Broad Species Tropism Research Articles

Nipah Virus Efficiently Replicates in Human Smooth Muscle Cells without Cytopathic Effect.

Nipah virus (NiV) is a highly pathogenic zoonotic virus with a broad species tropism, originating in pteropid bats. Human outbreaks of NiV disease occur almost annually, often with high case-fatality rates. The specific events that lead to pathogenesis are not well defined, but the disease has both respiratory and encephalitic components, with relapsing encephalitis occurring in some cases more than a year after initial infection. Several cell types are targets of NiV, dictated by the expression of the ephrin-B2/3 ligand on the cell’s outer membrane, which interact with the NiV surface proteins. Vascular endothelial cells (ECs) are major targets of infection. Cytopathic effects (CPE), characterized by syncytia formation and cell death, and an ensuing vasculitis, are a major feature of the disease. Smooth muscle cells (SMCs) of the tunica media that line small blood vessels are infected in humans and animal models of NiV disease, although pathology or histologic changes associated with antigen-positive SMCs have not been reported. To gain an understanding of the possible contributions that SMCs might have in the development of NiV disease, we investigated the susceptibility and potential cytopathogenic changes of human SMCs to NiV infection in vitro. SMCs were permissive for NiV infection and resulted in high titers and prolonged NiV production, despite a lack of cytopathogenicity, and in the absence of detectable ephrin-B2/3. These results indicate that SMC might be important contributors to disease by producing progeny NiV during an infection, without suffering cytopathogenic consequences.

A Review on Deadly Nipah Virus - Prevalence and its Management

NiV is an emerging infectious disease caused by infected bats from the family of Paramyxoviridae. From its secretions the disease can be spread to humans or through close contact with infected humans. NiV was detected for the first time in 1998 in Malaysia. NiV have broad species tropism and potential that may evolve life threatening respiratory and/ or neurologic disease in humans and as well as in animals which make them important trans-boundary biological threats. The disease presented mainly as acute encephalitis with a short incubation period of less than two weeks (4 to 18 days), with the main symptoms of fever, headache and giddiness followed by coma. The major involvement of the lung and brain in NiV infection often manifested as an acute severe respiratory syndrome, encephalitis etc. In case of Henipavirus the diagnosis of infection is mainly based on the details of contact with diseased animals, evidence of encephalitis and or pneumonia, with serologic evidence of infection using Enzyme Linked Immunosorbant (EIA) assay testing or polymerase chain reaction. The anti-viral drug Ribavirin is a well-known first line treatment strategy for suspected viral infections of unknown etiology. Based on a study conducted to discover whether combining monotherapeutic treatments with Ribavirin and Chloroquine would result in any protection indicative of favourable drug-drug interactions when treatment were initiated with lethal inoculums of NiV. The reason for multiple outbreaks may be due to low healthcare system capacity and robust surveillance strategy contributes to it. Multidisciplinary and multiple facet approach is vital in preventing the emergence of NiV. It is crucial to undertake rigorous research for developing vaccines and medicines to prevent and treat NiV.

Broad Cross-Species Infection of Cultured Cells by Bat HKU2-Related Swine Acute Diarrhea Syndrome Coronavirus and Identification of Its Replication in Murine Dendritic Cells In Vivo Highlight Its Potential for Diverse Interspecies Transmission.

Outbreaks of severe diarrhea in neonatal piglets in Guangdong, China, in 2017 resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (SeACoV) derived from the species Rhinolophus bat coronavirus HKU2 (Y. Pan, X. Tian, P. Qin, B. Wang, et al., Vet Microbiol 211:15-21, 2017). SeACoV was later referred to as swine acute diarrhea syndrome CoV (SADS-CoV) by another group (P. Zhou, H. Fan, T. Lan, X.-L. Yang, et al., Nature 556:255-258, 2018). The present study was set up to investigate the potential species barriers of SADS-CoV in vitro and in vivo We first demonstrated that SADS-CoV possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. Trypsin contributes to but is not essential for SADS-CoV propagation in vitro Furthermore, C57BL/6J mice were inoculated with the virus via oral or intraperitoneal routes. Although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. SADS-CoV nonstructural proteins and double-stranded RNA were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of SADS-CoV in the mouse model. We identified that splenic dendritic cells (DCs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. Finally, we demonstrated that SADS-CoV does not utilize known CoV receptors for cellular entry. The ability of SADS-CoV to replicate in various cells lines from a broad range of species and the unexpected tropism for murine DCs provide important insights into the biology of this bat-origin CoV, highlighting its possible ability to cross interspecies barriers.IMPORTANCE Infections with bat-origin coronaviruses (CoVs) (severe acute respiratory syndrome CoV [SARS-CoV] and Middle East respiratory syndrome CoV [MERS-CoV]) have caused severe illness in humans after "host jump" events. Recently, a novel bat-HKU2-like CoV named swine acute diarrhea syndrome CoV (SADS-CoV) has emerged in southern China, causing lethal diarrhea in newborn piglets. It is important to assess the species barriers of SADS-CoV infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. An in vitro susceptibility study revealed a broad species tropism of SADS-CoV, including various rodent and human cell lines. We established a mouse model of SADS-CoV infection, identifying its active replication in splenic dendritic cells, which suggests that SADS-CoV has the potential to infect rodents. These findings highlight the potential cross-species transmissibility of SADS-CoV, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-HKU2-origin CoV.

