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Received 5 Jan 2015 | Accepted 13 May 2015 | Published 24 Jun 2015

Benjamin A. Sattereld1,2, Robert W. Cross1,2, Karla A. Fenton1,2, Krystle N. Agans1,2, Christopher F. Basler3, Thomas W. Geisbert1,2 & Chad E. Mire1,2

The viral determinants that contribute to Nipah virus (NiV)-mediated disease are poorly understood compared with other paramyxoviruses. Here we use recombinant NiVs (rNiVs) to examine the contributions of the NiV V and W proteins to NiV pathogenesis in a ferret model. We show that a V-decient rNiV is susceptible to the innate immune response in vitro and behaves as a replicating non-lethal virus in vivo. Remarkably, rNiV lacking W expression results in a delayed and altered disease course with decreased respiratory disease and increased terminal neurological disease associated with altered in vitro inammatory cytokine production. This study conrms the V protein as the major determinant of pathogenesis, also being the rst in vivo study to show that the W protein modulates the inammatory host immune response in a manner that determines the disease course.

DOI: 10.1038/ncomms8483 OPEN

The immunomodulating V and W proteins of Nipah virus determine disease course

1 Galveston National Laboratory, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555, USA. 2 Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555, USA. 3 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA. Correspondence and requests for materials should be addressed to T.G. (email: mailto:[email protected]

Web End [email protected] ).

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Nipah virus (NiV) belongs to the family Paramyxoviridae and it emerged recently as a human pathogen causing acute respiratory distress and severe encephalitis with

systemic vasculitis1,2. Pulmonary or encephalitic NiV-mediated disease can be lethal with case fatality rates ranging from 38 to 92% (refs 3,4); many of the survivors experience long-term neurological sequelae5. Since the initial outbreak of NiV in 19981999 in Malaysia and Singapore6, outbreaks have occurred almost every year in Bangladesh and northeastern India712 and with a recent outbreak occurring in the Philippines13. Research with infectious NiV is restricted to Biosafety Level 4 (BSL-4) laboratories; therefore, much is still unknown about NiV pathogenesis14,15 including what viral determinates contribute to the pulmonary and encephalitic components of NiV disease.

The NiV P gene encodes not only the P protein but three additional proteins, namely C, W and V16. The C protein utilizes an alternative start codon and shares no homology with P16, while the V and W proteins are produced through mRNA editing17,18. This results in P, W and V proteins that share a common N-terminal domain (upstream of the editing site) with each having a unique C-terminal domain (downstream of the editing site).

NiV can naturally infect bats, humans, pigs, dogs, cats and other species1921. Current experimental animal models for NiV infection include hamsters22, cats23,24, pigs24,25, ferrets26, squirrel monkeys27 and African green monkeys28. Recently, interferon (IFN) receptor knockout (ko) mouse29 and human lung xenograph mouse30 models have been established, although non-modied mice only develop a mild, self-limited pulmonary infection31. Each model has certain advantages and disadvantages; among the small and medium-sized mammals, hamsters can develop meningoencephalitis (low dose) or respiratory disease (high dose)32. Acute respiratory disease occurs in the cat model23; however, the recently developed ferret model of NiV infection demonstrates both respiratory and neurological disease as well as systemic vasculitis26,33, thus accurately modelling all major aspects of NiV-mediated disease in humans2.

The IFN response through IFN-a and -b plays a critical role in controlling viral infections by signalling both infected and non-infected cells to enter into an antiviral state. This signalling occurs through the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) pathway. Many viruses have evolved means of inhibiting IFN signalling34. Paramyxoviruses have evolved mechanisms whereby the P gene products (P, V, W and C) inhibit the JAK/STAT signalling pathway35,36 through a common N-terminal domain that can bind to STAT1 and inhibit its phosphorylation37. The NiV V protein also inhibits the antiviral functions of RIG-I (ref. 38) and MDA5 (ref. 39) through phosphatase PP1 (ref. 40), and STAT2 (ref. 41) through direct sequestration. The NiV V and W proteins have also been shown to suppress expression of the IFN-b and IFN-stimulated gene 54 (ISG54) promoters in HEK 293T cells, with the W protein appearing the most potent42. The W protein potentially destabilizes IFN regulatory transcription factor 3, thereby blocking both Toll-like receptor 3 and Inhibitor of kB kinase e (IKKe) signalling pathways, whereas the V protein only blocks IKKe signalling42. Therefore, it has been suggested that both the V and W proteins play important roles in allowing NiV to evade the innate immune system and establish systemic infection with W likely playing a more prominent role43. However, it is unclear whether the W protein is able to function similarly in all cell types since evidence suggests that it is primarily found in the nucleus in some cell types42,44 but primarily in the cytoplasm of endothelial cells44. Much of the data are a result of overexpression studies and do not always align with infection-based studies45.

To investigate the role of the V and W proteins in NiV infection, recombinant NiV (rNiV) strains that lack the ability to express each of these proteins have been examined in vitro46 and in the hamster model47. However, the backbone used in the creation of the various rNiV-Malaysia (rNiVM) constructs to create the recombinant wild-type virus appeared attenuated in vivo when compared with the parental virus47,48, possibly because of additional restriction sites introduced for cloning. This attenuation makes the in vivo data difcult to interpret.

Here we utilize reverse genetics to produce rNiVM strains with no additional restriction sites in either a wild-type (rNiVM-wt) genotype or a knocked out genotype for the W protein (rNiVM-Wko) or V protein (rNiVM-Vko). These rNiVs are characterized in primary cells in vitro followed by studies in the ferret model in order to determine what role the V and W proteins play in the pathogenesis of NiV-mediated disease. Our results suggest that the V protein is the major determinate of pathogenesis and lethality, while the W protein contributes to the control of the inammatory response, with rNiVM-Wko altering the disease course from the pulmonary tissues to the neural tissues without altering disease lethality. To our knowledge, this is the rst report to show that ko of a paramyxovirus innate immune-modulatory protein, the NiV W protein, can alter disease course but not alter virulence in vivo.

ResultsrNiVM recovery and in vitro characterization. Through reverse genetics, rNiVM-wt, rNiVM-Wko and rNiVM-Vko strains were successfully recovered using previous methods43 (Fig. 1a,b). Western blot analysis of infected Vero cell lysates demonstrated that the W and V proteins were not being expressed by rNiVM-Wko and rNiVM-Vko, respectively (Fig. 1c). In addition, we noted increased production of C protein after removing W or V protein expression as has been noted elsewhere46.

Multicycle growth kinetics were determined for each rNiVM strain in Vero (Fig. 2a) and HEK 293T cells (Fig. 2b). In both cell lines the growth curves and peak titres were almost identical for the rNiVM-wt and rNiVM-Wko strains, while the rNiVM-Vko strain grew to moderately lower titres (0.250.5 logs) beginning at 36 h post infection (p.i.). The Vero cell data observed here appear to be closer to the rNiVM-wt than previously reported46,47. To examine whether the W and V proteins were important in the context of virus infection for overcoming the innate immune response, multicycle growth kinetics were assessed in Vero and HEK 293T cells pre-stimulated with 1,000 U ml 1 of Universal

IFN-a for 12 h before infection. The rNiVM-Wko strain again grew to similar titres as the rNiVM-wt strain, although both strains grew to moderately lower titres (0.250.5 logs) than in cells not pre-treated with IFN-a. The rNiVM-Vko strain, however, grew to markedly lower titres (1.01.75 logs) as compared with cells not pre-treated with IFN-a.

rNiVM-infected primary human endothelial cells. Endothelial cells in the brain and lungs are common targets for NiV infection2; therefore, primary human brain and lung microvascular endothelial cells were infected with the rNiVM strains to determine the multicycle growth kinetics. In human brain cerebrum microvascular endothelial cells (HBCMEC) rNiVM

Wko had similar kinetics to rNiVM-wt (Fig. 2c), but grew to moderately lower titres (0.5 logs) in human pulmonary microvascular endothelial cells (Fig. 2d).

To assess the effect of the innate immune response on multicycle growth kinetics in primary endothelial cells, HBCMECs and HPMECs were pre-treated with 1,000 U ml 1 of Universal IFN-a for 12 h before infection. IFN-a was very

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Common N-terminal

domain of P, V and W

407 a.a.

Unique C-terminal

Common N-terminal domain of P, V and W

domain of P, V and W

Unique C-terminal

domain of P, V and W

mRNA-editing site

202 a.a.

+1 ORF

+2 ORF

49 a.a.

43 a.a.

