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Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case

Abstract

BACKGROUND

According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak.

OBJECTIVE

To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies.

METHODS

We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM).

FINDINGS

We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM.

MAIN CONCLUSIONS

Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

Key words:
Mpox; monkeypox virus isolation; cell culture; transmission electron microscopy (TEM); ultrastructural studies


Monkeypox virus (MPXV) is a double-stranded DNA virus belonging to the family Poxviridae, genus Orthopoxvirus. Poxviruses have a brick- or oval-shaped structure measuring 200-400 nm.11. Louten J. Virus structure and classification. Essential human virology. Academic Press; 2016. p. 19-29.,22. Hasan S, Saeed S. Monkeypox disease: an emerging public health concern in the shadow of COVID-19 pandemic: an update. Trop Med Infect Dis. 2022; 7(10): 283. All poxviruses complete their replication cycle inside the infected cell’s cytoplasm. Due to the larger size of these viruses, makes it harder for viruses such as monkeypox to breach host defenses by passing through gap junctions. The larger size of the virus also makes it difficult for the virus to replicate rapidly and orthopoxviruses need a more comprehensive strategy to survive within the host. In the face of a hostile environment, poxviruses have developed an array of molecules that are encoded by virulence genes and designed to directly subvert the defenses mounted by the host.33. Kaler J, Hussain A, Flores G, Kheiri S, Desrosiers D. Monkeypox: a comprehensive review of transmission, pathogenesis, and manifestation. Cureus. 2022; 14(7): e26531.,44. Okyay RBE, Kaya E, Sahin A, Kocyigit B, Tasdogan A, Avci A, et al. Another epidemic in the shadow of Covid 19 pandemic: a review of monkeypox. EJMO. 2022; 6(2): 95-99.,55. Stanford MM, McFadden G, Karupiah G, Chaudhri G. Immunopathogenesis of poxvirus infections: forecasting the impending storm. Immunol Cell Biol. 2007; 85(2): 93-102.,66. Seet BT, Johnston JB, Brunetti CR, Barrett JW, Everett H, Cameron C, et al. Poxviruses and immune evasion. Annu Rev Immunol. 2003; 21: 377-423. Poxviruses have a very broad range of eukaryotic hosts, including mammals, birds, reptiles, and insects.77. Lefkowitz EJ, Wang C, Upton C. Poxviruses: past, present and future. Virus Res. 2006; 117(1): 105-18. The major mammals hosts of Poxviruses are rodents, rabbits, and non-human primates, which can occasionally be transmitted to humans, facilitating the occurrence of human-to-human transmission.33. Kaler J, Hussain A, Flores G, Kheiri S, Desrosiers D. Monkeypox: a comprehensive review of transmission, pathogenesis, and manifestation. Cureus. 2022; 14(7): e26531. Initially isolated in cynomolgus monkeys in Copenhagen in 1958, MPXV causes a human disease is currently named as Mpox (formerly monkeypox).88. Ulaeto D, Agafonov A, Burchfield J, Carter L, Happi C, Jakob R, et al. New nomenclature for mpox (monkeypox) and monkeypox virus clades. Lancet Infect Dis. 2023; 23(3): 273-5.,99. Oyebanji O, Ofonagoro U, Akande O, Nsofor I, Ukenedo C, Mohammed TB, et al. Lay media reporting of monkeypox in Nigeria. BMJ Glob Health. 2019; 4(6): e002019.