1790. Single-Center Experience and Lessons Learnt from Management of Nipah Virus Outbreak in India

BackgroundNipah virus (NiV) is re-emerging zoonotic RNA virus belonging to Paramyxoviridae family. Suspecting Nipah virus in a NiV naive tropical area is a challenge. NiV management is further confounded by acute presentation, high mortality, broad species tropism, multiple modes of transmission, difficulty to diagnose and lack of definitive treatment.MethodsRecent NiV outbreak that lasted for approximately 1 month (2–29 May 2018) and resulted in 23 cases with a case-fatality rate of 91%. We present clinical summary and management of five cases managed at Baby Memorial Hospital, Kozhikode, India from May 17, 2018 to May 30, 2018 and were epidemiologically linked to the index case. All patients presented with initial nonspecific prodromal symptoms of fever, muscle pain, watery diarrhea. Median age was 53 years, four were males, median hospital stay was 3 days, median incubation period of was days. Further complications, included encephalitis with viral bronchopneumonia/acute respiratory distress syndrome (ARDS) in 100 %, patients, encephalitis with viral bronchopneumonia/ARDS with myocarditis in 60 %patients, despite attempted therapy with ribavirin all patients developed cardiorespiratory arrest and succumbed to the illness. ResultsHematological Investigations showed normal TLC with a mean of 7,920 cells/ mm3, mild thrombocytopenia (mean 1,57,800) high Hb 16.12(SD1.10), ESR 19 mm/hr, DLC-N 82% high relative neutrophilic cytosis. Normal liver and renal function, Na+ 133 meq/L. CSF analysis showed high opening pressure, 100% lymphocytic pleocytosis, mean CSF sugar 118 mg/100mL, CSF protein 73.6. CT chest -bilateral airspace opacities and ground glassing. Brain FLAIR sequence showed nonspecific hyperintensities in white matter and brainstem correlating with vasculitic changes. Laboratory diagnosis of NiV was made by Real-Time RT–PCR on throat swab, blood, urine and cerebrospinal fluid by Manipal virus research center and National Institute of Virology. Pathological autopsy was done in 2 cases and found noncontributory.ConclusionWe report clinical and public health management experience from one of the three hospitals managing the patients affected with NiV. Managing outbreaks of high infectivity requires persistent organized and committed healthcare interventions Disclosures All authors: No reported disclosures.

Henipavirus Infection: Natural History and the Virus-Host Interplay

The review summarizes the history of the emergence of the Henipviruses, provides a general introduction of their molecular biology and the efforts to develop antiviral treatment strategies, including the successful development of a veterinary vaccine. Paramyxoviridae, genus Henipavirus comprises emerging pathogens detected in Southeast Asia, Australia and Africa. Henipaviruses are enveloped with non-segmented, negative-sense, single-stranded RNA molecules and are distinguished from other Paramyxoviruses by a substantially larger RNA genome. Hendra virus (HeV) and Nipah virus (NiV) are the causative agents of zoonotic disease outbreaks in Australia and Asia, respectively, and are indigenous to fruit bats. HeV and NiV can cause severe diseases with neurological and respiratory manifestations in humans and intermediate hosts but do not cause a disease in the natural bat host. The primary spill-over reservoir for humans to come into contact with HeV in Australia and NiV in Malaysia are horses and pigs, respectively. Direct bat-to-human transmissions are reported from NiV infections in Bangladesh. The life cycle of the Henipaviruses includes replication of the RNA genome in the cytoplasm, a nuclear phase of the viral matrix protein, and assembly and budding of infectious virions at the cell membrane. To prevent the establishment of an antiviral cellular state, Henipaviruses encode structural and non-structural viral proteins able to antagonize the interferon type I response. For the development of antiviral treatments, strategies are under investigation to interfere with viral replication and entry, and finally to generate antibodies suitable for passive immunotherapy. A veterinary vaccine against HeV has been in use since 2012 to protect horses, and hence interrupts the transmission pathway to humans. The successful development of a veterinary vaccine based on a soluble HeV envelope protein may set the path for the development of an optimal vaccine against the medically relevant NiV. Henipaviruses are widely distributed, predominantly using fruit bats as their natural hosts. Their broad species tropism, the capacity to exploit different transmission pathways, and the ability to cause severe diseases in humans and intermediate hosts highlight the need for potent antiviral strategies or vaccines. The characterization of the viral gene products, interacting host factors and immune targets is essential for the development of antiviral strategies to be able to overcome the threat of these emerging pathogens.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 Is an Important Surface Attachment Factor That Facilitates Entry of Middle East Respiratory Syndrome Coronavirus.