Mock

rNiV M-wt

rNiV M-V

rNiV M-W

+0 Gs

+2 Gs

P/V/W

IIe IIe

Pro Lys Lys Lys

Gly

AUUCCCAUU

AAAAAGGGC

AAAAAGGGGC

AAAAAGGGGGC

ACAGACGCGAAUA

3,610

mRNA-editing site

3,640

IIe IIe

Pro Lys Lys

Lys

Gly

+1 G

STOP

IIe IIe

Pro Lys Gly

Thr Asp Ala

His Arg Arg Glu

Ala Gln Thr Arg IIu

+2 Gs

Figure 1 | rNiVM genome design. (a) Schematic of the rNiVM genome with the name of each gene indicated (N, P, M, F, G and L). White segments indicate open reading frames (ORFs) and grey segments represent noncoding regions of the genome. Insert magnies the P gene, indicates the mRNA-editing site that allows for the production of the W and V proteins as well as the P protein from the P gene, shows the lengths of the common N-terminal domain and unique C-terminal domains and indicates the relative position of the ORFs of V and W after the mRNA-editing site. (b) The nucleotide and amino-acid sequences of the P, V and W ORFs around the mRNA-editing site are shown. Nucleotide changes, indicated by green and red arrows and bold letters, were incorporated into the P gene to introduce early stop codons in the V and W ORFs shortly after the mRNA-editing site (blue oval) to generate the rNiVM-Vko and rNiVM-Wko mutants, respectively. Numbers anking the nucleotide sequences indicate positive-sense antigenomic position. (c) Western blot analysis of Vero cell lysates either mock-infected, or infected with rNiVM-wt, rNiVM-Vko or rNiVM-Wko. NiV P-, V-, W- and C-specic polyclonal antibodies were used to detect the presence or absence of the respective proteins. * nonspecic binding of the V polyclonal antibody. Full blots can be seen in Supplementary Fig. 1.

potent at blocking rNiVM production in both HPMECs and HBCMECs; compared with untreated cells, cells pre-treated with IFN-a produced low levels of rNiVM-wt (2 logs lower) at 48 and 72 h p.i. in HPMECs, while no rNiVM-wt production was detected in HBCMECs pre-treated with IFN-a at any time points. No virus was detectable in either IFN-a pre-treated HBCMECs or HPMECs infected with either rNiVM-Wko or rNiVM-Vko.

To examine the chemokine/cytokine response after initial target cell infection, supernatants from the infected HPMECs were used to quantify the levels of 42 chemokines/cytokines using Bio-Plex or ELISA (Supplementary Table 1). Interestingly, many of the pro-inammatory and leukocyte-attracting chemokines including ENA-78, Eotaxin, Fractalkine, Gro-a, Gro-b, IL-8,

IP-10, I-TAC, MCP-1, MCP-4 and MIP-1d (Fig. 3a), and innate immune cytokines including TNF-a, IL-6 and IL-10 (Fig. 3b) were observed at higher levels in supernatants from HPMECs infected with rNiVM-Wko than in HPMECs infected with either rNiVM-wt or rNiVM-Vko.

Clinical disease in ferrets. Ferrets are considered an excellent model for studying NiV-mediated disease as they demonstrate respiratory, neurological, vascular and systemic disease similar to that seen in humans infected with NiV26,49,50. Three cohorts of ve ferrets each were challenged intranasally (i.n.) with B5,000 p.f.u. of rNiVM-wt, rNiVM-Wko or rNiVM-Vko. Post challenge, animal weight, temperature and clinical score were assessed daily, with blood drawn on days 3, 6, 10, 15 and 35, or on the day an animal succumbed to disease (Fig. 4a).

All animals in the rNiVM-wt cohort succumbed to rNiVM-mediated disease on days 78 p.i. (Fig. 4b, blue) with fever, severe respiratory disease and moderate neurological signs (Table 1). This is the same time to death as historical control ferrets infected with B5,000 p.f.u. of natural, non-recombinant

NiVM (Fig. 4b, light blue dotted line). All animals in the rNiVM-Wko cohort succumbed to disease on days 811 p.i. (Fig. 4b, red) with fever, moderate respiratory disease and severe neurological signs (Table 1). All animals in the rNiVM-Vko cohort developed fever but no respiratory signs, and all animals survived the challenge (Fig. 4b, dark green; Table 1). One of the animals, rNiVM-Vko-4, experienced some weight loss and developed mild neurological signs beginning on day 12 and resolving by day 23 p.i. (Fig. 4b, light green; Table 1). The other animals in the rNiVM-Vko cohort did not show any neurological signs at any point in the study.

Additional clinical signs observed in animals from the rNiVM-wt and rNiVM-Wko cohorts included depression;

lethargy; ocular, nasal and oral discharge; sneezing; nasal and oral frothing, dehydration; rales; ataxia; tremors and myoclonus. Some animals in the rNiVM-Wko cohort also had periodic seizures (Table 1).

Clinical biochemistry and haematological analysis was performed on all blood samples taken. As the disease progressed, common ndings in animals from the rNiVM-wt and rNiVM-Wko cohorts included thrombocytopaenia, lymphopenia, hypoalbuminemia, 43-fold increase in blood urea nitrogen and hyperglycaemia. These were also observed in a few of the animals from the rNiVM-Vko cohort, although not as consistently and to the degree as for the other cohorts (Table 1).

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a b

Vero 293T

HBCMEC HPMEC

rNiVM-wt -IFN

rNiVM-wt +IFN rNiVM-Wko -IFN

rNiVM-Vko +IFN

rNiVM-Vko -IFN

rNiVM-Wko +IFN

**** ***

*** *****

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p.f.u per ml

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24 36 48 72

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24 36 48 72

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p.f.u per ml

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p.f.u per ml

LOD

*** ***

***

** **

LOD

24 48 72

Hours post infection

Figure 2 | rNiVM in vitro growth kinetics. Growth kinetics in Vero cells (a), 293Tcells (b), HBCMECs (c) and HPMECs (d) either with or without 12-h pre-treatment of IFN-a. Blue bars indicate rNiVM-wt, red bars indicate rNiVM-Wko and green bars indicate rNiVM-Vko. Dark colours indicate no pre-treatment with IFN-a and light colours indicate 12-h pretreatment with IFN-a. Error bars show s.d. LOD: limit of detection of 25 p.f.u. per ml. Analysis of variance (ANOVA) with Dunnetts multiple comparison test; N 4. *P valueo0.05; **P valueo0.01; ***P valueo0.001 compared with non-IFN-treated cells

infected with the same rNiVM strain, or rNiVM-wt either with or without IFN treatment as indicated.

Neutralizing antibody response. To assess the humoral immune response to infection with all rNiVs, serum samples were measured for the amount of neutralizing antibody using a plaque reduction neutralization assay (PRNT) and reported as the dilution at which plaques were reduced by at least 50% compared with controls (PRNT50). No signicant neutralizing antibody was detected in any animals from the rNiVM-wt and rNiVM-Wko cohorts at any time points, while signicant levels of neutralizing antibody were detected in all animals from the rNiVM-Vko cohort beginning at day 10 p.i. with levels increasing on days 15 and 35 p.i. (Fig. 4D).

Gross pathology. Necropsies were performed on each animal after they succumbed to disease or when being euthanized at the study end point. The gross pathology ndings showed a marked difference in the lung lesions between the three cohorts, with all animals (5/5) in the rNiVM-wt cohort displaying multifocal to coalescing haemorrhagic and necrotizing pneumonia (Fig. 5a), all animals (5/5) in the rNiVM-Wko cohort had multifocal pinpoint haemorrhagic and necrotizing pneumonia (Fig. 5d), although with considerably less severe gross lesions than seen in the rNiVM-wt cohort, and the rNiVM-Vko cohort that showed no signicant gross pulmonary lesions (Fig. 5g). The spleens of all animals (5/5) in the rNiVM-wt cohort and all animals (5/5) in the rNiVM-Wko cohort were enlarged and mottled with multifocal white and dark red patches indicating splenomegaly and multifocal necrosis, respectively (Fig. 5b,e). In contrast, the spleens of the animals in the rNiVM-Vko cohort were uniformly

dark with no necrosis (Fig. 5h) and with some (3/5) animals showing minimal splenomegaly. Some animals (2/5) in the rNiVM-wt cohort had mucosal haemorrhagic lesions in the urinary bladder (Fig. 5c), all animals (5/5) in the rNiVM-Wko cohort had mucosal haemorrhagic lesions in the urinary bladder (Fig. 5f) and only a single animal (1/5) in the rNiVM-Vko cohort had multiple pinpoint mucosal reddening of the urinary bladder (Fig. 5i). Notably, this animal was rNiVM-Vko-4, which was the only animal in this cohort to demonstrate any clinical signs of disease other than fever.

The only obvious gross lesion in the brain of any animal in the rNiVM-wt cohort was the presence of a blood clot under the meninges and overlying the frontal lobe of one animal that was found dead. In contrast, all animals (5/5) in the rNiVM-Wko cohort had congestion of the meningeal blood vessels with a single animal having a focal pooling of blood under the meninges overlying the right lateral hemisphere. Poorly dened sulci and gyri of the brain, interpreted as oedema was the only gross brain lesion seen in any animal from the rNiVM-Vko cohort. Once again, this animal was rNiVM-Vko-4, which was the only animal in this cohort to demonstrate any clinical neurological signs of disease and have them resolved over the course of the experiment.