The first Mpox case in humans was reported in 1970 when the virus was isolated from a 9-month-old infant with suspected smallpox in a rural area of the Democratic Republic of Congo, following a potential zoonotic exposure.1010. Ladnyj ID, Ziegler P, Kima E. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull World Health Organ. 1972; 46(5): 593-7. Afterward, human-to-human Mpox transmission was identified, generally occurring through droplet exposure via large exhaled droplets or by direct contact with infected skin lesions or contagious materials, with an incubation period of 6 to 13 days (5 to 21 days).1111. Velavan TP, Meyer CG. Monkeypox 2022 outbreak: an update. Trop Med Int Health. 2022;27(7): 604-5. The disease has a similar clinical presentation as smallpox but is less severe, presenting as a maculopapular rash, frequently on the palms and soles.1212. Petersen E, Kantele A, Koopmans M, Asogun D, Yinka-Ogunleye A, Ihekweazu C, et al. Human Monkeypox: epidemiologic and clinical characteristics, diagnosis, and prevention. Infect Dis Clin North Am. 2019; 33(4): 1027-43. The most important distinguishing sign is lymphadenopathy with swollen lymph nodes, an early Mpox symptom.1313. Sah R, Siddiq A, Abdelaal A, Reda A, Lashin BI, Mohanty A, Alshahrani NZ, Rodriguez-Morales AJ. Public health emergency of international concern declared by the World Health Organization for Monkeypox. Global Security Health Science and Policy. 2022; 7(1): DOI:10.1080/23779497.2022.2124185.
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,1414. Alshahrani NZ, Alzahrani F, Alarifi AM, Algethami MR, Alhumam MN, Ayied HAM, et al. Assessment of knowledge of Monkeypox viral infection among the general population in Saudi Arabia. Pathogens. 2022; 11(8): 904.,1515. Alshahrani NZ, Algethami MR, Alarifi AM, Alzahrani F, Alshehri EA, Alshehri AM, et al. Knowledge and attitude regarding Monkeypox virus among physicians in Saudi Arabia: a cross-sectional study. Vaccines (Basel). 2022; 10(12): 2099.,1616. Alshahrani NZ, Assiri AM, Al-Tawfiq JA, Rodriguez-Morales AJ, Sah R. The human monkeypox in Saudi Arabia and global tourism. Ann Med Surg (Lond). 2022; 82: 104686. From the 1970s to 2021, Mpox have been considered endemic in selected African countries, with sporadic outbreaks identified in other continents, mainly associated with travellers returning from endemic regions.1717. Saied AA, Dhawan M, Metwally AA, Fahrni ML, Choudhary P, Choudhary OP. Disease history, pathogenesis, diagnostics, and therapeutics for human Monkeypox disease: a comprehensive review. Vaccines (Basel). 2022; 10(12): 2091.,1818. Gessain A, Nakoune E, Yazdanpanah Y. Monkeypox. N Engl J Med. 2022; 387(19): 1783-93.

On May 4, 2022, a patient with an unexplained rash and recent travel to Nigeria was admitted to an United Kingdom (UK) hospital. On May 6, 2022, MPXV was laboratory confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) testing of a vesicular swab conducted by the UK Health Security Agency (UKHSA) Rare and Imported Pathogens Laboratory.1919. WHO - World Health Organization. Disease outbreak news. Monkeypox - United Kingdom of Great Britain and Northern Ireland. 2022 [updated 2022 16 May 2022; cited 2022 03 February 2023]; Available from: https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON381.
https://www.who.int/emergencies/disease-...
Following this diagnosis, several other patients with no travel history to endemic countries were identified with Mpox in the UK and other countries from Europe and the Americas. Most of them were cisgender men, who presented with genital rash, genital oedema and proctitis.1818. Gessain A, Nakoune E, Yazdanpanah Y. Monkeypox. N Engl J Med. 2022; 387(19): 1783-93.,2020. Yinka-Ogunleye A, Aruna O, Dalhat M, Ogoina D, McCollum A, Disu Y, et al. Outbreak of human monkeypox in Nigeria in 2017-18: a clinical and epidemiological report. Lancet Infect Dis. 2019; 19(8): 872-9.,2121. Hobson G, Adamson J, Adler H, Firth R, Gould S, Houlihan C, et al. Family cluster of three cases of monkeypox imported from Nigeria to the United Kingdom, May 2021. Euro Surveill. 2021; 26(32): 2100745.,2222. Rao AK, Schulte J, Chen TH, Hughes CM, Davidson W, Neff JM, et al. Monkeypox in a traveller returning from Nigeria - Dallas, Texas, July 2021. MMWR Morb Mortal Wkly Rep. 2022; 71(14): 509-16. The situation quickly evolved into a multi-national outbreak, resulting in the declaration of Mpox as a public health emergency of international concern in July 2022.2323. WHO - World Health Organization. WHO director-general declares the ongoing monkeypox outbreak a public health emergency of international concern. 2022 [updated 2022 23 July 2022; cited 2022 13 February 2023]; Available from: https://www.who.int/europe/news/item/23-07-2022-who-director-general-declares-the-ongoing-monkeypox-outbreak-a-public-health-event-of-international-concern.
https://www.who.int/europe/news/item/23-...