The spike proteins of coronaviruses are capable of binding to a wide range of cellular targets, which contributes to the broad species tropism of coronaviruses. Previous reports have demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) predominantly utilizes dipeptidyl peptidase 4 (DPP4) for cell entry. However, additional cellular binding targets of the MERS-CoV spike protein that may augment MERS-CoV infection have not been further explored. In the current study, using the virus overlay protein binding assay (VOPBA), we identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV. CEACAM5 coimmunoprecipitated with the spike protein of MERS-CoV in both overexpressed and endogenous settings. Disrupting the interaction between CEACAM5 and MERS-CoV spike with anti-CEACAM5 antibody, recombinant CEACAM5 protein, or small interfering RNA (siRNA) knockdown of CEACAM5 significantly inhibited the entry of MERS-CoV. Recombinant expression of CEACAM5 did not render nonpermissive baby hamster kidney (BHK21) cells susceptible to MERS-CoV infection. Instead, CEACAM5 overexpression significantly enhanced the attachment of MERS-CoV to the BHK21 cells. More importantly, the entry of MERS-CoV was increased when CEACAM5 was overexpressed in permissive cells, which suggested that CEACAM5 could facilitate MERS-CoV entry in conjunction with DPP4 despite not being able to support MERS-CoV entry independently. Taken together, the results of our study identified CEACAM5 as a novel cell surface binding target of MERS-CoV that facilitates MERS-CoV infection by augmenting the attachment of the virus to the host cell surface. Infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with the highest mortality rate among all known human-pathogenic coronaviruses. Currently, there are no approved vaccines or therapeutics against MERS-CoV infection. The identification of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV advanced our knowledge on the cell binding biology of MERS-CoV. Importantly, CEACAM5 could potentiate the entry of MERS-CoV by functioning as an attachment factor. In this regard, CEACAM5 could serve as a novel target, in addition to dipeptidyl peptidase-4 (DPP4), in the development of antiviral strategies for MERS-CoV.

Ebola virus entry into host cells: identifying therapeutic strategies.

Filoviruses cause severe hemorrhagic fever in humans. The archetypal virus of this group, Ebola virus, is responsible for the current filovirus epidemic in West Africa. Filoviruses infect most mammalian cells, resulting in broad species tropism and likely contributing to rapid spread of virus throughout the body. A thorough understanding of filovirus entry events will facilitate the development of therapeutics against these critical steps in the viral life cycle. This review summarizes the current understanding of filovirus entry and discusses some of the recent advancements in therapeutic strategies that target entry.

SARS-CoV ancestor found in Chinese bats

A new study reports the isolation of a novel coronavirus (CoV) from Chinese horseshoe bats, which is more closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) than any previously identified bat CoV, providing strong evidence that SARS-CoV might have originated in bats. Whole-genome sequencing of the novel CoV revealed that it shares 95% sequence identity with human SARS-CoV, with the receptor-binding domain (RBD) in the spike protein from both viruses showing an identical amino acid sequence. The RBD of human SARS-CoV uses the cellular receptor angiotensin-converting enzyme 2 (ACE2) for cell entry, and infectivity assays confirmed that the novel CoV also uses human ACE2 for cell entry, suggesting that it might be capable of directly infecting humans. Furthermore, preliminary in vitro data suggest that the new virus has broad species tropism.

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect. ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP(2,9). Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect. ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9. Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Production of the matrix protein of Nipah virus in Escherichia coli: Virus-like particles and possible application for diagnosis

The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass ( M r) of about 43 kDa was detected by anti-NiV serum and anti- myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 °C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.

Potent Neutralization of Hendra and Nipah Viruses by Human Monoclonal Antibodies

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 microg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 microg/ml, and 98% neutralization required only 1.6 microg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.