Histopathology and immunohistochemistry. Tissues were examined with haematoxylin and eosin (H&E) staining and with immunohistochemistry (IHC) using antibodies specic to the NiV N protein. Hepatic histopathologic lesions for all animals in the rNiVM-wt (Fig. 6a) and rNiVM-Wko (Fig. 6e) cohorts

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ENA-78

Eotaxin Fractalkine Gro-

I-TAC

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pg I1

2,000

1,500

1,000

500

0 1 24 48 72

Hours post infection

pg I1

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pg I1

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Gro-

IL-8

IP-10

300 200 100

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pg I1

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MCP-1 MCP-4 MIP-1

IL-10

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IL-6

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0 1 24 48 72

Hours post infection

Figure 3 | Selected chemokine/cytokine levels in supernatants of infected HPMECs. Inammatory chemokine levels (a) and innate immune cytokine levels (b) of HPMECs infected with rNiVM-wt (blue), rNiVM-Wko (red) or rNiVM-Vko (green). Error bars show s.d. ANOVA with Dunnetts multiple comparison test; N 4. *P valueo0.05; **P valueo0.01; ***P valueo0.001 compared with rNiVM-wt, FI, uorescence intensity.

included minimal to moderate hepatocellular degeneration/ necrosis with minimal to mild vacuolar change, moderate congestion with occasional sinusoidal leukocytosis (neutrophilia) and moderate to severe periportal lymphoplasmacytic inltrates. Strong immunolabelling for NiV antigen was present in all animals in the rNiVM-wt (Fig. 6b) and rNiVM-Wko (Fig. 6f) cohorts.

Immunolabelling for NiV was present in multifocal regions of the sinusoidal lining cells, few scattered mononuclear cells within the sinusoids (Kupffer cells), the endothelium of medium to large caliber vessels and scattered mononuclear inammatory cells, that were largely centred around areas of necrosis. In animals from the rNiVM-Vko (Fig. 6i) cohort, there was no hepatocellular degeneration/necrosis noted, and no immunolabelling for NiV antigen (Fig. 6j). Splenic histopathologic lesions for all animals in the rNiVM-wt (Fig. 6c) and rNiVM-Wko (Fig. 6g) cohorts included moderate to marked (rNiVM-wt) or mild to moderate (rNiVM-Wko) diffuse lymphoid depletion of lymphoid follicles, syncytial cell formation, haemorrhage and brin deposition of the white pulp with accumulation of viable neutrophils, degenerative neutrophils and cellular debris. Strong immunolabelling for NiV antigen was present in all animals in the rNiVM-wt (Fig. 6d) and rNiVM-Wko (Fig. 6h) cohorts. Immunolabelling for NiV was present in scattered mononuclear cells (largely centred on lymphoid follicles/germinal centre remnants), syncytial cells and

scattered endothelium. No splenic histopathologic lesions were noted in animals in the rNiVM-Vko cohort (Fig. 6k) and no immunolabelling for NiV antigen was noted (Fig. 6l).

Pulmonary histopathologic lesions in all lung lobes of animals in the rNiVM-wt (Fig. 7a) cohort and to a lesser extent all animals in the rNiVM-Wko (Fig. 7e) cohort included interstitial pneumonia with nodular inammation and necrosis of the alveolar septae near the terminal bronchioles and occasional, small syncytial cell formation of endothelium and/or respiratory epithelial cells. Strong immunolabelling for NiV antigen in the rNiVM-wt (Fig. 7b) and rNiVM-Wko (Fig. 7f) cohorts was present in scattered mononuclear, endothelial and respiratory epithelium. Overall, the nodular inammation and necrosis of the terminal bronchioles appeared more discrete, larger and more haemorrhagic in sections from the rNiVM-Wko cohort compared with rNiVM-wt; however, there was more diffuse oedema and haemorrhage noted throughout the lung lobes from the rNiVM-wt cohort. The rNiVM-Vko (Fig. 7i) cohort had rare areas of minimal interstitial pneumonia or oedema; however, no other signicant histopathologic lesions were noted and no immunolabelling for NiV antigen was identied (Fig. 7j).

No histopathologic lesions were noted on routine H&E staining of the brain for all animals in the rNiVM-wt (Fig. 7c)

cohort. However, strong immunolabelling for NiV antigen was

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a b

5,000 PFU i.n.

Day 0 3 6 10 15 35

P P P

N = 5 rNiVM-wt

N = 5 rNiVM-Wko N = 5 rNiVM-Vko

Percent survival

100

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rNiVM-Vko

Days post challenge

c rNiVM-wt rNiVM-Wko

rNiVM-Vko (1 2 3 5)

rNiVM-Vko 4

140

120

100

60 0 5 10 15 20 25 30 35

Percent weight change

Days post infection

20,480

10,240

5,120

2,560

1,280

640

320

160

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rNiVM-Wko

rNiVM-Vko (1 2 3 5)

rNiVM-Vko 4

Days post infection

PRNT 50

0 3 6 7 8 91011 15 35

Figure 4 | Clinical disease in ferrets after experimental infection with rNiVM. (a) Flow chart showing the day of infection (triangles) and days of sample collection (arrows). P, Pilot animals (numbers -1 and -2 from each cohort) were not sampled on these days. (b) KaplanMeier survival curve for ferrets infected with rNiVM-wt (blue circles), rNiVM-Wko (red triangles) and rNiVM-Vko (green squares); historical natural, non-recombinant NiVM controls have been included (light blue dotted line, open circles; N 10). Log-rank (MantelCox) test; N 5 for all rNiVM ferret cohorts. *P valueo0.05; **P valueo0.01

compared with rNiVM-wt. (c) Weight change for animals from the rNiVM-wt cohort (blue circles), rNiVM-Wko cohort (red triangles), rNiVM-Vko cohort animals 1, 2, 3 and 5 (dark green squares) and animal rNiVM-Vko-4 (light green diamonds). Ferret rNiVM-Vko-4 is separated from the rest of the

rNiVM-Vko cohort because it showed distinct clinical signs. (d) Neutralizing antibody titres for each ferret cohort. Error bars show s.d.

present in the endothelium of small caliber vessels within multiple sections of the brain (cerebrum, brainstem, choroid plexus and meninges) in all animal of the rNiVM-wt (Fig. 7d)

cohort. No histopathologic lesions were noted in the brain of animals in the NiVM-Wko (Fig. 7g) cohort; however, congestion of vessels and haemorrhage was noted expanding the meninges. Animals in the rNiVM-Wko cohort had multifocal areas with strong immunolabelling for NiV antigen within the endothelium and neurons in the cerebrum, cerebellum/brainstem and/or hippocampus (Fig. 7h). No animals in the rNiVM-Vko cohort showed any histologic lesions (Fig. 7k) or immunolabelling for NiV antigen (Fig. 7l) in the brain, including the endothelium.

Viral load. To assess the viral load in animals, virus isolation and quantitative real-time PCR (qRTPCR) were attempted with qRTPCR used to quantify the amount of viral genomes present in whole blood (Fig. 8a). All rNiVM-wt cohort animals had detectable levels of viral genome on day 6 p.i. and remained

viraemic until they succumbed to disease. Three of the animals in the rNiVM-Wko cohort had detectable levels of viral genome in their blood on day 6 p.i., and all animals became viraemic before succumbing to disease. Only two animals in the rNiVM-Vko cohort (rNiVM-Vko-2 and -5) had detectable level of viral genome in whole blood on day 6 p.i. and on day 10 p.i. for rNiVM-Vko-5.

Viral loads were also measured in 10% weight/volume tissue homogenates taken at necropsy; RNA was extracted and the presence of viral genomes was detected and quantied using qRTPCR (Fig. 8b). High levels of viral genome were detected in all tissues assayed for all animals in the rNiVM-wt and rNiVM-Wko cohorts; only three animals from the rNiVM-Vko cohort had detectable levels of viral genome in some tissues, and these were considerably lower in value (56 logs lower). Virus isolation was attempted from the liver, spleen, kidney and adrenal gland in PCR-positive samples, and virus was isolated from all PCR-positive samples from animals in the rNiVM-wt and rNiVM-Wko cohorts, with the exception of some of the liver samples in the rNiVM-Wko cohort; however, virus was not isolated from any samples of the rNiVM-Vko cohort (Fig. 8c).

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Table 1 | Clinical disease in ferrets after experimental infection with rNiVM.

Ferret no.

