Human SLAM-adapted canine distemper virus can enter human peripheral blood mononuclear cells and replicate in mice expressing human SLAM and defective for STAT1 expression

ABSTRACT

Canine distemper virus (CDV) is a member of the genus Morbillivirus with a worldwide distribution that causes fatal diseases in canids and marine mammals. In recent years, CDV has demonstrated the remarkable ability of pathogens to cross species barriers. The natural host range of CDV has expanded from Canidae to Primates, presumably attributed to ecological shifts and the emergence of viral variants. Therefore, it is important to investigate whether CDV can infect humans by adapting to the human signalling lymphocyte activation molecule (hSLAM) receptor to cross the species barrier. Through successive passaging and plaque cloning of a CDV wild-type strain (5804PeH) in Vero cells expressing hSLAM (Vero-hSLAM), we obtained an hSLAM adaptive strain, 5804PeH-VhS. The adapted CDV strain exhibited a D540G mutation within the receptor-binding domain (RBD) of the haemagglutinin (H) protein. The H^D540G mutation has enhanced cell-cell fusion activity in Vero-hSLAM cells. This adaptation allowed the CDV strain to infect human peripheral blood mononuclear cells (PBMCs), particularly T lymphocytes and inhibited lymphocyte proliferation. Additionally, this strain could replicate in the lymphoid tissues of transgenic mice that express the hSLAM receptor, causing viraemia. However, the adapted strain did not spread to the epithelial cells or the central nervous system of the mice. While this adaptation indicates a potential risk, there is no definitive evidence that the virus can spread among humans.

Introduction

Morbilliviruses belong to the Paramyxoviridae family and include a number of highly pathogenic viruses, such as measles virus (MeV), canine distemper virus (CDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), cetacean morbillivirus (CeMV), and porpoise morbillivirus (PMV), which cause devastating diseases in humans and animals. Notably, all Morbilliviruses except CDV have a tightly restricted host range, whereas CDV have a broad host range. Over the past 20 years, CDV has emerged as a causative agent of mortality in various mammalian families, including Canidae, Felidae, Ursidae, Hyaenidae, Mustelidae, Procyonidae, and Viverridae [1–5].

Two cellular receptors have been identified for morbilliviruses: signalling lymphocyte activation molecule (SLAM or CD150) and nectin-4 [6–8]. SLAM is the key “entry receptor,” allowing morbilliviruses to invade immune cells in the upper respiratory tract initially. In the later stages of infection, nectin-4 serves as the “exit receptor,” facilitating the release of the virus. Morbilliviruses employ H proteins that recognize and interact with SLAM to enter target cells [8,9]. Mutations in the H protein affect virulence and host receptor binding ability, leading to changes in species susceptibility [10–13], resulting in the continual production of new CDV strains, making CDV outbreaks difficult to control. In addition, the CDV H protein has been identified as a major determinant of induced neutralizing antibodies [14].

Recently, CDV has been shown to cause disease and lethal outbreaks in nonhuman primates [15–18]. In 1989, the first cases of natural CDV infection in Japanese macaques (Macaca fuscata) were reported [15]. In 2006, an outbreak of canine distemper (CD) occurred among captive macaques in Guangxi Province (China), resulting in approximately 10,000 macaques contracting the disease and 4,250 deaths [16]. In 2008, monkeys (Macaca mulatta) from the Beijing Laboratory Animal Center of China experienced natural CDV infection, with a mortality rate of 60% [17]. In the same year, 46 Japanese crab-eating macaques (Macaca fascicularis) that died from severe pneumonia during the quarantine period were associated with CDV infection [18]. The expansion of host species to include primates has raised concerns regarding the potential risk of CDV infection in humans. The high identity of amino acids between Macaca mulatta SLAM (maSLAM) and hSLAM is 96.7%; therefore, CDV has the potential to overcome this restriction and spread to humans through macaques [8,19], which could be attributed to mutations in CDV H. Morbilliviruses can adapt to new hosts by mutating the H gene. The functional adaptation of CDV and PPRV to hSLAM requires only a single amino acid exchange at the RBD of the H protein (CDV-H^D540G, H^P541S; PPRV-H^R191P) [20–22].

Considering these concerns, we conducted gain-of-function (GOF) experiments via serial passaging of the CDV 5804PeH strain in vitro in cells expressing human SLAM (Vero-hSLAM). This virus strain was designated 5804PeH-VhS. We identified mutated target amino acid positions within the RBD of the H protein and assessed their ability to overcome the restriction of the human SLAM receptor.

Materials and methods

Cells and viruses

Vero cells stably expressing dog SLAM (Vero-dSLAM), human SLAM (Vero-hSLAM), human nectin-4 (Vero-hNectin-4), and dog nectin-4 (Vero-dNectin-4), used in the present study, were frozen and stored in our laboratory (Animal Molecular Pathology and Vaccine Research Laboratory of Heilongjiang Bayi Agricultural University, Department of Microbiology and Immunology Laboratory of Dalhousie University) [6,23]. All the cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (331–010-CL; Wisent) supplemented with 5% heat-inactivated foetal bovine serum (FBS) (086–150; Wisent). The recombinant 5804P wild-type CDV isolates expressing the enhanced green fluorescent protein (EGFP) reporter gene (5804PeH) was obtained from Professor Veronika von Messling, Paul-Ehrlich Institute, Germany [24].

Adaptation of CDV 5804PeH to the human slam receptor

To obtain a human SLAM-adapted CDV strain, propagation of the 5804PeH strain was performed in Vero-hSLAM cells [20]. Briefly, Vero-hSLAM cells were infected with 5804PeH at an MOI of 1. After five days, when apparent syncytia were visible, the virus was harvested (1^st passage, P1). The virus was titrated with Vero-dSLAM cells, and Vero-hSLAM cells were infected at an MOI of 0.01. When large syncytia were observed, the virus was harvested and titrated again with Vero-dSLAM cells (2^nd passage, P2). Vero-hSLAM cells were then infected with this virus at an MOI of 0.01 and harvested at three days post-infection (3^rd passage, P3). Further passages of the virus were produced accordingly until later passages when virus titres did not further increase.

Virus plaque assays were performed as previously described [20]. Briefly, 70% confluent Vero-hSLAM cell monolayers in six-well cell culture plates were infected with a tenfold dilution series of 5804PeH in DMEM containing 0% foetal calf serum (FCS). Further incubation at 37°C for 1 h was followed by overlaying the wells with 3 ml of double DMEM supplemented with 5% FCS and 2% Noble agar (A5431; Sigma‒Aldrich). Plaques were visualized after five days of incubation at 37°C by staining with 3% neutral red (553-24-2; Sigma). Virus clones were randomly selected and picked from the plaques, purified twice from Vero-hSLAM cells and named 5804PeH-VhS.

Confocal microscopy

Vero-hSLAM cells were infected with 5804PeH-VhS at an MOI of 0.01. Three days after incubation, the cells were fixed in 4% paraformaldehyde (10 min) and blocked with 5% FCS in PBS for 1 h at room temperature. Human SLAM was detected by incubating the cells with the mouse anti-human SLAM antibody IPO-3 (ab2604, Abcam) at 5 μg/ml in PBS containing 5% FCS for 45 min at room temperature. The cells were subsequently stained with an Alexa Fluor® 568-conjugated antibody (ab175473, Abcam) for 30 min at room temperature, followed by staining with 4′,6-diamidino-2-phenylindole (DAPI) in PBS containing 0.1% Triton X-100. Images were acquired via ZEN2008 imaging software on a Zeiss LSM510 upright laser scanning confocal microscope (Zeiss, Germany).

