Cedar virus: a novel Henipavirus isolated from Australian bats

Abstract

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin-B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.

Figure 1. Comparison of genome size and organization of CedPV to those of the prototype viruses of the five existing genera in the subfamily Paramyxovirinae.

Each of the coding and non-coding regions is drawn to scale. The six major genes present in all paramyxovirus genomes are indicated as follows: light shaded = RNA polymerase and nucleocapsid genes (N, P and L); slanted = envelope membrane protein genes (F and attachment protein); dotted = matrix protein (M). The dark shaded box represents the gene (SH) not commonly shared among members of the subfamily.

Figure 2. Phylogenetic tree based on the N protein sequences of selected paramyxoviruses.

Virus name (abbreviation) and GenBank accession numbers are as follows: Avian paramyxovirus 6 (APMV6) AY029299; Atlantic salmon paramyxovirus (AsaPV) EU156171; Beilong virus (BeiPV) DQ100461; Bovine parainfluenza virus 3 (bPIV3) AF178654; Canine distemper virus (CDV) AF014953; Cedar virus (CedPV) JQ001776; Fer-de-lance virus (FdlPV) AY141760; Hendra virus (HeV) AF017149; Human parainfluenza virus 2 (hPIV2) AF533010; Human parainfluenza virus 3 (hPIV3) Z11575; Human parainfluenza virus 4a (hPIV4a) AB543336; Human parainfluenza virus 4b (hPIV4b) EU627591; J virus (JPV) AY900001; Menangle virus (MenPV) AF326114; Measles virus (MeV) AB016162; Mossman virus (MosPV) AY286409; Mapeura virus (MprPV) EF095490; Mumps virus (MuV) AB000388; Newcastle disease virus (NDV) AF077761; Nipah virus, Bangladesh strain (NiV-B) AY988601; Nipah virus, Malaysian strain (NiV-M) AJ627196; Parainfluenza virus 5 (PIV5) AF052755; Peste-des-petits-ruminants (PPRV) X74443; Porcine rubulavirus (PorPV) BK005918; Rinderpest virus (RPV) Z30697; Salem virus (SalPV) AF237881; Sendai virus (SeV) M19661; Simian virus 41 (SV41) X64275; Tioman virus (TioPV) AF298895; Tupaia paramyxovirus (TupPV) AF079780.

Figure 3. Antigenic cross reactivity between CedPV and HeV.

Vero cells infected with CedPV and HeV, respectively, were stained with rabbit sera raised against recombinant N proteins of each virus.

Figure 4. Functional testing of ephrin-B2 and -B3 as an entry receptor for CedPV.

Infection of HeLa-USU cells by CedPV in the presence and absence of ephrin gene products. The susceptibility of infection, as an indirect measurement of receptor function, is demonstrated by the formation of syncytial CPE.

Figure 5. Immunohistochemical analysis of bronchial lymph node of CedPV infected ferrets.

Bronchial lymph node of ferret #2, euthanized on day 6 pi, was stained with rabbit antiserum against recombinant N protein of CedPV (B) and NiV (D), respectively. Bronchial lymph node of an unrelated ferret (infected with influenza H5N1 from another experiment) was used as negative control and stained with the same anti-CedPV (A) and anti-NiV (C) antisera under identical conditions.

Figure 6. Induction of IFN responses upon henipavirus infection.

HeLa cells were infected at an MOI 0.5 for 24 hours. Total RNA was isolated, and quantitative real-time PCR for IFN-α and IFN-β was performed. n = 2, with error bars indicating SEM.