Clinical outcome

Respa Neurob Hemc Fever Clinical disease

rNiVM-

wt-1

F.D. d8 d47 Thrombocytopaenia (d6); hypoalbuminemia (d6); hyperglycaemia (d6); depression (d67);

lethargy (d67); inappetence (d7); ocular and nasal discharge (d7); myoclonus (d7); ataxia (d7); loss of 20% body weight

rNiVM-

wt-2

EU d8 d47 Thrombocytopaenia (d6,8); lymphopaenia (d6,8); hypoalbuminemia (d6,8); 43-fold increase in

BUN (d8); hyperglycaemia (d6); depression (d68); lethargy (d68); sneezing (d78); ocular, nasal and oral discharge (d78); severely obtunded (d8)

rNiVM-

wt-3

F.D. d8 d56 Thrombocytopaenia (d6); lymphopaenia (d6); hypoalbuminemia (d6); hyperglycaemia (d6);

depression (d57); lethargy (d57); inappetence (d67); (d7); ocular and nasal discharge (d67); ataxia (d7); hindlimb myoclonus (d7)

rNiVM-

wt-4

EU d7 d46 Thrombocytopaenia (d67); lymphopaenia (d67); hypoalbuminemia (d6,7); 43-fold increase

in BUN (d7); hyperglycaemia (d7); depression (d67); lethargy (d57); inappetence (d67); ocular and nasal discharge (d67); nasal/oral frothing (d7); periorbital/facial oedema (d7); ataxia (d67); myoclonus (d7); violent tremors (d7); hypothermia (d7)

rNiVM-

wt-5

EU d8 d47 Thrombocytopaenia (d6,8); lymphopaenia (d6); hypoalbuminemia (d6,8); 43-fold increase in

BUN (d8); depression (d57); lethargy (d57); inappetence (d68); dehydration (d8); rales (d68); ocular, nasal and oral discharge (d6); ataxia (d68); severe hypothermia (d8)

rNiVM-

Wko-1

EU d9 d78 Thrombocytopaenia (d9); lymphopaenia (d9); hypoalbuminemia (d9); 43-fold increase in BUN

(d6,9); hyperglycaemia (d9); depression (d89); lethargy (d79); dehydration (d9); sneezing (d89); rales (d9); nasal and oral frothing (d9); severe ataxia (d9); periodic seizures (d9); hypothermia (dy9)

rNiVM-

Wko-2

EU d10 d69 Thrombocytopaenia (d10); hypoalbuminemia (d10); 43-fold increase in BUN (d10); depression

(d810); lethargy (d710); inappetence (d810); hypersalivation (d10); sneezing (d8); ataxia (d810); full-body tremors (d10); periodic severe seizures with random vocalization (d10); severe hypothermia (d10)

rNiVM-

Wko-3

EU d11 d79 Thrombocytopaenia (d1011); lymphopaenia (d10); hypoalbuminemia (d10,11); 43-fold increase

in BUN (d11); hyperglycaemia (d10); depression (d811); lethargy (d711); sneezing (d810); ocular and nasal discharge (d1011); severe hypothermia (d11); obtunded (d11)

rNiVM-

Wko-4

EU d8 d68 Thrombocytopaenia (d8); lymphopaenia (d8); hypoalbuminemia (d8); depression (d78);

lethargy (d78); sneezing (d78); nasal discharge (d7); heavy nasal and oral frothing (d8); severe ataxia (d8); severely obtunded (d8)

rNiVM-

Wko-5

EU d9 d68 Thrombocytopaenia (d6, 9); lymphopaenia (d9); hypoalbuminemia (d9); 43-fold increase in

BUN (d9); hyperglycaemia (d9); depression (d89); lethargy (d79); dehydration (d9); sneezing (d78); rales (d9); nasal discharge (d7); nasal and oral frothing (d9); severe ataxia (d9)

rNiVM-

Vko-1

s.e. d35 d69 lymphopaenia (d6)

rNiVM

Vko-2

s.e. d35 d6

Thrombocytopaenia (d6); lymphopaenia (d6); lethargy (d8, 11)

rNiVM

Vko-3

s.e. d35 d67 Thrombocytopaenia (d6); lymphopaenia (d6)

rNiVM

Vko-4

s.e. d35 d711 lymphopaenia (d6,10,15); hypoalbuminemia (d6,10,15); hyperglycaemia (d15); depression (d12

16); lethargy (d1216); nasal discharge (d12); tremors in head and neck with occasional facial and ear twitching (d1223); mild ataxia (d1923)

rNiVM-

Vko-5

s.e. d35 d6

Thrombocytopaenia (d6,10); lymphopaenia (d10); hypoalbuminemia (d10)

BUN, blood urea nitrogen; EU, euthanized due to rNiV -mediated disease; d, day p.i.; F.D., found dead; hem, hemorrhage; neuro, neurologic involvement; resp, respiratory involvement; s.e., euthanized at study end point.

aThe absence ( ) or presence ( ) of increased respiratory effort and/or rate.

bThe absence ( ) or presence of minor ( ), moderate ( ) or severe ( ) neurological signs.

cExtensive perioribtal, facial and ventral neck oedema with subcutaneous haemorrhages.

DiscussionPrevious studies using in vitro expression systems of NiV W and V proteins suggested that both proteins were able to antagonize multiple aspects of IFN production and signalling, with the observation that the W protein was the more potent antagonist37,38,4143. However, one study examined this in vivo47 and showed that a Vko strain of rNiVM, called rNiV(V-) was completely attenuated in hamsters with no hamsters succumbing to disease, while hamsters infected with the Wko strain, termed rNiV(W-), succumbed to disease in a time frame indistinguishable from their rNiVM wild-type strain. In that study, a Cko strain was also completely attenuated with no hamsters showing signs of disease. However, the rNiVM strains examined contained, by design, restriction sites in all intergenic regions, resulting in a rNiVM wild type that was slightly

attenuated and not uniformly lethal. This attenuation was critically noted when another group produced wild-type and Cko strains of rNiVM without designed intergenic restriction sites and subsequently infected hamsters51. This wild-type rNiVM was uniformly lethal while the Cko strain was partially attenuated with some hamsters surviving. It is likely that the attenuation observed in the rst groups rNiV wild type, and therefore effecting the ko strains, was due to the inserted restriction sites, therefore, partially confounding the hamster in vivo data.

While not intending to compare backbones, we were concerned about this potential confounding attenuation; therefore, we produced rNiVM-wt, rNiVM-Wko and rNiVM-Vko strains that do not contain designed restriction sites in the intergenic regions. The multicycle virus growth kinetics in both Vero and 293T (Fig. 2a,b) cell lines were more similar when

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a b c

d e f

g h i

Figure 5 | Gross pathology of lungs, spleens and urinary bladders from rNiVM-infected ferrets. Representative gross pathology of lungs (a,d,g), spleen (b,e,h) and urinary bladders (c,f,i) taken from ferrets infected with rNiVM-wt (ac), rNiVM-Wko (df) and rNiVM-Vko (gi). Inserts show magnied regions of each specimen. Multifocal to coalescing haemorrhage and necrosis of all lung lobes is seen in rNiVM-wt-infected ferrets (a), while much fewer and smaller numbers of haemorrhagic and necrotic foci are seen in rNiVM-Wko-infected ferrets (d). Splenomegaly and multifocal necrosis are seen in spleens from rNiVM-wt (b)- and rNiVM-Wko (e)-infected ferrets. Kidneys from ferrets infected with rNiVM-Wko showed multifocal haemorrhage and necrosis (e, black arrow) and livers were diffusely pale and reticulated (e, white arrow). Similar lesions were seen in rNiVM-wt-infected ferrets (not shown). Very minimal to no pathology was observed in lungs (e) and spleens (f) from rNiVM-Vko-infected ferrets. Urinary bladders showed large haemorrhagic lesions in ferrets infected with rNiVM-wt (c) and rNiVM-Wko (f); very small, multifocal haemorrhagic lesions seen in the urinary bladder of ferret rNiVM-Vko-4 (i); no lesions were observed in the urinary bladders of the other ferrets from the rNiVM-Vko cohort.

compared with previous reports46,47. The rNiVM-Wko strain grew to comparable titres as rNiVM-wt, with rNiVM-Vko growing to slightly lower titres. Pre-treatment of cells with universal IFN-a revealed a marked difference in titre from rNiVM-Vko-infected cells but no difference between rNiVM-wt or rNiVM-Wko. To the best of our knowledge, these data are the rst ever to suggest that the V protein could play a more prominent role than the W protein in regard to the ability of NiV to antagonize the innate immune response in these established cell lines. In addition, these data suggest that the severe attenuation observed in the ferrets challenged with rNiVM-Vko was a result of a more robust innate immune response, thereby allowing an effective adaptive immune response to clear the infection when compared with the rNiVM-

wt and rNiVM-Wko cohorts (Fig. 4d).