CDV RNA isolation, RT-PCR, and genome sequencing

To determine the genomic sequence alterations required for CDV adaptation, sequences were determined from the original parental virus 5804PeH and six virus clones obtained by plaque picking from the independently adapted viruses 5804PeH-VhS. The viral RNA of 5804PeH-VhS was extracted via the QIAmp Viral RNA Mini Kit (52904; Qiagen). cDNA was synthesized via random priming via the Primer Script^TM II 1st Strand cDNA Synthesis Kit (6210A; TaKaRa), followed by PCR amplification of the CDV genome via specific primers (Supplemental Table 1). The purified DNA fragments were subsequently cloned and inserted into the pGEM-T vector and sequenced via dideoxynucleotide chain termination by Shanghai Bioengineering Co., Ltd. (Shanghai, China).

Antibody block virus infection assay

To confirm that the specific adaptation of 5804PeH-VhS to the human SLAM receptor occurred, a blocking experiment was performed with anti-hSLAM antibodies. Vero-hSLAM cells and Vero-dSLAM cells as controls were incubated with the anti-hSLAM monoclonal antibody IPO-3 (ab2604; Abcam) in 0% FCS DMEM for 1 h at 37°C. Purified mouse IgG1 (clone MOPC-31C, BD Biosciences) was used as an isotype control. Antibody-treated Vero-hSLAM and Vero-dSLAM cells were then infected with 5804PeH-VhS at an MOI of 0.1 for 1 h before the medium was changed to 5% FCS-containing medium and incubated for 72 h at 37°C. Fluorescence images were captured at each time point. EGFP intensity was analysed via ImageJ software (National Institutes of Health, Bethesda, United States of America) by calculating the corrected total cellular fluorescence (CTCF). CTCF = integrated fluorescence density − (area of cells × mean background fluorescence) [25],

Determination of replication kinetics of CDV

To determine whether 5804PeH-VhS caused any alterations in growth compared with the parental 5804PeH strain, we observed the viral growth curves of 5804PeH and 5804PeH-VhS in Vero-dSLAM, Vero-hSLAM, Vero-dNectin-4, and Vero-hNectin-4 cells. These cells were infected at an MOI of 0.01, and the supernatant and cells were harvested every day and stored at − 80°C. The virus titres at the respective time points were determined in Vero-dSLAM cells by limiting dilution and expressed as 50% tissue culture infectious doses (TCID₅₀), which were calculated as described by Reed and Muench [26].

Cell-cell fusion induced by the CDV-H and -F expression plasmids

The full-length H gene of the 5804PeH and 5804PeH-VhS strains was cloned and inserted into a pCI expression vector (E1721, E1731; Promega) carrying a FLAG tag (DYKDDDDK) located at the 3’ end of the gene to generate pCI-5804PeH-Hflag and pCI-5804PeH-VhS-Hflag. For the fusion experiments, Vero-dSLAM and Vero-hSLAM cells in six-well plates were transfected with 2 µg of the plasmid pCI-5804PeH-F expressing the F protein of 5804PeH and pCI-5804PeH-Hflag or pCI-5804PeH-VhS-Hflag via Lipofectamine™ 3000 Transfection Reagent (L3000150; Thermo Fisher) according to the manufacturer’s protocol. Fusion activity was evaluated 24 hours post-transfection via a microscope (CKX53, OLYMPUS, Japan).

Cell surface protein biotinylation assay

The cell surface protein biotinylation assay was performed via an EZ-Lipofectamine™ Sulfo NHS-SS Biotinization Kit (21442; Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, 100-mm-diameter cell dishes plated with HEK293T cells were transfected with pCI-5804PeH-Hflag, pCI-5804PeH-VhS-Hflag, or pCI vector plasmids. At 36 hours post-transfection, 1/2 of the cells were used for total protein expression detection, and the rest were used for the cell-surface biotinylation assay [27,28]. The cells were incubated for 30 min at 4°C with 2 mm EZ-Link Sulfo-NHS-SS for biotinization (21442; Thermo Fisher) in PBS. A 5 min incubation of the mixture in PBS supplemented with 100 mm glycine terminated the biotinylation reaction. The cells were then lysed with 500 μL of RIPA buffer (89900; Thermo Fisher) supplemented with protease inhibitors. Biotinylated proteins were pulled down by overnight incubation of protein samples with Pierce Streptavidin Agarose Resin (20353; Thermo Fisher). The beads were washed, and the biotinylated proteins were eluted with 50 mm DTT. The eluent was then subjected to 4–12% SDS‒PAGE and western blotting with a rabbit anti-FLAG polyclonal antibody (F7425; Sigma‒Aldrich) to quantify the H proteins.

For total CDV H protein expression assays [27,28], the collected 1/2 of the cells were lysed with RIPA buffer containing a protease inhibitor. After the protein concentration was assessed via a Pierce BCA protein assay kit (23225; Thermo Fisher Scientific), the same amount of protein was allocated to each sample. Protein samples were combined with Bolt sample buffer supplemented with reducing agent (B0009; Thermo Fisher) and boiled for 10 min at 70°C. The proteins were separated by SDS‒PAGE and western blotting with a rabbit anti-Flag polyclonal antibody (F7425; Sigma‒Aldrich) to quantify the total CDV H protein concentration. The proteins in the membranes were detected with Luminata Crescendo Western HRP substrate (Merck KGaA, Darmstadt, Germany) via an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). Protein expression was analysed via ImageJ software (National Institutes of Health, Bethesda, USA).

Structure modeling and binding site prediction of H^wt/H^D540G and hSLAM/dSLAM

To predict the interaction sites between 5804PeH-H^wt/5804PeH-VhS-H^D540G and hSLAM/dSLAM, the complete amino acid sequences of 5804PeH-H^wt, 5804PeH-VhS-H^D540G, hSLAM and dSLAM were input into the AlphaFold 2.3.1(DeepMind, London, The United Kingdom of Great Britain and Northern Ireland, UK) [29–31] for structural modelling. (The GenBank accession numbers of the sequences used for modeling are as follows: AY386315 for 5804PeH-H^wt, AY040554 for hSLAM, and MG669628 for dSLAM. The sequenced data for 5804PeH-VhS-H^D540G has not been uploaded to NCBI.) The amino acid sequences of the two proteins to be analyzed were concatenated with a linker (five glycines) inserted between the two sequences. Upon running the AlphaFold 2 script, the system automatically performed multiple sequence alignment (MSA), template retrieval, and structural prediction. The quality of the resulting predictions was evaluated using predicted Local Distance Difference Test (pLDDT) scores [30,32]. Protein models were analyzed with PyMOL 2.5.1 (Schrödinger LLC, Delano, USA). H^wt-hSLAM and H^D540G-dSLAM served as controls to compare the changes in interaction sites between the RBD region of the mutated H^D540G and hSLAM. The atomic coordinates and pLDDT scores in PDB format for the structural models of 5804PeH-H^wt/5804PeH-VhS-H^D540G and hSLAM/dSLAM, along with MSA sequence hits, are available in the Zenodo database at https://zenodo.org/records/14504235.

