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Monkeypox (MPX) is an emerging zoonotic disease caused by the MPX virus (MPXV), which is recognized as the most important Orthopoxvirus infection in humans in the smallpox post-eradication era (1). The unexpected increase in human MPX cases in non-endemic countries raises concerns regarding a novel global public health threat (2). Between January 1 and November 10, 2022, a cumulative total of 79,151 laboratory-confirmed cases of MPX and 49 related deaths were reported to the World Health Organization by 110 countries or areas worldwide (3).
The first imported case of MPX in the mainland of China was confirmed on September 16, 2022, in a 29-year-old salesman of Chinese nationality who visited Germany (4). On September 9, the patient had a dry and itchy throat, a fever, and red rashes and pustules displayed on the right thigh. Clinical manifestations were reported, and complete genomes of the MPXV from clinical samples suggest that the MPXV strain in this case belongs to the B.1 branch of the West African lineage (4). The response to this public health emergency must include rapid isolation and identification of MPXV and development of novel assays for MPXV (5). To date, MPXV isolation and characterization have not been reported in the mainland of China. Additionally, limited information is available regarding a plaque assay for an infectious titration of MPXV and a plaque reduction neutralization test (PRNT) for MPXV-neutralizing antibody (nAb) detection on the basis of the MPXV-B.1 strains of the current epidemic.
In this study, the isolation and characteristics of MPXV in the first imported case of MPX in the mainland of China are reported. A plaque assay for MPXV infectious titration and a PRNT for MPXV nAb detection were developed.
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Swabs from the skin lesion, oropharynx, nasopharynx, and whole blood samples were used for viral isolation in the Vero cells. Viral replication was confirmed by qPCR assay based on conserved F3L genes that specifically detect MPXV with DNA from cell cultures collected at 48, 96, 120, and 144 h after inoculation (Table 1). The cycle threshold (Ct) values for the nasopharyngeal swabs and blood incubations were negative at 48 h and 144 h. In contrast, for the blister fluid swab inoculation, Ct values of 27.88, 19.05, and 18.15 were detected at 48 h, 96 h, and 120 h after the inoculation. For the oropharyngeal swab inoculation, Ct values were detected as 31.00, 27.28, and 25.68 at 48 h, 96 h, and 144 h, respectively. These results indicated that MPXV replicated in the blister fluid and oropharyngeal incubation.
Specimen Ct values* CPEs at harvest time MPXV isolation Clinical sample 48 h 96 h 120 h 144 h Blister fluid swab 20.76 27.88 19.05 18.15† ND ++++ Positive Oropharyngeal swab 27.81 31.00 27.28 ND 25.68† + Positive Nasopharyngeal swab 31.00 ND ND ND ND† - Negative Blood 33.65 ND ND ND ND† - Negative Note: CPEs: -, Negative or no CPEs; +, 0–25%; ++, 25%–50%; +++, 50%–75%; ++++, 75%–100%.
Abbreviation: MPXV=monkeypox virus; qPCR=quantitative real-time polymerase chain reaction; ND=not determined; CPEs=cytopathic effects; Ct=cycle threshold.
* For the blister fluid swab, viral DNAs from the supernatant of 48 h and 96 h were detected, while DNA from cell lysates of 120 h was detected. For the oropharyngeal swab, nasopharyngeal swab and blood samples, viral DNAs from the supernatant of 48 h, 96 h, 120 h were detected, while DNA from cell lysates of 144 h was detected.
† Harvest cell lysate.Table 1. Detection of monkeypox virus (MPXV) DNA using quantitative real-time polymerase chain reaction in Vero cell cultures with different specimens from the MPX case.