To further characterize the sensitivity of the recombinants to the innate immune response beyond established cell lines, in vivo target cells, primary human endothelial cells of brain or pulmonary origin, were used for multicycle virus growth kinetics (Fig. 2c,d). Interestingly, although rNiVM-Wko had similar growth kinetics to rNiVM-wt in the brain HBCMECs, it grew to lower titres in pulmonary HPMECs. These in vitro data correlate with the in vivo data, where we observed severe neurological signs in ferrets infected with rNiVM-Wko along with reduced respiratory signs and pathology in the lungs when compared with rNiVM-wt. The minimal detectable virus produced from the

mutlicycle growth kinetics study in rNiVM-Vko-infected HBCMECs and HPMECs with intact innate immune responses support the suggestion from the observation made in IFN pre-treated cell lines. In addition, IFN pre-treatment led to stronger inhibition of rNiVM-wt replication in HBCMECs and

HPMECs when compared with the cell lines examined. This sensitivity to IFN in primary cells, and the fact that NiV W and V proteins have been shown to antagonize both IFN production and IFN-induced signalling, suggests that a reduced inhibition of the innate immune response leads to the altered pathogenesis observed in ferrets infected with rNiVM-Wko and rNiVM-Vko.

Each of the rNiVM strains grew to considerably lower titres in HPMECs and HBCMECs than in the established Vero and 293T cell lines. This is likely because of the fact that established cell lines often have deciencies in innate immune responses, whereas the primary cells have intact immune responses. By infecting these cells with a low multiplicity of infection (MOI) of 0.01 and observing multicycle growth kinetics, the effects of the innate immune signalling responses can be observed as opposed to cells infected with a high MOI such as observed elsewhere46.

All ferrets in the rNiVM-wt cohort succumbed within the same time frame observed in previous studies for non-recombinant NiVM (7-8 days p.i.)33, demonstrating that the rNiVM-wt used in this study is not attenuated compared with non-recombinant NiVM. However, ferrets infected with the rNiVM-Wko and

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a b c d

e f g h

Figure 6 | H&E and immunohistochemistry of the ferret liver and spleen. Representative H&E (a,c,e,g,i,k) and immunohistochemistry labelled with a NiV N protein-specic polyclonal rabbit antibody (b,d,f,h,j,l). The liver (a,b,e,f,i,j) and spleen (c,d,g,h,k,l) from representative ferrets infected with rNiVM-wt (ad), rNiVM-Wko (eh) and rNiVM-Vko (il). Images taken, liver 20, spleen 20; scale bar, 100 mm.

a b c d

e f g h

Figure 7 | H&E and immunohistochemistry of the ferret lung and brain. Representative H&E (a,c,e,g,i,k) and immunohistochemistry labelled with a NiV N protein-specic polyclonal rabbit antibody (b,d,f,h,j,l). The lung (a,b,e,f,i,j) and brain (c,d,g,h,k,l) from representative ferrets infected with rNiVM-wt (ad), rNiVM-Wko (eh) and rNiVM-Vko (il). White arrows and magnied inserts indicate syncytial cells (a,b,h) and black arrow indicates neuronwith immunolabelling (h). Images taken: lung, 20, brain, 40; scale bar, 100 mm for the lung and 50 mm for the brain.

rNiVM-Vko strains exhibited altered disease courses. When compared with the rNiVM-wt cohort (Table 1), the ferrets in the rNiVM-Wko cohort developed onset of all clinical signs at later time points (B24 days later). Although the rNiVM-Wko cohort succumbed to disease, it was at later time points (B13 days). Remarkably, we observed that knocking out the innate immunomodulatory W protein, despite having no effect on disease outcome, dramatically altered the disease course. In the

rNiVM-Wko cohort respiratory signs and histopathology were less severe, while neurological signs (including seizures) and histopathology were more severe when compared with cohort rNiVM-wt. This aligned with the striking observation of NiV antigen detected in neurons (Fig. 7h, black arrow) in the rNiVM-Wko cohort. These observations from the rNiVM-Wko cohort demonstrate that the W protein does contribute to NiV pathogenesis in ferrets, compared with the study in hamsters47.

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1012

1011

1010

109

108

107

106

105

104

103

102

101

100

1012

1011

1010

109

108

107

106

105

104

103

102

101

100

107

106

105

104

103

102

101

100

Total genome equivalents per ml Total genome equivalents per g

1014

1013

* *

* * *

rNiVM-wt-1

rNiVM-wt

rNiVM-wt-2 rNiVM-wt-3 rNiVM-wt-4 rNiVM-wt-5

rNiVM-Wko-1

rNiVM-Wko

rNiVM-Wko-2 rNiVM-Wko-3 rNiVM-Wko-4 rNiVM-Wko-5

0 3 6 Terminal 79 Terminal 11

Days post infection

rNiVM-Vko-1 rNiVM-Vko-2 rNiVM-Vko-3 rNiVM-Vko-4 rNiVM-Vko-5

Adrenal Gland

Lung RU

Liver

Spleen

Kidney

Lung RL

Lung RM

Lung LU

Lung LM

Lung LL

Brain frontal

Pancreas

p.f.u. per g

Liver

Spleen

Kidney

Adrenal gland

Figure 8 | Viral load in rNiVM-infected ferrets. Viral load in ferrets as detected by GEq using qRTPCR from (a) whole blood as GEq per ml and from (b) tissues as GEq per g (c) p.f.u. per g of rNiVM isolated from tissues of ferrets after necropsy. Hashed blue and red lines represent the average of all animals in the rNiVM-wt and rNiVM-Wko cohorts, respectively. Error bars show s.d. N 5 for hashed lines; N 2 for all other lines. Right upper (R.U.),

right middle (R.M.), right lower (R.L.), left upper (L.U.), left middle (L.M.), left upper (L.U.) *indicates samples in which rNiVM was successfully isolated from whole-blood samples. wAnimal was found dead, blood was examined using qRTPCR but no attempt was made to isolate virus. zAnimal was

found dead and no attempt was made to examine blood using qPCR or to isolate virus.

On the basis of our results, the W protein has a novel, yet to be appreciated effect on lung pathogenesis in ferrets, because of its role in antagonizing the inammatory immune response. While the V protein prominently affects lethal outcome, the W protein appears to contribute to lung pathology with rNiVM-Wko resulting in delayed onset of clinical signs, altered disease course and increased time to death. This delay does not appear to be because of any defect inherent in viral replication since rNiVM-Wko grows equally well as rNiVM-wt in established cell lines as shown in Fig. 2a,b as well as other work46,47. Rather, this seems to be because of its inability to fully antagonize innate immune cytokine production (as discussed below) thus allowing for decreased replication in HPMECs (Fig. 2d) and the likely inux of innate immune cells due to the cytokine production.

Unlike hamsters that develop either respiratory (high dose) or neurological (low dose) disease dependent on the challenge dose32, ferrets appear to either develop both respiratory and neurological disease or no disease at all depending on the challenge dose26. However, the numbers involved in that study were small, and we cannot rule out that the apparent control by the innate immune system to rNiVM-Wko infection allows for the disease course to be altered, similar to the low dose, neurological disease observed in hamsters. Alternatively, or in combination with this, increased cytokine levels released by rNiVM-Wko-infected endothelial or other cells in the central nervous system might lead to some of the increase in neurological signs and pathology observed in the rNiVM-Wko cohort compared with the rNiVM-wt cohort.

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The ferrets in the rNiVM-Vko cohort developed fever, with some animals also having detectable levels of NiV RNA in whole blood and tissues; however, the lack of respiratory and neurological signs in all animals, except for the transient, mild neurological signs in rNiVM-Vko-4, was remarkable. This was not due to the lack of virus replication as circulating viral RNA was detected in two of the animals (Fig. 8a), low levels of viral RNA were detected in the tissues of some animals (Fig. 8b) and transient abnormalities occurred in the clinical chemistry and haematological values of some of the animals (Table 1); however, the lack of serious disease in the animals infected with rNiVM-Vko demonstrates the prominent role of the V protein in NiV pathogenesis in ferrets. In humans, NiV can cause recrudescence in survivors months to years after the initial infection19. It is unknown whether this phenomenon can also occur in ferrets. However, the possibility of this occurring cannot be ruled out for rNiVM-Vko-infected animals.