H-hSLAM coimmunoprecipitation assay

The interaction between the H proteins of 5804PeH or 5804PeH-VhS and hSLAM was evaluated via coimmunoprecipitation (co-IP) assays with minor modifications [33]. Briefly, HEK293T cells in a six-well plate were transfected with 4 μg of pCI-5804PeH-Hflag or pCI-5804PeH-VhS-Hflag plasmid and 4 µg of pDisplay-hSLAM (human SLAM expression plasmid with an HA tag) via Lipofectamine™ 3000 Transfection Reagent (L3000150; Thermo Fisher). At 36 hours post-transfection, the cells were washed three times with ice-cold PBS, and the cells were collected in RIPA buffer (10 mm Tris, pH 7.4; 150 mm NaCl; 1% deoxycholate; 1% Triton X-100; 0.1% SDS) containing complete protease inhibitor mixture (Roche). The cell lysates were transferred into tubes and centrifuged at 4°C at 12,000 × g for 30 min. The supernatants were transferred into new tubes, and 15 μl of DynaGreen™ protein A magnetic beads (80103 G; Invitrogen) was added, as was a 1:1,000 dilution of anti-HA MAb (66006–2-LG; Proteintech) overnight at 4°C for the immunoprecipitation of hSLAM. The cells were then washed five times with NP40 buffer (P0013F; Beyotime). The immunoprecipitated proteins were separated via SDS‒PAGE and analysed via western blot analysis via a rabbit anti-FLAG polyclonal antibody (F7425, Sigma) or anti-HA MAb (66006–2-LG; Proteintech).

In vitro CDV infection of human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood [the blood of healthy adults was donated without compensation by the author Jianjun Zhao (from the Animal Molecular Pathology and Vaccine Research Laboratory of Heilongjiang Bayi Agricultural University); this study adheres to the Declaration of Helsinki; Committee on Science and Technology Ethics of the College of Animal Science and Veterinary Medicine in Heilongjiang Bayi Agricultural University (BYAU), DWKJXY2020062] via density gradient centrifugation via Ficoll‒Paque (GE Healthcare, Sweden). The cells were cultured in six-well plates in RPMI 1640 supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin containing 10 μg/ml phytohaemagglutinin (PHA) (11249738001; Roche) to induce the expression of SLAM [34]. Twenty-four hours after PHA stimulation, the PBMCs were infected with the cell-free virus 5804PeH or 5804PeH-VhS at an MOI of 0.1. At 96 hours post-infection, the PBMCs were observed under a fluorescence microscope (DMB3000, Leica, Germany) [35].

PBMCs infected with CDV were harvested and analysed via flow cytometry (Monisight, Gaugene, China) to test the efficiency of CDV infection of different immune cells. The cells were washed in 200 μl of ice-cold PBS and incubated for 1 h in 500 μl of PBS containing 5 μl of phycoerythrin-conjugated CD3- (ab155344, Abcam), CD19- (A22820, ABclonal), or CD14- (ab157300, Abcam) specific monoclonal antibodies. The cells were washed three times with 200 μl of ice-cold PBS. Marker and GFP expression in 10⁴ cells was analysed via two-colour fluorescence-activated cell sorting. The background was determined using mock-infected cells [27]. Simultaneously, PBMCs were seeded with Vero-dSLAM cells in 96-well plates by limiting dilution. PBMC-associated virus titres were determined and expressed as TCID₅₀/10^6.0 PBMCs and calculated as described by Reed and Muench [26].

In addition, the in vitro proliferation of human PBMCs inoculated with 5804PeH or 5804PeH-VhS (MOI = 0.1) in 96-well plates was also assessed via stimulation with PHA (1 μg/ml), and bromodeoxyuridine (BrdU) incorporation into proliferating cells was subsequently detected via the Cell Proliferation ELISA BrdU Kit (11647229001; Roche) according to the manufacturer’s instructions.

Mouse infections

Twelve 6-week-old hSLAM^(+/+)/Stat1^(-/-) transgenic mice used in this study were provided by the Dalhousie University Faculty of Medicine [36], which were generated via the cloning of human genomic DNA into a bacterial artificial chromosome (BAC), resulting in a construct containing the complete hSLAM gene, including its endogenous promoter for transcription. Additionally, to improve the efficiency of MeV production, hSLAM⁺ mice were bred with Stat1-deficient mice. The hSLAM receptor is expressed on activated B, T, and dendritic cells with an expression profile equivalent to that in humans in hSLAM^(+/+)/Stat1^(-/-) mice [36]. The transgenic mice were anesthetized intraperitoneally with ketamine and xylazine (Ket/Xyl) (Bayer, Toronto, Canada). Five animals in each group were infected intranasally with 10^6.0 TCID₅₀ of 5804PeH or 5804PeH-VhS in 50 μl of DMEM divided into two doses (25 μl for each nare). Two mock-infected mice were inoculated intranasally with 50 μl of DMEM. The animals were observed daily for signs of disease until 14 d post-infection [37]. All experimental animal procedures were conducted following a protocol previously approved (Dalhousie University Committee on Laboratory Animals, 16–003; Committee on Science and Technology Ethics of the College of Animal Science and Veterinary Medicine in BYAU, DWKJXY2020062).

Blood and organ collection

Blood samples from 5804PeH-infected (n = 3) and 5804PeH-VhS-infected (n = 4) mice at seven days post-infection, which were obtained via terminal bleeding via cardiac puncture, were collected in a microcontainer tube with lithium heparin. PBMCs were isolated via density gradient centrifugation via Ficoll-Paque (GE Healthcare, Sweden) according to the manufacturer’s protocol. The mice were euthanized by anaesthesia, and the organs were collected seven- and fourteen-days post-infection [37]. Lymphoid-associated organs, including the spleen, lymph nodes, thymus, lungs, kidneys, and brain, were also collected aseptically. One part of the tissue from each organ was transferred into a 15 ml tube containing PBS with 75% sucrose overnight at 4°C, embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, Canada), and frozen at − 80°C until immunofluorescence assays were performed. The remaining tissue samples were placed individually in tubes and stored at − 80°C to determine the viral loads and viral RNA isolation.

Quantification of the virus load in collected lymphoid organs and PBMCs

Serial 10-fold dilutions of PBMCs were prepared in DMEM supplemented with 5% FBS and 1% penicillin/streptomycin. Four replicates of 10¹–10⁵ PBMCs were cocultured with 10⁵ Vero-dSLAM cells in 96-well plates. Cultures were monitored for cytopathic effects five days later. The virus titres are expressed as the TCID₅₀ per 10⁶ PBMCs.

One hundred milligrams of each kind of collected organ (spleen, lymph node, thymus, or lung) was homogenized, and cell debris was removed by 15 min of centrifugation at 8000 r/min at 4°C. The virus titres of the supernatants were determined in Vero-dSLAM cells in 96-well plates and expressed as TCID₅₀/mg of the organ. Additionally, viral RNA from homogenized samples of the kidney and brain was extracted and quantified via CDV RT-PCR via primers for the CDV H RBD.

CDV RNA extraction and RT-PCR

Viral RNA from each homogenized organ was extracted via a QIAmp Viral RNA Mini Kit (52904; Qiagen). Reverse transcription was performed to generate cDNA via Multiscript High-Capacity Reverse Transcriptase (4374967; Thermo Fisher Scientific) and random primers. The cDNA was then diluted 5 × with ultrapure water and stored at − 20°C. CDV RNA analysis was performed via RT-PCR. Primers specific to the cDNA of the CDV H RBD were used to amplify by PCR.

