Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real-time monitoring of viral spread in small animal models of henipavirus infection.
Nipah virus (NiV) and Hendra virus (HeV) are closely related henipaviruses of the Paramyxovirinae. Spillover from their fruit bat reservoirs can cause severe disease in humans and livestock. Despite their high sequence similarity, NiV and HeV exhibit apparent differences in receptor and tissue tropism, envelope-mediated fusogenicity, replicative fitness, and other pathophysiologic manifestations. To investigate the molecular basis for these differences, we first established a highly efficient reverse genetics system that increased rescue titers by ≥3 log units, which offset the difficulty of generating multiple recombinants under constraining biosafety level 4 (BSL-4) conditions. We then replaced, singly and in combination, the matrix (M), fusion (F), and attachment glycoprotein (G) genes in mCherry-expressing recombinant NiV (rNiV) with their HeV counterparts. These chimeric but isogenic rNiVs replicated well in primary human endothelial and neuronal cells, indicating efficient heterotypic complementation. The determinants of budding efficiency, fusogenicity, and replicative fitness were dissociable: HeV-M budded more efficiently than NiV-M, accounting for the higher replicative titers of HeV-M-bearing chimeras at early times, while the enhanced fusogenicity of NiV-G-bearing chimeras did not correlate with increased replicative fitness. Furthermore, to facilitate spatiotemporal studies on henipavirus pathogenesis, we generated a firefly luciferase-expressing NiV and monitored virus replication and spread in infected interferon alpha/beta receptor knockout mice via bioluminescence imaging. While intraperitoneal inoculation resulted in neuroinvasion following systemic spread and replication in the respiratory tract, intranasal inoculation resulted in confined spread to regions corresponding to olfactory bulbs and salivary glands before subsequent neuroinvasion. This optimized henipavirus reverse genetics system will facilitate future investigations into the growing numbers of novel henipavirus-like viruses.


OBJECTIVE
Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions.
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, USA.
Tatyana
T
Yun
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, California, USA Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Arnold
A
Park
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, USA.
Terence E
TE
Hill
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, California, USA.
Olivier
O
Pernet
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, California, USA Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Shannon M
SM
Beaty
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, USA.
Terry L
TL
Juelich
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, USA.
Jennifer K
JK
Smith
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, USA.
Lihong
L
Zhang
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, California, USA.
Yao E
YE
Wang
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, California, USA Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Frederic
F
Vigant
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, USA.
Junling
J
Gao
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, USA.
Ping
P
Wu
Department of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, Los Angeles, California, USA Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York, USA Benhur.Lee@mssm.edu anfreibe@utmb.edu.
Benhur
B
Lee
Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA Galveston National Laboratory, University of Texas Medical Branch, Galveston, Texas, USA Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas, USA Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, USA Benhur.Lee@mssm.edu anfreibe@utmb.edu.
Alexander N
AN
Freiberg
D000818:Animals; D004195:Disease Models, Animal; D005816:Genetic Complementation Test; D045404:Hendra Virus / Q000502:physiology*; D045464:Henipavirus Infections / Q000821:virology*; D006801:Humans; D018345:Mice, Knockout; D045405:Nipah Virus / Q000502:physiology*; D011995:Recombination, Genetic; D059386:Reverse Genetics; D056189:Viral Tropism; D053586:Virus Internalization; D057074:Virus Release