. The clustering of rabies virus strains from jackals with those from domestic animals provides evidence of similar virus strains circulating in both wildlife and domestic animals, and that the jackal might be one of the potential reservoirs of rabies virus infection. In this study, no strains circulating in Zimbabwe were detected in Zambia.
Assuntos
Variação Genética , Filogeografia , Vírus da Raiva/classificação , Vírus da Raiva/genética , Raiva/virologia , Animais , Humanos , Reação em Cadeia da Polimerase , Raiva/veterinária , Vírus da Raiva/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Proteínas Estruturais Virais/genética , Zâmbia , ZimbábueRESUMO
Bat trypanosomes consist of more than 30 trypanosome species from over 70 species of bats. Recent studies suggest that bats play a role in disseminating trypanosomes from African continent to the terrestrial mammals both in the Afrotropic-Palearctic Ecozones and Nearctic Ecozone. However, the diversity, distribution, and evolution of bat trypanosomes are still unclear. To better understand their evolution, more genetic data of bat trypanosomes from a variety of locations are required. During a survey of Borrelia spp. of bats inhabiting a cave in Zambia, we observed flagellate parasites from 5 of 43 hemocultures. Sequence and phylogenetic analyses of the glycosomal glyceraldehyde 3-phosphate dehydrogenase gene (gGAPDH; 572 bp) and the 18S ribosomal RNA gene (18S rRNA gene; 1,079-1,091 bp) revealed that all were Trypanosoma spp. belonged to the Trypanosoma cruzi clade. Three and two of them exhibited the similarity with T. conorhini and T. dionisii, respectively. The present study provides the first genetic data on Trypanosoma spp. of bats inhabiting Zambia.