While the rNiVs in this study had innate immunomodulatory proteins knocked out, we were interested whether the apparent lack of innate immune control for each led to an effective adaptive immune response in either case. This is of interest because monoclonal antibody therapy has proven effective at preventing NiV-mediated disease in ferrets and non-human primates26,52. As expected, the animals infected with rNiVM-wt did not develop neutralizing antibody before they succumbed (Fig. 4d). Similar results have been described in ferrets infected with NiVM (ref. 33). To date, it is unclear whether the acute time to death prevented the production of neutralizing antibody (78 days p.i.) or whether the lack of neutralizing antibodies was due to a NiV-mediated mechanism. The data from the rNiVM-Wko cohort revealed that, even with an extended time to death, neutralizing antibodies were not developed after the challenge, even in those surviving to day 10 or 11 p.i. However, all animals infected with rNiVM-Vko developed neutralizing antibodies by day 10. Therefore, the innate immunomodulatory V protein of NiV appears to play a role in preventing neutralizing antibody production rather than the short time to death resulting in a lack of neutralizing antibody. Another potential mechanism for the lack of neutralizing antibody production could be because of the stark lymphoid depletion observed in the splenic germinal centres of animals in the rNiVM-wt and rNiVM-Wko cohorts, but not the rNiVM-Vko cohort (Fig. 6c,g,k). This agrees with a similar report of canine distemper virus-infected ferrets where wild-type and Cko viruses were 100% lethal and did not develop neutralizing antibody responses, whereas a Vko virus was attenuated and led to a potent neutralizing antibody response53; however, that study did not determine any potential mechanism for this phenomenon.

While differences in circulating neutralizing antibodies did not appear to be responsible for differences between the rNiVM-wt and rNiVM-Wko cohorts, we attempted to understand why the rNiVM-Wko cohort exhibited less lung pathology than the rNiVM-wt cohort by examining the chemokine/cytokine proles of infected HPMECs, the presumed early target cells in NiV infections. It was notable that many of the pro-inammatory and leukocyte-attracting cytokines were at higher levels in supernatants from HPMECs infected with rNiVM-Wko than in

HPMECs infected with either rNiVM-wt or rNiVM-Vko (Fig. 3 and Supplementary Table 1). Because the HPMECs were infected with a low MOI of 0.01 and presumably rNiVM-Vko was unable to spread effectively to other cells (Fig. 2d, green bars), the lower cytokine levels found in many of these supernatants is likely due to only a small proportion of the cells that were being infected and stimulated to produce the cytokines limiting the peak levels. However, both rNiVM-wt and rNiVM-Wko were presumably able to infect most or all of the cells in the well (Fig. 2d, blue and red

bars, respectively) with rNiVM-Wko at slightly lower levels. Therefore, the higher levels of chemokines/cytokines produced by the rNiVM-Wko-infected HPMECs is likely due to the ability of the W protein to suppress and control chemokine/cytokine response in rNiVM-wt-infected HPMECs. These data suggest that the rNiVM-Wko-infected ferret cohort had an increased pulmonary chemokine/cytokine response, allowing for greater leukocyte recruitment to the lungs early in infection diminishing the severe insult to the lungs. In addition, this may explain the increased number and larger inammatory nodules seen on histopathological examination for the rNiVM-Wko cohort (Fig. 7e,f) compared with the rNiVM-wt cohort (Fig. 7a,b) and why viral antigen was much more localized to only the endothelium and these inammatory nodules in the rNiVM-Wko cohort. This may have some similarities to what was described with Cko rNiV-infected hamsters51, although this leads to some hamsters surviving instead of developing more severe neurological disease as was observed with our rNiVM-Wko ferret cohort. We have captured our overall interpretation of these data in Fig. 9.

While we show the V protein is the prominent determinate of pathogenesis (Fig. 9c), strikingly we also show the W protein as a minor determinate of pathogenesis for NiV-mediated disease in ferrets causing a delayed disease course with less severe respiratory signs and lung pathology, as knocking out this protein completely alters the nal disease course to the neural tissue (Fig. 9b). This is likely because rNiVM-Wko has a reduced ability to suppress the initial chemokine/cytokine response resulting in less severe insult to the lungs allowing for a prolonged disease course for the virus to cause disease within the central nervous system. This is probably the reason for the more severe neurological signs observed in these ferrets. Despite the slower disease course, lymphoid depletion within the splenic germinal centres occurred, no neutralizing antibody response was detected and all animals succumbed to NiV-mediated neurological disease.

Previously, innate immunomodulatory proteins of paramyxoviruses have been studied, including the V and C proteins of measles virus in rhesus macaques54, the STAT1-binding domain of the measles virus P and V proteins in rhesus macaques55 and the V and C proteins of canine distemper virus in ferrets53, without drastically altering the disease course and were touted as potential solutions to vaccines for immunocompromised individuals. We have reported here the rst observation of mutating a paramyxovirus immunomodulatory protein, as seen with the Wko NiV recombinant, which alters the disease course but not lethality. On the basis of our ndings, further examination of viral innate immunomodulatory proteins is warranted to observe whether this phenomenon is common; this especially should be considered for those who are considering designing vaccines on the basis of attenuating immunomodulatory proteins or therapeutics targeting such proteins found in pathogenic viruses.

Overall, this study reveals the crucial contribution of the NiV V and W proteins to the pathogenesis and disease course in the ferret model with evidence pointing to the V protein being the major determinant with its ability to antagonize the innate immune response in target endothelial cells resulting in attenuated pathogenesis in vivo. The W protein on the other hand appears to have the ability to control the chemokine/ cytokine response of target endothelial cells, which has an effect on the disease course, although not lethality. The specic mechanisms that the V and W proteins use to contribute to pathogenesis in vivo and in vitro are yet to be elucidated along with the contribution of other possible pathogenic determinates. These include the role of the C protein in pathogenesis in comparison with the data available from the hamster model, and

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Lung endothelial cells Lungs

rNiVM-wt

Spleen Brain

Germinal centre necrosis

Germinal centre intact

No neutralizing antibody response

Endothelium

Endothelium Neurons

rNiVM-Wko

rNiVM-Vko

Leukocyte recruitment

Inflammatory chemokine cloud

Innate cytokine cloud

Potent neutralizing antibody response

Figure 9 | Model of the immunomodulating roles of the NiV W and V proteins in ferret pathogenesis. rNiVM-wt (a) spreads unhindered in early target lung endothelial cells (Fig. 2d) leading to extensive lung injury (Fig. 5a) followed by spread to the spleen (Fig. 5b) causing germinal centre necrosis (Fig. 6c) leading to a lack of neutralizing antibody response (Fig. 4d); there is also spread to the endothelium of the brain (Fig. 7d) and moderate neurological signs develop before the ferret succumbs to pulmonary disease. rNiVM-Wko (b) allows lung endothelial cells to generate an inammatory chemokine cloud (Fig. 3 and Supplementary Table 1) somewhat hindering viral titres (Fig. 2d) and presumably allowing increased leukocyte recruitment to the lungs, limiting lung injury (Fig. 5d) and sequestering most viral spread to the endothelium and distinctive inammatory nodules (Fig. 7f), thus causing milder respiratory signs; rNiVM-Wko is still able to spread to the spleen (Fig. 5e) causing germinal centre necrosis (Fig. 7g) and leading to a lack of neutralizing antibody response (Fig. 4d); there is also spread to the brain endothelium and, owing to prolonged time to death, eventually neurons as well leading to severe neurological signs and succumbing to neurological disease. rNiVM-Vko (C) in unable to grow efciently in lung endothelial cells (Fig. 2d)

possibly due to the production of an innate cytokine cloud (Fig. 3 and Supplementary Table 1) and is unable to efciently spread or cause injury in the lungs (Fig. 5g), spleen (Fig. 5h) or brain (Fig. 7l); the splenic germinal centres remain intact (Fig. 6k) and a potent neutralizing antibody response develops (Fig. 4d) that leads to viral clearance and 100% ferret survival.

the contribution of the STAT1-binding domain shared by the P, V and W proteins. Studies looking deeper into these mechanisms are warranted and necessary to ultimately understand how NiV evades the host immune response contributing to pathogenesis.

Methods

Cell lines. BSR-T7/5 cells, a BHK-21 cell line stably expressing T7 RNA polymerase56, were maintained in Dulbeccos modied Eagle medium (DMEM; Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco),100 U ml 1 penicillin, 100 g ml 1 streptomycin and 0.5 mg ml 1 Geneticin (Gibco). Vero 76 cells (ATCC CRL-1587) were maintained in Eagles Minimum

Essential Medium supplemented with 10% FBS and 100 U ml 1 penicillin (Gibco), 100 g ml 1 streptomycin (Gibco). HEK 293T/17 cells (ATCC CRL-11268) were maintained in DMEM supplemented with 10% FBS, 100 U ml 1 penicillin and 100 g ml 1 streptomycin.

Primary cells. HPMECs (Lonza, Basel, Switzerland) were maintained in EBM-2MV media supplemented with 5% FBS and proprietary concentrations of human epidermal growth factor, broblast growth factor, vascular endothelial growth factor, insulin-like growth factor, hydrocortisone and gentamicin, all provided by Lonza. HBCMECs were obtained from Sciencell (Carlsbad, CA) and were maintained in EBM-2 containing 5% FBS, 1.4 mM hydrocortisone, 5 mg ml 1 ascorbic acid, 1 ng ml 1 basic broblast growth factor (Sigma, St Louis, MO), 1X chemically dened lipid concentrate, 10 mM HEPES and PenicillinStreptomycin

(Life Technologies, Carlsbad, CA). Media were changed every 23 days and cells were used up to passage 7.