Immunofluorescence assay

Frozen sections of the collected tissues were obtained using a Leica CM1900 (Leica, Germany). Tissue sections 4 to 6 μm in thickness were obtained, fixed in 99.5% histological-grade acetone (534064; Sigma‒Aldrich), and stored at 80°C until further use. The sections were thawed at room temperature for 5 min and fixed in 2% paraformaldehyde solution (USB Corporation, Cleveland, OH, USA) for 10 min. For immunofluorescence staining, the sections were permeabilized for 3 min at RT in PBS with 0.1% (v/v) Triton X-100 (9036-19-5; Sigma‒Aldrich). The sections were incubated with 5% goat serum blocking solution for 30 min at RT. The sections for confocal microscopy were prepared as previously described. For hSLAM staining, tissue sections were incubated with a 5% mouse anti-SLAM/CD150 antibody [IPO-3] (ab2604, Abcam) for 1 h at room temperature, followed by incubation with an Alexa Fluor 568-conjugated antibody (ab303465; Abcam) for 30 min at room temperature. All the sections were counterstained with DAPI to distinguish the cell nuclei. Lung, spleen, and lymph node sections were visualized, and images were acquired via ZEN2008 imaging software on a Zeiss LSM510 upright laser scanning confocal microscope (Zeiss, Germany).

Sera cross-protective neutralization assay

To measure 5804PeH and 5804PeH-VhS strain virus neutralization, Vero-dSLAM (VdS) cells were seeded in 96-well flat bottom plates and cultured for 48 h. Fourteen anti-MeV-positive sera were collected from MeV vaccine-vaccinated healthy adults [the 14 healthy adults (from the Animal Molecular Pathology and Vaccine Research Laboratory of Heilongjiang Bayi Agricultural University) signed a voluntary blood donation consent form, agreeing that their serum samples would be used in this study; this study adheres to the Declaration of Helsinki; Committee on Science and Technology Ethics of the College of Animal Science and Veterinary Medicine in BYAU, DWKJXY2020062], and nine CDV-antibody-positive sera were collected from CDV-vaccinated raccoon dogs, foxes, and dogs (five foxes, one raccoon, and three dogs were from the fur animal breeding farm of the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences; Committee on Science and Technology Ethics of the College of Animal Science and Veterinary Medicine in BYAU, DWKJXY2020062). Two-fold serial dilutions of serum samples (final serum dilutions of 1:4–512) were mixed with 100 TCID₅₀ of 5804PeH or 5804PeH-VhS for 1 h at 37°C in triplicate. The VdS cells were then added to 96-well white flat-bottom plates. The plates were incubated for five days at 37°C, and the wells were examined via microscopy for CPE. Virus neutralization titres were calculated as 50% endpoint measurements via the Reed and Muench method [26].

Statistical analysis

All the data were analysed via GraphPad Prism software (version 8.0; USA) and are expressed as the mean and standard error of the mean unless otherwise indicated. Significance was tested via one-way ANOVA followed by Tukey’s multiple comparison test. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001.

Results

Adaptation of 5804PeH with a D504G mutation in the H protein to Vero-hSLAM cells

5804PeH was cultured on Vero-hSLAM (VhS) cells for five consecutive passages. The adapted virus formed similar syncytia with Vero-hSLAM cells as the parental virus with Vero-dSLAM cells, demonstrating that receptor interactions, including fusion helper functions, appear to be fully adapted. The fluorescence intensity and range of EGFP produced by virus-infected cells increased with passage time (Figure 1a), indicating that the number of virus-infected cells also increased with passage time. During the first 3 passages, the viral titre increased to approximately 10^6.2 TCID₅₀/ml. In later passages, the virus titres did not increase further (Figure 1c).

Figure 1.

Infection of Vero-hSLAM with the CDV 5804PeH Strain. (a) Vero-hSLAM cells were infected with the 5804PeH strain for the first passages at an MOI of 1.0 and incubated for five days post-infection. The virus was harvested, and the TCID₅₀ was determined in Vero-dSLAM cells. Vero-hSLAM cells were infected with this virus at an MOI of 0.01 from the second to fifth passages (P1-P5). Cells were photographed at × 100 magnification. (b) Expression of the CDV EGFP and human SLAM receptor in Vero-hSLAM cells was determined via confocal microscopy. 5804PeH is shown in green, hSLAM labelled with the mouse anti-hSLAM/CD150 antibody [IPO-3] is shown in red, and nuclei stained with DAPI are shown in blue. Cells were photographed at × 100 magnification. (c) Viral titres in Vero-dSLAM cells harvested from the first to the 5th passages were determined. (d) 5804PeH was compared with the whole genome of the adapted strain 5804PeH-VhS. Sequence variations were identified in six clones derived from independently adapted viruses (5804PeH-VhS). The results for the five out of six virus clones with consistent amino acid (aa) mutations are shown. The mutation aa are highlighted in different colours. The labels N, H, and L at the top correspond to the regions encoding the nucleocapsid protein, haemagglutinin protein and large protein, respectively.

Since 5804PeH is derived from the parent strain 5804, which was first isolated from Vero cells from a CDV-infected dog, it may also adapt to receptors expressed in this cell [12,38]. We assessed by confocal microscopy whether the H of 5804PeH-VhS interacts with hSLAM. The Vero-hSLAM cells were infected with 5804PeH-VhS at an MOI of 0.01, and three days later, they were incubated with an anti-hSLAM antibody at room temperature for 45 min. Confocal microscopy images demonstrated that 5804PeH-VhS colocalized with hSLAM in Vero-hSLAM cells (Figure 1b).

To determine the sequence alterations required for this adaptation, CDV genomic sequences were determined from the original parental virus (5804PeH) and six virus clones obtained by plaque picking from the independently adapted viruses (5804PeH-VhS). In five out of the six sequenced clones of adapted viruses, we reproducibly found the same sequence alteration. In 5804PeH-VhS, one mutation was found in each of the N, H, and L genes, namely, N (S513P), H (D540G), and L (L973F) (Figure 1d).

To demonstrate that the adaptation occurred to hSLAM and not to another receptor, we performed an infection inhibition experiment using hSLAM-specific antibodies, which recognize hSLAM but not dSLAM (Figure 2a). Vero-hSLAM cells were incubated with different concentrations of the anti-hSLAM antibody (IPO3) or control isotype antibody. The infect ion was inhibited in a dose-dependent manner with 1 and 10 μg/ml IPO-3 but not with the isotype antibody, indicating that the virus indeed adapted to use hSLAM as a receptor (Figure 2b). As a further control, Vero-dSLAM cells were preincubated with IPO-3 and infected subsequently (Figure 2c). As expected, the antibody did not inhibit the infection of Vero-dSLAM cells.

Figure 2.

Infection with human slam-adapted 5804PeH-VhS is specifically inhibited by anti-hSLAM antibodies. (a-left, b) Vero-hSLAM cells were incubated with an IPO-3 monoclonal antibody against hSLAM at different concentrations or with IgG and Vero-hSLAM cells for 1 h. An MOI = 0.1 was used to infect Vero-hSLAM cells harboring the hSLAM adaptive strain 5804PeH-VhS. (a-right, c) as a control, Vero-dSLAM cells were infected with the 5804PeH-VhS strain in the presence or absence of IPO-3 (ns, no significance; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Cells were photographed at × 100 magnification.