Further phylogenetic analysis pointed to a new MPXV subclade involved in the 2022 Mpox multi-national outbreak and clade II was divided into subclades IIa and IIb, with the latter associated with current cases.2424. Mitjà O, Ogoina D, Titanji BK, Galvan C, Muyembe J-J, Marks M, et al. Monkeypox. Lancet. 2023; 401(10370): 60-74. Mutations and natural selection have increased the transmissibility of MPXV. Recently, it has been indicated that three positive amino acid substitutions (T/A426V in MPXVgp010, A423D in MPXVgp012, and S105L in MPXVgp191), which appeared in 2019 or 2022, would be crucial for the eventual virus adaptation to humans in the current outbreak.2525. Zhan XY, Zha GF, He Y. Evolutionary dissection of monkeypox virus: positive Darwinian selection drives the adaptation of virus-host interaction proteins. Front Cell Infect Microbiol. 2023; 12: 1083234. From May 2022 to July 21 2023, more than 88,540 cases and 152 deaths were reported, most of them (97.98% of cases and 86.84% of deaths) in locations that have not historically reported Mpox.2626. CDC - Centers for Disease Control and Prevention. 2022 Mpox Outbreak Global Map. 2022 [updated 17 July 2023; cited 21 July 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/response/2022/world-map.html.
https://www.cdc.gov/poxvirus/monkeypox/r...
The United States of America (USA) leads the ranking with more than 30,600 cases, followed by Brazil with more than 10,900 cases.2626. CDC - Centers for Disease Control and Prevention. 2022 Mpox Outbreak Global Map. 2022 [updated 17 July 2023; cited 21 July 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/response/2022/world-map.html.
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,2727. Tiecco G, Antoni MD, Storti S, Tomasoni LR, Castelli F, Quiros-Roldan E. Monkeypox, a literature review: what is new and where does this concerning virus come from? Viruses. 2022; 14(9): 1894. Since May 2022, the number of cases diagnosed outside Africa has surpassed the total number of cases detected outside the continent since 1970.2626. CDC - Centers for Disease Control and Prevention. 2022 Mpox Outbreak Global Map. 2022 [updated 17 July 2023; cited 21 July 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/response/2022/world-map.html.
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,2828. Kozlov M. Monkeypox goes global: why scientists are on alert. Nature. 2022; 606(7912): 15-6. The increasing number and rapid spread of the cases have raised concerns that MPXV may fill the ecological niche once occupied by the smallpox virus. The combined effects of deforestation, population growth, and destruction of natural reservoir habitats have recently heightened this concern.2929. Bunge EM, Hoet B, Chen L, Lienert F, Weidenthaler H, Baer LR, et al. The changing epidemiology of human monkeypox-A potential threat? A systematic review. PLoS Negl Trop Dis. 2022; 16(2): e0010141.,3030. Titanji BK, Tegomoh B, Nematollahi S, Konomos M, Kulkarni PA. Monkeypox: a contemporary review for healthcare professionals. Open Forum Infect Dis. 2022; 9(7): ofac310. On May 11 2023, WHO declares global Monkeypox emergence over, but the virus is still around and further waves and outbreaks could continue.3131. WHO - World Health Organization. Virtual press conference on COVID-19 and other global health issues transcript - 11 May 2023. 2023 [updated 2023 11 May 2023; cited 2023 15 May 2023]; Available from: https://www.who.int/publications/m/item/virtual-press-conference-on-covid-19-and-other-global-health-issues-transcript---11-may-2023.
https://www.who.int/publications/m/item/...