Plasmid construction and generation of recombinant NiVs. The NiV genomic sequence used to construct the rNiVs in this study was UMMC1 (GenBank accession no. AY029767), an isolate cultured from the cerebrospinal uid of an encephalitic human patient in the initial outbreak in Malaysia. This NiVM genome was assembled into three segments, A (nt 16,780), B (nt 6,78010,404) and C (nt 10,40418,246), as described previously43. These fragments could then be mutated followed by assembly into full-length cDNA clones in pSL1180 cloning vectors containing T7 promoter and terminator sequences and a hepatitis delta virus ribozyme sequence. Three NiVM full-length cDNA clones (pFL-NiVM-wt, pFL-NiVM-Wko and pFL-NiVM-Vko) were constructed. Site-directed mutagenesis was performed by Mutagenex Inc (Piscataway, NJ) to introduce the Wko (c3628t) and Vko (a3629t) mutations in the A segment (Fig. 1b). These mutations introduced stop codons shortly after the editing site in the W and V open reading frames, respectively.

The helper plasmid pTM1-HA NiVM P was constructed as described previously43; the P gene was haemagglutinin (HA)-tagged at the amino terminus and subcloned into the pTM1 expression plasmid. The helper plasmids pTM1. W-NiVM N and pTM1.W-NiVM L were constructed by amplifying the sequences for the N and L genes from the A and C segments, respectively, with PCR (Supplementary Table 2) using Platinum PFX polymerase (Life Technologies) and were cloned into a pTM1.W expression vector.

BSR-T7/5 cells were seeded in six-well plates and were co-transfected with 3.5 mg of NiVM full-length cDNA clone, 0.2 mg of pTM1-HA NiVM P, 0.75 mg of pTM1.

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W-NiVM N and 0.4 mg of pTM1.W-NiVM L per well in Optimem (Gibco) with Lipofectamine 2,000 (Life Technologies) according to the manufacturers protocol. At 72 h post transfection, the medium and cells were collected and passaged on Vero cells. Cytopathic effect (CPE) was typically observed to be beginning between days 4 and 8 p.i. The medium was then collected and passed on Vero cells for plaque purication of the virus. A small quantity (P1) of the plaque-puried virus was then grown in Vero cells followed by a larger quantity (P2) in Vero cells infected with a MOI of 0.01. At 48 h p.i. the virus-containing medium was harvested, claried by low-speed centrifugation, aliquoted and stored at 80 C. The presence of

introduced mutations was conrmed by sequencing of RTPCR fragments amplied from virus RNA isolated from virus stocks with Trizol LS (Ambion, Carlsbad, CA). Virus titres were determined by standard plaque assay using 5% neutral red. All experiments using full-length clones or infectious rNiVM were performed using protocols approved by the Institutional Biosafety Committee (IBC) and National Institutes of Health recombinant advisory committee (NIH RAC) in BSL-4 containment at the Galveston National Laboratory in Galveston, TX.

Western blot analysis. Polyclonal rabbit antisera against the unique C-terminal domains of the NiV P, W and V proteins and to the NiV C protein were produced by GenScript (Piscataway, NJ). Overall, 1.2 106 Vero cells per well were seeded in

a six-well plate and infected with rNiVM-wt, rNiVM-Wko or rNiVM-Vko at an MOI of 0.01. The cells were harvested at 40 h.p.i. in 1 ml of 2 Laemmli sample buffer

(Bio-Rad, Hercules, CA) and heated to 95 C for 20 min. Samples were then run on a denaturing 412% SDSPAGE gel (Bio-Rad). Proteins were transferred from the gel on polyvinilidene uoride (PVDF) membranes and blocked in TTBS (100 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.1% Tween 20) with 5% skim milk. PVDF membranes were incubated with polyclonal rabbit antisera against P, V, W and C described above, diluted in TTBS with 5% milk (P: 1:10,000; V: 1:5,000; W: 1:5,000; C: 1:500) for 1 h at room temperature and washed three times in TTBS. The membranes were then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich; 1:20,000 dilution) for 1 h at room temperature, washed three times in TTBS, incubated with ECL reagent (Promega) for 5 min and imaged with a VersaDoc (Bio-Rad).

Virus growth kinetics. Overall, 1.2 106 cells per well of 293T or Vero cells were

seeded in six-well plates and incubated at 37 C for 12 h with either regular medium, or medium containing 1,000 U ml 1 of Universal IFN-a (PBL Assay

Science, Piscataway, NJ). The cells were then infected at an MOI of 0.01 with rNiVM-wt, rNiVM-Wko or rNiVM-Vko for 1 h followed by the removal of the inoculum, four washes with PBS and the addition of fresh medium. Supernatants were collected at 1, 6, 12, 24, 36, 48 and 72 h p.i., claried by centrifugation, aliquoted and stored at 80 C. All infections were performed in duplicate.

Samples were then titred on Vero cells using standard plaque assays. Limit of detection was 25 p.f.u. per ml.

Similarly, 2.5 105 cells per well of HPMEC or HBCMEC were seeded in

24-well plates and were allowed to polarize for 48 h at 37 C, followed by incubation at 37 C for 12 h with either regular medium, or medium containing 1,000 U ml 1 of Universal IFN-a (PBL). The cells were then infected at an MOI of 0.01 with rNiVM-wt, rNiVM-Wko or rNiVM-Vko for 1 h followed by the removal of the inoculum, four washes and the addition of fresh medium. Supernatants were collected at 1, 24, 48 and 72 h p.i., claried by centrifugation, aliquoted and stored at 80 C. Additional aliquots were stored for use in chemokine/cytokine

analysis described below. All infections were performed in duplicate. Samples were then titred on Vero cells using standard plaque assays. Limit of detection was25 p.f.u. per ml.

Chemokine/cytokine analysis. HPMECs were infected and supernatants ali-quoted and stored as described above. Levels of 6Ckine/CCL21, BCA-1/CXCL13, CTACK/CCL27, ENA-78/CXCL5, Eotaxin/CCL11, Eotaxin-2/CCL24, Eotaxin-3/ CCL26, Fractalkine/CX3CL1, GCP-2/CXCL6, GM-CSF, Gro-a/CXCL1, Gro-b/

CXCL2, I-309/CCL1, IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8/CXCL8, IL-10, IL-16, IP-10/CXCL10, I-TAC/CXCL11, MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, MDC/CCL22, MIF, MIG/CXCL9, MIP-1a/CCL3, MIP-1d/CCL15,

MIP-3a/CCL20, MIP-3b/CCL19, MPIF-1/CCL23, SCYB16/CXCL16, SDF-1a b/

CXCL12, TARC/CCL17, TECK/CCL25 and TNF-a were quantied in super-natants from infected HPMECs. Briey, gamma-irradiated supernatants were diluted to 1:4, and 50 ml of each sample was quantied using a Bio-Plex Pro Human

Chemokine Panel, 40-Plex (Bio-Rad) according to the manufacturers instructions. Infections and supernatant sample collection were performed in duplicate, and each sample was quantied in duplicate. Samples were assayed across at least a 100-bead region performed on the Bio-Plex-200 machine and analysed using the Bio-Plex Manager Software (v 6.1; Bio-Rad).

Levels of IFN-a and IFN-b were quantied using 25 ml of undiluted supernatant in either a Human IFN alpha (Multi-Subtype) ELISA kit or Human IFN beta ELISA kit, respectively (Thermo Scientic, Waltham, MA) according to the manufacturers instructions. All samples were read for dilution end points at405 nm on a Molecular Devices Emax system microplate reader (Molecular Devices, Sunnyvale, CA).

Statistics. Conducting animal studies in BSL-4 severely restricts the number of animal subjects, the volume of biological samples that can be obtained andthe ability to repeat assays independently and thus limit statistical analysis. Consequently, data are presented as the mean calculated from replicate samples, not replicate assays, and error bars represent the s.d. across replicates.

The Prism 5 software was used to calculate statistical signicance throughout this study using the following tests: log-rank (MantelCox) test for KaplanMeier survival curves; analysis of variance (ANOVA) with Dunnetts multiple comparison test for viral growth kinetics and chemokine/cytokine analysis.