Adapting 5804PeH-VhS to the hSLAM receptor did not impair its ability to infect susceptible cells

To compare the growth characteristics of the 5804PeH and 5804PeH-VhS strains in vitro, Vero cells with different receptors [Vero-dSLAM (VdS), Vero-hSLAM (VhS), Vero-dNectin-4 (VdN), and Vero-hNectin-4 (VhN)] were infected with 5804PeH and 5804PeH-VhS at an MOI of 0.01 (Figure 3). Compared to 5804PeH, the 5804PeH-VhS strain exhibited higher titres only at three days post-infection in Vero-dNectin-4 cells. At other time points, no significant differences were observed between the 5804PeH and 5804PeH-VhS strains in their replication efficiency in Vero-dSLAM, Vero-dNectin-4, or Vero-hNectin-4 cells. Interestingly, compared with the other groups, the 5804PeH group had a potential ability to infect Vero-hSLAM cells, but its virus titres were reduced by 2–3 logs compared with 5804PeH-VhS. These results indicate that we obtained a complete adaptation of 5804PeH-VhS to the human SLAM receptor. The adaptation to hSLAM did not impair the replications of this virus on dSLAM-, dNectin-4-, or hNectin-4-expressing cells.

Figure 3.

Replication kinetics of 5804PeH-VhS in susceptible cells. Vero cells expressing SLAM (a) or nectin-4 (b) receptors were infected with 5804PeH and 5804PeH-VhS at an MOI of 0.01 and incubated for the indicated times before harvesting cell-bound plus released virus. Viral titres were detected in Vero-dSLAM cells via the TCID₅₀ method to yield growth curves. The asterisks indicate a significant difference between the 5804PeH-VhS on VdN group and the 5804PeH on VdN group (***, p < 0.001).

The D540G mutation enhanced cell-cell fusion but did not change the expression level of the H protein

To determine the functional consequences of the detected D540G mutation in the CDV H gene, both Vero-dSLAM and Vero-hSLAM cells were transfected with the plasmids pCI-5804PeH-Hflag and pCI-5804PeH-F or with pCI-5804PeH-VhS-Hflag and pCI-5804PeH-F. In Vero-dSLAM cells, large syncytia were observed at 24 hours post-transfection with both combinations of plasmids. In contrast, in Vero-hSLAM cells, syncytia were observed only after transfection with the plasmids encoding H^D540G and F but not after transfection with the plasmids encoding H^wt and F (Figure 4a). These findings demonstrate that the H^D540G mutation in 5804PeH-VhS is necessary to mediate a functional interaction with hSLAM, contributing to membrane fusion capacity of the viral H and F envelope glycoprotein complex.

Figure 4.

Induction of cell-to-cell fusion in Vero-hSLAM cells expressing the CDV H and F proteins. (a) Vero-dSLAM and Vero-hSLAM cells were transfected with the pCI-5804PeH-Hflag or pCI-5804PeH-VhS-Hflag plasmid and the pCI-5804PeH-F plasmid and the images were observed via a microscope. Cells were photographed at × 100 magnification. (b and c) Cell surface biotinylation of 5804PeH and 5804PeH-VhS H proteins. (b) Cells transfected with pCI-5804PeH-Hflag, pCI-5804PeH-VhS-Hflag, or pCI vector plasmids were labelled with NHS-LC-LC-biotin, immunoprecipitated with an rabbit anti-flag polyclonal antibody, and detected with a streptavidin-hrp conjugate. Western blots of cell lysates from the same experiment were analysed for total H protein expression. (c) The relative surface expression represents the ratio between the biotinylation signal and the Western blot signal for each replicate. Average relative surface expression levels were calculated from three independent experiments via ImageJ (ns, no significance).

Next, to determine whether the D540G mutation affects the expression of the CDV H protein in the cell membrane, HEK293T cells were transfected the pCI-5804PeH-Hflag and pCI-5804PeH-VhS-Hflag plasmids. Prior to lysis, the transfected cells were biotinylated at 4°C to label surface-exposed proteins. Total H expression was studied by Western blot analysis of cell extracts (Figure 4b, lower panel), and surface-exposed H protein was analysed by immunoprecipitation of biotinylated protein (Figure 4b, upper panel). The expression level of the CDV H protein in the 5804PeH and the5804PeH-VhS did not show any significant difference (Figure 4b–c). The results showed that D540G mutation in the CDV H protein did not change the expression of the H protein.

The interaction between H^wt/H^D540G and hSLAM/dSLAM

Interaction models between 5804PeH-VhS-H^D540G (H^D540G) and hSLAM, 5804PeH-H^wt (H^wt) and hSLAM, as well as H^D540G and dSLAM were predicted using AlphaFold. Analysis of the predicted models with PyMOL revealed that the binding interface in all three models (H^D540G-hSLAM, H^wt-hSLAM and H^D540G-dSLAM) were similar, localized to the V-like domain of SLAM and the RBD of the H protein (Figure 5a). The PLDDT scores for the interaction regions were all above 80% (data not shown). The prediction results revealed three components of the binding interface between H^wt/H^D540G and hSLAM/dSLAM (sites 1–3, the distances of interacting residues varied between 2.1 Å and 3.6 Å). Site 1 is formed by hydrogen bonds of Y186, S497, I527, S528, D530, Y539, R543, S546, and T548 of H^wt/H^D540G with S28, S59, R90, S73, E/K75, N125, and R/H130 of hSLAM/dSLAM. Site 2 is formed by salt bridges of D501, D503, D526, R529, and R552 of H^wt/H^D540G with K77 and E123 of hSLAM/dSLAM. Site 3 is an intermolecular β-sheet [39] formed by the polypeptide backbones of the T192–G196 strand of CDV-H (β6 sheet) and the V/F126–R/H130 strand of hSLAM/dSLAM (Figure 5b–d). By comparing the predicted interaction sites between the RBD of H^wt/H^D540G and hSLAM/dSLAM, it was observed that the H^D540G mutation introduced a new interaction between Y539 of H^D540G and S73 of hSLAM (Figure 5e). This additional interaction site may enhance the adaptation of 5804PeH-VhS-H to the hSLAM receptor, thereby facilitating effective infection of Vero-hSLAM cells.

Figure 5.

Interaction between H^wt/H^D540G and hSLAM/dSLAMbased on structural model. (a) Side view of the 5804PeH-H^wt/5804PeH-VhS-H^D540G monomer (blue) bound to hSLAM/dSLAM (green), depicted in a ribbon diagram. (b – d) Detailed views of the interaction sites [SLAM, green; CDV-H, blue; interatomic distances (Å), yellow dashed lines]. The three components of the binding interface are indicated (sites 1–3). Predicted interaction sites between (b) 5804PeH-VhS-H^D540G and hSLAM; (c) 5804PeH-H^wt and hSLAM; (d) 5804PeH-VhS-H^D540G and dSLAM. (e) Comparison of amino acid residues involved in interactions within the RBD region of the H protein based on prediction results (left panels: 5804PeH-VhS-H^D540G and hSLAM; middle panels: 5804PeH-H^wt and hSLAM; right panels: 5804PeH-VhS-H^D540G and dSLAM). Key amino acid residue variations in the interactions are highlighted in yellow.