Multiple USA federal agencies, including the Administration for Strategic Preparedness and Response (ASPR), US Food and Drug Administration (FDA), National Institutes of Health (NIH), and the Centres for Disease Control and Prevention (CDC) are coordinating efforts to implement a vaccination strategy with JYNNEOS vaccine to prevent MPXV infection by demonstrating an immune response of persons considered to be at high risk for infection.3232. CDC - Centers for Disease Control and Prevention. Interim clinical considerations for use of JYNNEOS and ACAM2000 vaccines during the 2022 U.S. Mpox outbreak. 2022 [updated 2022 19 October 2022; cited 2023 03 February 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/health-departments/vaccine-considerations.html.
https://www.cdc.gov/poxvirus/monkeypox/h...
The JYNNEOS vaccine is licensed as a series of two doses administered with a 28 days (four weeks) interval.3333. CDC - Centers for Disease Control and Prevention. JYNNEOS vaccine. 2022 [updated 2022 22 December 2022; cited 2023 03 February 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/interim-considerations/jynneos-vaccine.html.
https://www.cdc.gov/poxvirus/monkeypox/i...
In addition to this, another vaccine (ACAM2000) is licensed as a single dose for immunisation against smallpox and made available for use against Mpox under an Expanded Access Investigational New Drug (EA-IND) protocol.3232. CDC - Centers for Disease Control and Prevention. Interim clinical considerations for use of JYNNEOS and ACAM2000 vaccines during the 2022 U.S. Mpox outbreak. 2022 [updated 2022 19 October 2022; cited 2023 03 February 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/health-departments/vaccine-considerations.html.
https://www.cdc.gov/poxvirus/monkeypox/h...
,3434. CDC - Centers for Disease Control and Prevention. ACAM2000 vaccine. 2022 [updated 2022 21 October 2022; cited 2023 03 February 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/interim-considerations/acam2000-vaccine.html.
https://www.cdc.gov/poxvirus/monkeypox/i...
Although no specific antiviral Mpox treatment is available, tecovirimat and brincidofovir have been indicated for treatment.3535. Adler H, Gould S, Hine P, Snell LB, Wong W, Houlihan CF, et al. Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect Dis. 2022; 22(8): 1153-62.,3636. Grosenbach DW, Honeychurch K, Rose EA, Chinsangaram J, Frimm A, Maiti B, et al. Oral tecovirimat for the treatment of smallpox. N Engl J Med. 2018; 379(1): 44-53.,3737. Chittick G, Morrison M, Brundage T, Nichols WG. Short-term clinical safety profile of brincidofovir: a favorable benefit-risk proposition in the treatment of smallpox. Antiviral Res. 2017; 143: 269-77.,3838. FDA - US Food and Drug Administration. FDA approves drug to treat smallpox. 2021 [updated 2021 06 April 2021; cited 2023 14 April 2023]; Available from: https://www.fda.gov/drugs/news-events-human-drugs/fda-approves-drug-treat-smallpox.
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However, these drugs were approved for the management of smallpox based on animal models.3535. Adler H, Gould S, Hine P, Snell LB, Wong W, Houlihan CF, et al. Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect Dis. 2022; 22(8): 1153-62.,3939. Rizk JG, Lippi G, Henry BM, Forthal DN, Rizk Y. Correction to: prevention and treatment of Monkeypox. Drugs. 2022; 82(12): 1343. Dose studies for these drugs have been conducted in humans, but the efficacy of these agents has not been thoroughly defined, and their use remains under monitoring.3535. Adler H, Gould S, Hine P, Snell LB, Wong W, Houlihan CF, et al. Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect Dis. 2022; 22(8): 1153-62.,4040. Sherwat A, Brooks JT, Birnkrant D, Kim P. Tecovirimat and the treatment of Monkeypox - past, present, and future considerations. N Engl J Med. 2022; 387(7): 579-81. For example, brincidofovir may cause an increase in serum transaminases and serum bilirubin.3535. Adler H, Gould S, Hine P, Snell LB, Wong W, Houlihan CF, et al. Clinical features and management of human monkeypox: a retrospective observational study in the UK. Lancet Infect Dis. 2022; 22(8): 1153-62. Therefore, the development of vaccines and antiviral molecules for Mpox treatment and prevention remains a pivotal strategy to control and combat the disease.

Viral isolation in a biologically safe place and the performance of in vitro studies are extremely important for a better understanding of virus-cell interaction and mechanisms of infection.4141. Rosa RB, de Castro EF, da Silva MV, Ferreira DCP, Jardim ACG, Santos IA, et al. In vitro and in vivo models for monkeypox. iScience. 2023; 26(1): 105702. This knowledge is strategic to antiviral targets identification for new molecules development and drugs repurposing. Herein, we describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, RT-PCR and transmission electron microscopy (TEM).