Animals. Animal studies were performed in BSL-4 biocontainment at the GNL at UTMB in Galveston and were approved by the UTMB Institutional Animal Care and Use Committee (IACUC). Animal research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals, and adheres to the principles stated in the eighth edition of the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011 (ref. 57). The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Fifteen female, 12-month-old ferrets (Mustela putorius furo) weighing0.751.0 kg were housed in groups of two (pilot animals) or three animals per virus cohort. Before infection, subjects were anaesthetized by 5% isouorane and had transponder chips (BioMedic Data Systems, Seaford, DE) implanted subcutaneously for animal identication and temperature monitoring. For challenge and procedures, animals were anaesthetized with a ketamine acepromazine xylazine cocktail and inoculated i.n. with B5,000 p.f.u. of rNiVM-wt, rNiVM-Wko or rNiVM-Vko in 0.5 ml of 10% FBS Hanks Balanced Salt Solution (Gibco). Animals were anaesthetized for clinical examination, respiration quality and blood collection on days 0, 6 and 35 p.i. or terminal end point collection for pilot animals, and days 0, 3, 6, 15 and 35 p.i. or terminal end point for all other animals (Fig. 4a). After the challenge, animals were assessed daily for weight and temperature, and scored on a scale of 012 for clinical observations on the basis of coat appearance, social behaviour and provoked behaviour; animals scoring 9 or more were euthanized as per the IACUC protocol. Subjects in the rNiVM-Vko cohort were euthanized at the study end point on day 35 p.i., whereas the subjects in the rNiVM-wt and rNiVM-Wko cohorts reached euthanasia criteria and were euthanized according to approved humane end points on days 711 p.i.

rNiVM serum neutralization assays. PRNT50s were determined using a conventional serum neutralization assay. Briey, sera were serially diluted twofold and incubated with B100 p.f.u. of rNiVM-wt for 1 h at 37 C. Virus and antibodies were then added to individual wells of six-well plates of conuent Vero cell monolayers in duplicate. Plates were stained with neutral red 2 days after infection and plaques were counted 24 h after staining. The 50% neutralization titre (PRNT50) was determined as the serum dilution at which there was a 50%

reduction in plaque counts versus control wells.

Specimen collection and processing in rNiVM-infected ferrets. Blood was collected and placed in MiniCollect EDTA tubes or serum tubes (Greiner Bio One, Monroe, NC). Immediately following sampling, 100 ml of whole blood was added to 600 ml of AVL viral lysis buffer with carrier RNA (Qiagen) for RNA extraction. For tissues, B100 mg was stored in 1 ml RNAlater (Qiagen) for 96 h to stabilize RNA. RNAlater was completely removed, and tissues were homogenized in 600 ml RLT buffer (Qiagen) in a 2-ml cryovial using a tissue lyser (Qiagen) and 1.4-mm ceramic beads (Precellys, Saint-Quentin-en-Yvelines, France). The tissue samples included the right lung upper lobe, right lung middle lobe, right lung lower lobe, left lung upper lobe, left lung middle lobe, left lung lower lobe, liver, spleen, kidney, adrenal gland, pancreas and brain (frontal cortex). All whole-blood samples were inactivated in AVL viral lysis buffer with carrier RNA, and tissue samples were homogenized and inactivated in RLT buffer before removal from the BSL-4 laboratory. Subsequently, RNA was isolated from whole blood and swabs using the QIAamp viral RNA kit (Qiagen) from tissues using the RNeasy minikit (Qiagen) according to the manufacturers instructions supplied with each kit.

Haematology and serum biochemistry. Blood was collected on days 0, 6 and 35 p.i. or on terminal end point for pilot animals, and days 0, 3, 6, 15and 35 p.i. or on terminal end point for all other animals (Fig. 4a). Complete blood counts of total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, haematocrit values, total haemoglobin concentrations, mean cell volumes, mean corpuscular volumes and mean corpuscular haemoglobin concentrations were analysed from blood collected in MiniCollect EDTA tubes (Greiner Bio One) using a Hemavet HV950FS instrument as per the manufacturers instructions (Drew Scientic, Oxford, CT). Serum was centrifuged at 400g (rcf) for 10 min, and analysis of blood chemistries was performed using a VetScan classic analyser and comprehensive diagnostic prole rotors measuring of albumin, amylase, alanine aminotransferase, alkaline phosphatase, calcium, glucose, total protein, total bilirubin, blood urea nitrogen, creatinine, phosphorus, sodium and total protein (Abaxis, Union City, CA). All blood and serum samples were processed and analysed immediately after collection.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8483

Histopathology and IHC. Necropsy was performed on all subjects. Tissue samples of all major organs were collected for histopathologic and immunohistochemical examination and were immersion-xed in 10% neutral buffered formalin for at least 21 days in BSL-4. Subsequently, formalin was changed; specimens were removed from BSL-4, processed in BSL-2 by conventional methods and embedded in parafn and sectioned at 5-mm thickness as previously described33. Briey,for IHC, specic anti-NiV immunoreactivity was detected using an anti-NiV N protein rabbit polyclonal primary antibody28 (kindly provided by Dr Christopher Broder, Uniformed Services University of the Health Sciences, Bethesda, MD) at a 1:5,000 dilution for 30 min. The tissue sections were processed for IHC using the Dako Autostainer (Dako, Carpinteria, CA). Secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:200 for 30 min followed by Dako LSAB2 streptavidin-HRP (Dako) for 15 min. Slides were developed with Dako DAB chromagen (Dako) for 5 min and counterstained with haematoxylin for 1 min. Non-immune rabbit IgG was used as a negative staining control.

Detection of rNiVM load. RNA was isolated from whole blood or tissues and analysed using primers/probe targeting the N gene and intergenic region between N and P of NiV for qRTPCR with the probe used here being 6-carboxyuorescein-50-CGTCACACATCAGCTCTGACGA-30-6 carboxytetramethylrhodamine (Life Technologies). rNiV RNA was detected using the CFX96 detection system (Bio-Rad) in One-step probe qRT-PCR kits (Qiagen) with the following cycle conditions: 50 C for 10 min, 95 C for 10 s and 40 cycles of 95 C for 10 s and 59 C for 30 s. Threshold cycle (Ct) values representing rNiV genomes were analysed with the CFX Manager Software, and data are shown as GEq. To create the GEq standard, RNA from NiV challenge stocks was extracted and the number of NiV genomes was calculated using Avogadros number and the molecular weight of the NiV genome. Virus titration was performed by plaque assay with Vero cells from all whole blood and tissue samples. Briey, increasing 10-fold dilutions of the samples were adsorbed to Vero cell monolayers in duplicate wells (200 ml); the limit of detection was 25 p.f.u. per ml.

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Acknowledgements

We thank Daniel Deer, Joan Geisbert and Courtney Williams for study support, Jessica

Graber for expert technical assistance with the ABSL-4 sample collection during the

infection studies, the staff of the UTMB Animal Resources Center for animal care and

Kenneth Escobar, Kerry Graves and Natalie Dobias for their technical expertise in

histology. This study was supported by the Department of Health and Human Services,

National Institutes of Health grant number AI082121 to T.W.G., AI109945-01 to C.F.B.

and UC7 AI094660 for BSL-4 operations support of the Galveston National Laboratory.

Author contributions

B.A.S., C.F.B., T.W.G. and C.E.M. conceived and designed the in vitro work. B.A.S.,

T.W.G. and C.E.M. conceived and designed the ferret study. B.A.S. designed, cloned,

recovered, characterized and propagated the rNiVs used in this study. R.W.C. and B.A.S.

contributed to the primary human endothelial cells work. B.A.S., R.W.C., K.A.F. and

C.E.M. performed ferret infection studies, carried out clinical observations and processed

animal tissues and blood. B.A.S. performed virus isolation, neutralizing antibody titres

and clinical pathology assays. K.A.F. provided veterinary pathology expertise and

performed histologic and immunohistochemical analysis. K.N.A. performed qRTPCR

and BIO-PLEX assays. B.A.S., R.W.C., K.A.F., K.N.A., C.F.B., T.W.G. and C.E.M.

analysed the data. B.A.S., C.F.B., T.W.G. and C.E.M. wrote the paper. All authors had

access to all of the data and approved the nal version of the manuscript. Opinions,

interpretations, conclusions and recommendations are those of the authors and are not

necessarily endorsed by UTMB.

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Abstract

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The viral determinants that contribute to Nipah virus (NiV)-mediated disease are poorly understood compared with other paramyxoviruses. Here we use recombinant NiVs (rNiVs) to examine the contributions of the NiV V and W proteins to NiV pathogenesis in a ferret model. We show that a V-deficient rNiV is susceptible to the innate immune response in vitro and behaves as a replicating non-lethal virus in vivo. Remarkably, rNiV lacking W expression results in a delayed and altered disease course with decreased respiratory disease and increased terminal neurological disease associated with altered in vitro inflammatory cytokine production. This study confirms the V protein as the major determinant of pathogenesis, also being the first in vivo study to show that the W protein modulates the inflammatory host immune response in a manner that determines the disease course.

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Title

The immunomodulating V and W proteins of Nipah virus determine disease course

Author

Satterfield, Benjamin A; Cross, Robert W; Fenton, Karla A; Agans, Krystle N; Basler, Christopher F; Geisbert, Thomas W; Mire, Chad E

Pages

7483

Publication year

2015

Publication date

Jun 2015

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms8483

ProQuest document ID

1690654957

The immunomodulating V and W proteins of Nipah virus determine disease course

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