D540G did not significantly enhance the interaction between H and hSLAM

The H^D540G mutation is necessary for syncytial lesions in Vero-hSLAM cells. We next conducted co-IP experiments to investigate the underlying mechanism by which the H^D540G mutation strains formed syncytia in Vero-hSLAM cells. To achieve this aim, we coexpressed the full-length versions of the hSLAM and H^wt/H^D540G proteins in HEK293T cells and pulled down the potential complexes via an anti-HA MAb that targeted hSLAM. Finally, bound H proteins were detected via an anti-FLAG PAb. Unexpectedly, as shown by the results in Figure 6a, H^wt and the H^D540G mutant could efficiently bind to the full-length hSLAM protein and exhibited significant physical interactions. The experiment was repeated three times, and the same results were obtained. Furthermore, we measured the interaction between H^wt/H^D540G and hSLAM via a semiquantitative assay [40]. The results revealed that the interaction between H^wt and H^D540G with hSLAM were at similar level (Figure 6b). The hSLAM appeared to bind to H^wt or H^G540G with a similar affinity, with no significant difference (Figure 6c). Analysis of the CDV-H/hSLAM interaction via coimmunoprecipitation protein assays may be limited due to its low sensitivity, and exact quantifying the affinity of the H/hSLAM interaction is also challenging. More advanced protein-membrane resonance (SPR) technology may help clarify the impact of the D540G mutation on H-hSLAM affinity [33].

Figure 6.

Immunoprecipitation analysis of the interaction between CDV and hSLAM. (a) HA-tagged hSLAM and Flag-tagged 5804PeH-H or 5804PeH-VhS-H were coexpressed in HEK293T cells, which were subsequently lysed with RIPA buffer 36 hours post-transfection. The H protein-hSLAM complexes were then immunoprecipitated (IP) with anti-ha MAb and DynaGreen™ protein A magnetic bead treatment. Proteins were boiled and subjected to immunoblotting (IB) via an anti-flag polyclonal antibody to detect H antigenic materials (co-ip). co-ip CDV H proteins were detected in comparison with CDV H proteins present in cell lysates prior to IP by immunoblotting with the same anti-flag antibody. Gels illustrating total expression and immunoprecipitation of SLAM molecules are also shown (detected via an anti-ha polyclonal antibody). The specific MAbs used for the immunoprecipitation (IP) or immunoblotting (IB) steps are indicated on the left of the gels (three-colour prestain protein marker, WJ103; Epizyme Biotech). (b) The signals in each H protein and hSLAM band were quantified, and the avidity of H-hSLAM interactions is represented by the ratio of the amount of co-ip H over the product of H in the cell lysates divided by the ratio of the amount of immunoprecipitated hSLAM over the product of hSLAM in the cell lysate [(co-ip H/Input H)/(IP hSLAM/Input SLAM)]. (c) All ratios were subsequently normalized to the ratio of the 5804PeH H-hSLAM interactions, which was set to 1. The values are averages of results from at least three independent experiments. H/hSLAM avidity of interactions is shown (ns, no significance).

5804PeH-VhS enters human PBMCs in vitro and inhibits their proliferation

We determined the ability of 5804PeH and 5804PeH-VhS to infect the PBMCs of healthy adult donors. PHA-stimulated human PBMCs were infected with 5804PeH or 5804PeH-VhS at an MOI of 0.1 for 96 hours. In human PBMCs, specific EGFP fluorescence was almost exclusively detected in the 5804PeH-VhS group (Figure 7a). The cells and the supernatant were collected, and the virus titres were measured. The titre of 5804PeH-VhS was approximately 10-fold greater than that of the 5804PeH strain, with a significant difference (Figure 7b). Next, we determined the phenotype of 5804 PeH-VhS-infected cells in peripheral blood and lymphoid tissues via FACS analysis. Between 0.3% and 0.1% of T lymphocytes and monocytes were 5804PeH-VhS-infected, whereas 5804PeH-VhS replication was virtually absent in B lymphocytes. As expected, we observed no positive human PBMCs at 36 hours post-infection with the 5804PeH strain (Figure 7c). Human PBMC proliferation was measured via BrdU proliferation ELISA, which revealed that 5804PeH was not significantly different from the control, whereas the OD450 nm light uptake value of 5804PeH-VhS was significantly lower than that of the control (Figure 7d). These data indicated that the 5804PeH-VhS strain could suppress their proliferation. These findings demonstrate that 5804PeH-VhS could infect human T lymphocytes and monocytes and effectively inhibit the proliferation of human PBMCs.

Figure 7.

Comparison of flow cytometry, viral load, and proliferation results between 5804PeH and 5804PeH-VhS infections in human PBMCs. (a) 5804PeH- and 5804PeH-VhS-infected human PBMCs for 96 h. Slides were photographed at × 100 magnification. (b) 5804PeH- and 5804PeH-VhS-infected human PBMCs. (c) Efficiency of 5804PeH- and 5804PEH-VhS-infected T lymphocytes, B lymphocytes, and monocytes. (d) Comparison of the proliferative activity of human PBMCs infected with 5804PeH and 5804PeH-VhS (ns, no significance; *, p < 0.05; **, p < 0.01).

5804PeH-VhS replication in lymphatic organs and PBMCs of hSLAM transgenic mice

In vitro experiments revealed that the 5804PeH-VhS strain has been fully adapted to the hSLAM receptor. We next determined whether the 5804PeH-VhS strain adapts to the hSLAM receptor in vivo by infecting transgenic mice expressing hSLAM receptors with 5804PeH and 5804PeH-VhS. The blood and organs of the transgenic mice were collected seven days post-infection, and the spleen weights and organ virus titres were analysed. The spleens of the transgenic mice infected with 5804PeH-VhS were marked tumescence, and the spleen weight was increased (Figure 8a–b). Examination of CDV loads revealed that 5804PeH-VhS-infected mice presented significantly higher viral loads compared with 5804PeH-VhS-infected mice in the PBMCs, spleens, lymph nodes, thymus, and lungs at seven days post-infection (Figure 8c), indicating that infection of these mice with 5804PeH-VhS induced viraemia. RT-PCR for CDV RNA isolated from the spleen, lymph nodes, thymus, lung, kidney, and brain from infected mice was performed to monitor the level of infection at seven days post-infection. After infection with 5804PeH-VhS, CDV RNA were detected in the spleen, lymph nodes, thymus, lungs, and kidneys but not in the brains of the hSLAM mice. Higher level of CDV H RNA was detected in the spleen and lung than in the other organs. However, the 5804PeH strain was unable to replicate effectively and spread in transgenic mice (Figure 8d). The results revealed that 5804PeH-VhS replicated in epithelial tissue but had not yet replicated in the brain.

Figure 8.

CDV infected hSLAM transgenic mice with 5804PeH and 5804PeH-VhS strain. (a) Size of the spleen of infected mice; (b) average weight of the spleen of infected mice; (c) viral load in peripheral blood monocytes, spleen, lymph nodes, thymus, and lungs; and (d) viral RNA in the spleen, lymph nodes, thymus, lungs, kidneys, and cerebrum were detected after hSLAM transgenic mice were infected with CDV for seven dpi (AccurateRun^TM prestained 100 bp-ii DNA ladder, GsDL1002, Generay Biotech) (**, p < 0.01; ***, p < 0.001).