The virus-induced changes in CPE-related cell morphology were observed daily. The nasopharyngeal and blood-incubated cells were CPE-negative. In contrast, CPEs were observed within 48 h for the skin blister fluid-incubated cells, exhibiting cell rounding, detachment, or even death (Figure 1A). The degree of visible damage to the cells increased at 72 h, 96 h, 120 h, and 144 h, resulting in >85% CPEs (Table 1). Meanwhile, minor CPEs were also observed after 48 h for the oropharyngeal incubated cells, resulting in <20% CPEs at 144 h post-incubation (Table 1).
Figure 1.Characterization of MPXV-B.1-China-C-Tan-CQ01 in the Vero cell. (A) Cytopathic effects of monkeypox virus (MPXV) in the Vero cell; (B) Immunofluorescence analysis of Vero cell infected or uninfected with MPXV-B.1-China-C-Tan-CQ01 using the serum of the MPX case; (C) Transmission electron microscopy micrographs of MPXV.
Note: In panel A, the typical cytopathic effects are observed in the Vero cells 48 h after the first passage, thereby exhibiting cell detaching and rounding. In panel B, the nuclei were stained with DAPI (blue), while the MPXV infected cell showed green fluorescence. In panel C, the left panel showed the negative staining of purified MPXV particles with a typical brick shape observed in a diameter that varies between 200 nm and 300 nm and irregular tubular strips on the surface. The right panel showed the ultrathin section of MPXV infected Vero cell and mature virus particles in cytoplasm that show typical morphology including a dumbbell shaped core and the outer membrane on longitudinal orientation.
Abbreviation: MPXV=monkeypox virus; MPX=monkeypox; DAPI=4'6-diamidino-2-phenylindole.
Immunofluorescence assay with the patient’s serum was performed to confirm MPXV antigen expression, which showed the infected foci, visualized with green fluorescence in the cell, reacted with the serum of the patient with MPX (Figure 1B). IFA with anti-rabbit antibody against orthopoxviruses was also positive around the infected foci (data not shown).
Negative stained virus particles present a typical brick shape about 200 nm × 200 nm × 250 nm in size with irregular tubular strips on the surface. On sections, mature virus particles in cytoplasm show typical morphology including a dumbbell-shaped core and the outer membrane on longitudinal orientation (Figure 1C).
Plaque assays indicated that the infectious MPXV titers in the first passage of blister fluid or oropharyngeal swab incubation were 6 × 104 PFU/mL and 10 PFU/mL, increased to 1 × 105 PFU/mL and 1 × 104 PFU/mL for the second passage, and to >106 PFU/mL after the third passage (Figure 2A).
Figure 2.Plaque assays and PRNT based on MPXV-B.1-China-C-Tan-CQ01. (A) Quantification of infectious monkeypox virus from clinical samples (the first passage of isolation, P1, to the third passage of isolation, P3, using plaque assay). (B) Plaque reduction neutralization test to detect neutralizing antibodies (nAb) against MPXV in the serum of the MPX case (patient) and two 30-year-old healthy donors (H1 and H2).
Note: In panel A, the plates are fixed and stained with crystal violet 72 h to 96 h post-infection. Representative plates for the MPXV titration from blister fluid are shown. Plaques for P1 with a dilution of 10−2 (−2) and mock, for P2 with a dilution from 10−3 to 10−5 (−3, −4, −5) in double well, and for P3 with a dilution from 10−2 to 10−6 (−2, −3, −4, −5, −6) and mock are all shown. In panel B, the representative plate and neutralization cures are presented.
Abbreviation: MPXV=monkeypox virus; MPX=monkeypox; P1=the first passage of isolation; P2=the second passage of isolation; P3=the third passage of isolation; PRNT=The plaque reduction neutralization test; nAb=the neutralizing antibody; H1=healthy donor 1; H2=healthy donor 2.
A PRNT in the Vero cells with the MPXV-B.1-China-C-Tan-CQ01 was established. Serum 50% neutralization titer (NT50) was detected as 35 for the MPXV case 6 days after symptom onset, while negative for the 30-year-old healthy donors (Figure 2B).
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