5804PeH-VhS can replicate in the immune tissues of hSLAM transgenic mice

As the spleen, lung, and lymph nodes seem to be affected during 5840PeH-VhS infection in hSLAM transgenic mice, we analysed the intracellular distribution of 5840PeH-VhS and 5804PeH in these organs after intranasal infection. At seven days post-infection, frozen tissue sections were stained with anti-hSLAM antibody (red) and DAPI (blue). In addition, 5804PeH and 5804PeH-VhS expressed EGFP (green) for monitoring of virus replication. EGFP/hSLAM colocalization was examined via confocal microscopy. The immunofluorescence images revealed the colocalization of 5804PeH-VhS and hSLAM at the cell membrane (Figure 9, indicated with arrows). Additionally, and the positive expression of hSLAM but EGFP was detectable in the lymph nodes of the mice only infected with 5804PeH (Figure 9). These results indicated that 5804PeH-VhS could replicate in transgenic mice immune tissues and has acquired the ability to bind to hSLAM receptors in vivo.

Figure 9.

5804PeH-VhS replicated in the immune tissues of hSLAM transgenic mice. Transgenic mice expressing hSLAM receptors were intranasally infected with 10⁶ TCID₅₀ of either 5804PeH or 5804PeH-VhS. At seven days post-infection the organs were collected, tissue sectioned (4–6 μm), and analysed via laser scanning confocal microscopy. Tissue sections were stained with a primary antibody against hSLAM [IPO-3] followed by a secondary antibody conjugated with Alexa Fluor 568 (red). Virus replication was visualized using EGFP expression (green), and nuclei were counterstained with DAPI (blue). In the upper panels, colocalization of 5804PeH-VhS and hSLAM receptors was observed on the cell membrane (yellow, arrows) in the lung, spleen, and lymph nodes (L.N.). In contrast, the lower panels showed 5804PeH-infected lymph nodes (L.N.) as a control, where no apparent EGFP was detected. Images were acquired using a Zeiss LSM510 confocal microscope at × 400 magnification.

The MeV vaccine cannot provide a cross protection against CDV 5804PeH-VhS infection

To determine the cross-protective effects of MeV -vaccinated sera against the CDV H^D540G mutation strain (5804PeH-VhS), we next examined the cross-neutralizing activities of 5804PeH and 5804PeH-VhS against the vaccine vaccinated sera of humans and animals (fox, dog, and raccoon dog) respectively. Using virus cross-neutralization test, we investigated that all the 14 MeV-positive human sera showed negative neutralizing titres against the CDV 5804PeH and 5804PeH-VhS strain (Figure 10a). In contrast, CDV positive sera collected from vaccinated animals showed higher neutralizing titres against 5804PeH-VhS than 5804PeH (p < 0.01), suggesting the H^D540G mutant may cause the structural changes of the H protein and alters its neutralizing epitopes in 5804PeH-VhS (Figure 10b).

Figure 10.

Neutralization is affected by the D540G mutation within CDV H protein. (a and b) Neutralization of 5804PeH and 5804PeH-VhS by sera from humans (MeV specific; left panel; n = 14) (a) and animals (CDV specific, right pane; fox = 5, raccoon dog = 1, dog = 3) (b). Neutralizing antibody titres (color-matched by serum) are shown (**, p < 0.01).

Discussion

Within the family Paramyxoviridae, morbilliviruses pose potential pandemic threats [22]. Recent campaigns for the eradication of MeV have raised significant concerns. When the goal of measles eradication is achieved, and measles vaccination is stopped, the remaining morbilliviruses may emerge in vacated ecological niches. They might eventually cross the species barrier to humans and emerge as a new human pathogen, best exemplified by reverse zoonotic outbreaks caused by CDV in macaques in China and Japan [16–18]. The zoonotic potential of CDV is functionally supported by their ability to utilize noncognate SLAM receptors [41–43]. CDV initially infect immune cells expressing the SLAM receptor, which exhibits significant interspecies variation within the host [44,45].

Despite the high amino acid sequence identity of SLAM receptors between macaques and humans (96.7%), there have been no reports of CDV infection in humans. In contrast, the amino acid sequence of nectin-4, another morbillivirus receptor, is highly conserved among different mammals [46]. Our present study demonstrated that 5804PeH also has an intrinsic ability to use human nectin-4, and the adapted virus, 5804PeH-VhS, was able to utilize hSLAM and human nectin-4 (hNectin4) as efficiently as canine SLAM (dSLAM) and canine nectin-4 (dNectin4), respectively (Figure 3). Thus, nectin-4 may play only a minor role in determining the host specificity of CDV. In contrast, the blockade of hSLAM significantly affected the replication of 5804PeH-VhS in Vero-hSLAM cells but not in Vero-dSLAM cells, suggesting that 5804PeH-VhS is adapted to the hSLAM receptor and not to other receptors in Vero cells. Interestingly, 5804PeH exhibited reduced infectivity within VhS cells, approximately 100–1000-fold lower than that of the 5804PeH-VhS strain, rather than being completely absent. This might be attributed to CDV utilizing other receptors present within VhS cells, such as the recently reported LRP6 receptor [38].

By generating highly diverse genome sets, RNA viruses adapt rapidly to new host environments, so-called “quasispecies.” Minor genetic variants promote their rapid adaptation, allowing for the emergence of drug-resistant or immune-escape mutants [47]. Only one amino acid exchange in the RBD of the 5804PeH protein at position D540G was required for functional adaptation to human SLAM, which is similar to the findings of an adaptive study of the CDV A75/17 strain by Bieringer [20]. Our protein interaction analysis revealed that the interaction model composed of hSLAM and 5804PeH-VhS-H^D540G had different interaction sites with A75/17-VhS-H. The H^D540G mutation of the adapted strain A75/17-VhS interacted with residue hSLAM-E71, whereas the H^D540G mutation of 5804PeH-VhS did not interact with residue hSLAM-E71, but its neighbouring amino acid residue H^D540G-Y539 interacted with residue hSLAM-S73. The reason for the different interaction sites might be the different molecular structures of the H proteins of different CDV strains. Interestingly, residue H^D540G-Y539 is conserved among morbillivirus, and S73 is conserved in hSLAM and dSLAM. The residues at position 539 of H^D540G and 73 of hSLAM are both noncharged amino acids. This interaction probably exerts an adaptive constraint for amino acid 540 in the H^D540G protein, which leads to the observed mutation from a charged to a noncharged residue. Thus, the D540G mutation likely provides a balanced charge distribution in the H^D540G-hSLAM complex, altering the α-helix conformation and affecting the protein secondary structure, which in turn facilitates the interaction between H^D540G-Y539 and hSLAM-S73 amino acid residues. Similar interactions may be sufficient for the CDV H protein to adapt to the hSLAM receptor. The amino acid residue Y543 of the MeV H protein (equivalent to Y539 of the CDV H protein) forms a salt bridge with residue E75 of the marmoset SLAM (maSLAM) receptor and interacts hydrophobically with residues M72 and V74 [39], similar to what we found: H-Y539 interacts with hSLAM-S73. As a result, 5804PeH-VhS strains may replicate in human PBMCs and transgenic mice through hSLAM receptors.

A CDV strain, CYN07-dV, associated with a lethal outbreak in monkeys, uses hSLAM as a receptor only poorly but is readily adapted to use it following a P541S substitution in the H protein [21]. However, unlike the CYN07-dV strain, the P541S mutation did not confer hSLAM-using ability on the H proteins of Ac96I and MSA5 (both wild-type CDV strains) [21]. These results were different from our results. Different CDV strains may mutate different amino acid sites in the H protein to adapt to hSLAM. Both amino acids at positions 540 and 541 are located at the edge of the hydrophobic region [22], which may facilitate adsorption for extensive replication in Vero-hSLAM cells.

On the other hand, the H^D540G mutation may enhance the interaction between the CDV H and F proteins, leading to cell fusion in Vero-hSLAM cells. Therefore, the D540G mutation may not play a crucial role in the interaction with the hSLAM receptor in vitro. In the future, we will continue to investigate the molecular mechanisms of CDV adaptation to the hSLAM receptor by examining how the CDV H^D540G mutation alters CDV H-F interactions and affinity.

Recent studies have shown that in vitro infection of human PBMCs with MeV, the positivity rate for B lymphocytes and monocytes can exceed 20%, while the positivity rate for T lymphocytes is approximately 5% [27,35]. In our present study, although the 5804PeH strain failed to infect human PBMCs in vitro, our flow cytometry results indicate that the 5804PeH-VhS not only infected human PBMCs in vitro but also decreased their proliferation ability. Moreover, 5804PeH-VhS was inclined to infect of T lymphocytes and monocytes although it is less efficient than MeV in vitro. Vero-hSLAM cells differ from PBMCs in that they are immunodeficient and cannot produce IFN [48], which enables the 5804PeH strain to replicate more effectively after adapting to the hSLAM receptor. After viral inoculation, the 5804PeH-VhS may require an extended period to induce substantial infection in human PBMCs in vitro. However, extended infection time also leads to a significant reduction in immune cells, subsequently reducing the number of positive PBMCs detected [49,50]. Additionally, we did not use infection-enhancing agents, such as GM-CSF, which resulted in a poor quantity of positive PBMCs. B lymphocytes are less susceptible to the 5804PeH-VhS strain, possibly because B lymphocytes require sufficient stimulation conditions to express the SLAM receptor, which may not provide adequate in vitro stimulation [51,52].

The initially reported hSLAM transgenic mouse model expressed hSLAM under the control of the T-cell-specific lck promoter [53]. The second model involves a mouse expressing hSLAM under the control of the promoter of the ubiquitously expressed hydroxymethylglutaryl coenzyme A (HMGCoA) reductase protein [54]. The third model is controlled by the CD11c promoter [55]. Compared with these models, hSLAM^(+/+)/Stat1^(-/-) mice express hSLAM with human-like tissue specificity and better simulation of human MeV infection, which we used in the present study. As expected, the use of hSLAM^(+/+)/Stat1^(-/-) mice [36] allowed the replication of 5804PeH-VhS in vivo. The observed splenomegaly in the infected mice is in line with previous research on MeV infection in hSLAM transgenic mice. This phenomenon is associated with the antiviral response triggered by extensive replication of the virus in vivo. Upon infection, MeV initially disseminate to the lungs and can only be detected in mediastinal lymph nodes. It then spreads to other sites, including the spleen, PBMCs, and submandibular lymph nodes [37], explaining the positive results observed in the lungs and spleen (Figure 8c–d). The colocalization of the virus with hSLAM expressed in the lungs, spleen, and lymph nodes suggests that the virus propagates through SLAM receptors (Figure 9). Several instances of inadequate colocalization may result from decreased hSLAM expression following CDV infection. The limitation of this study is that it was performed in Stat1^(-/-) mice to achieve replication, like the other components involved in the adaptation of CDV to human cells. While the use of a mouse model for experiments has several limitations, we compared infection with the parental and adapted CDV strains, which can, to some extent, eliminate the impact of Stat1⁻ on CDV replication efficiency. While CDV can adapt to Vero-hSLAM and replicate in hSLAM transgenic mice, this adaptation represents only an initial stage of full adaptation to human target cells and does not necessarily imply the ability to spread in humans. Further experiments are needed to clarify whether 5804PeH-VhS can cause clinical symptoms in humans and be effectively transmitted.

A recent study has suggested that partial protection against CDV was observed in measles-vaccinated macaques, as demonstrated by the accelerated control of virus replication and limited shedding from the upper respiratory tract [15]. However, neither CDV infection nor MeV vaccination induced cross-reactive neutralizing antibodies. MeV neutralizing antibody levels in MeV-vaccinated macaques are boosted after CDV challenge [15]. We utilized sera from CDV-vaccinated animals and MeV-vaccinated humans for neutralization assays and found that anti-MeV antibodies cannot effectively neutralized the CDV 5804PeH and 5084PeH-VhS strains. In contrast, the R191P mutation in the H protein of PPRV adapts to the hSLAM receptor and influences cross-protective neutralizing activity against anti-MeV antibodies [22]. Our results revealed that D540G mutation in the H protein of 5804PeH-VhS strain did not change the cross-protective activity to anti-MeV antibodies but affected its neutralizing ability against-CDV-vaccine induced antibodies. These results indicate that the H^D540G mutation in CDV raising the possibility of MeV-vaccinated humans may be inefficiency antibody-protection against CDV-hSLAM adapted strain, such as 5804Pe-VhS.

Although CDV adapts rapidly to Vero-hSLAM and can replicate in hSLAM transgenic mice, other human target cells are not susceptible to infection. Morbillivirus cross-specific immunity of the human population restricts the possibilities of CDV replicating in or adapting to humans. Adaptation of the CDV to the hSLAM receptor is the first step in fully adapting to the target cell of the humans and other viral genetic changes may be necessary to achieve full virulence within human.

Funding Statement

This work was supported by the Natural Science Foundation of China under Grant [31972714]; the State Key Laboratory of Veterinary Biotechnology under Grant [SKLVBF202004]; the Heilongjiang Bayi Agricultural University under Grant[XY201912].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

JZ and BZ: Conceptualization, writing-original draft, formal analysis, investigation, methodology, software. WR and YS: Investigation, methodology, software, formal analysis, and data curation. AA, ML, JC: Resources. DS and CDR: Resources, project administration, writing, review, and editing. JZ: The final approval of the version to be published and that all authors agree to be accountable for all aspects of the work.

Availability of data and materials

The data generated during the study is available at [Zenodo] with the DOI [https://doi.org/10.5281/zenodo.14504235] and the reference number [14504235].

Consent for publication

Written informed consent for publication was obtained from all participants.

Health and Safety

All mandatory laboratory health and safety procedures have been complied with in the course of conducting any experimental work.

Ethics approval and consent to participate

All experimental animal procedures were conducted following a protocol previously approved by the Dalhousie University Committee on Laboratory Animals, Dalhousie University, Canada (The study of measles virus and canine distemper virus interactions with the innate immune system and the role of the immune system in virus receptor switching, 16–003) and the Committee on Science and Technology Ethics of the College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, China (The replication of CDV-hSLAM adapted strains in human PBMC and the cross-protection effect of MEV-specific antibodies against CDV-hSLAM adapted strains, DWKJXY2020062). We adhered to the ARRIVE guidelines. The studies involving human participants adhere to the Declaration of Helsinki.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2025.2457967