MATERIALS AND METHODS

Human sample and clinical description - In the present study, we used one sample from an observational cohort study that enrolled patients with suspected Mpox virus infection between June 12 and August 19, 2022, in Rio de Janeiro, Brazil.4242. Silva CCMST, Torres TS, Peixoto E, Ismério R, Lessa F, INI-Fiocruz Mpox Study Group, et al. Ambulatory and hospitalized patients with suspected and confirmed mpox: an observational cohort study from Brazil. Lancet Reg Health Am. 2022; 17: 100406. The selected patient was a cisgender man with no comorbidities who was hospitalised in July 2022 due to severe proctitis requiring intravenous analgesia. The clinical characterisation of this patient has been previously described.4242. Silva CCMST, Torres TS, Peixoto E, Ismério R, Lessa F, INI-Fiocruz Mpox Study Group, et al. Ambulatory and hospitalized patients with suspected and confirmed mpox: an observational cohort study from Brazil. Lancet Reg Health Am. 2022; 17: 100406. He presented with fever, adenomegaly, headache, asthenia, and disseminated mucocutaneous lesions initiated six days before the medical assessment. He had sexual contact with two confirmed Mpox cases, and none of them reported travel history, pointing to a community transmission. MPXV was detected in swabs collected from penile and buttock lesions an oropharyngeal swab. These samples were received by the National Reference Laboratory in Enteroviruses and Reference Laboratory Diagnosis in MPXV (Laboratório de Enterovírus, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil). The virus DNA was detected by a specific MPXV qPCR protocol in an AB 7500 Real-time PCR system (Applied Biosystems).4343. Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA. J Virol Methods. 2010; 169(1): 223-7. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Review Board at Instituto Nacional de Infectologia (INI)-Fiocruz CAAE # 61290422.0.0000.5262.

MPXV isolation - Virus isolation was performed in the sample from the oropharyngeal swab. For virus isolation, Cercopithecus aethiops monkey kidney cells (Vero, BCRJ code: 0245) in a 12-well plate (2.0 x 105 cells/well) were incubated with a 150 µL/well of an MPXV positive sample at two different dilutions (1:10 and 1:100) in Dulbecco’s Modified Eagle Medium (DMEM - high glucose with sodium pyruvate; Life Technologies, Grand Island, NY, USA) supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin (Sigma-Aldrich, Burlington, MA, USA), and maintained at 37ºC in 5% CO2 atmosphere. After a 1-h incubation period, 850 µL/well of a new medium supplemented with 2% Foetal Bovine Serum (FBS; Life Technologies, South American, Brazil) was added. Cell monolayers were inspected daily under photonic microscopy for development of cytopathic effect (CPE), until six days post-infection. Then, supernatants were harvested for virus titration and genome detection, and the monolayers were collected for ultrastructural analysis by TEM. The access to the genetic heritage was approved by the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen; approval number: A19C586). All procedures were performed at a biosafety level 3 laboratory (BSL3), according to WHO biosafety guidelines and Biosafety in Microbiological and Biomedical Laboratories 6th Edition (CDC/NIH).4444. CDC - Centers for Disease Control and Prevention. Biosafety in microbiological and biomedical laboratories. 6th ed. Bethesda: Public Health Service/Centers for Disease Control and Prevention/National Institutes of Health; 2020. Available from: https://www.cdc.gov/labs/pdf/SF__19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf.

Virus titration - Titration was performed using a 50% Tissue Culture Infectious Dose (TCID50) method. Monolayers of Vero cells (1,5 x 104 cells/well) were incubated with 50 µL of infected cell culture supernatants, formerly starting for virus isolation, in serial 10-fold dilutions (102 - 105) with 10 replicates. After 1 h at 37ºC in 5% CO2, 50 µL of medium (DMEM high glucose supplemented with sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin and 2% FBS) was added. After 72 h of incubation, the CPE was analysed by photonic microscopy and 100 µL of 4% formalin was added to fix the cells. After 3 h, this solution was harvested, and the cell monolayers were stained with 0.04% solution of crystal violet in 20% ethanol for 1 h, as described before.4545. Sacramento CQ, Fintelman-Rodrigues N, Dias SSG, Temerozo JR, Da Silva APD, da Silva CS, et al. Unlike chloroquine, mefloquine inhibits SARS-CoV-2 infection in physiologically relevant cells. Viruses. 2022; 14(2): 374.,4646. Frias-De-Diego A, Crisci E. Use of crystal violet to improve visual sytopathic effect-based reading for viral titration using TCID50 assays. J Vis Exp. 2022; 12(180). doi: 10.3791/63063. Then, the wells containing virus plaques units were counted and virus titers determined by TCID50/mL, according to the Reed and Muench method.4747. Reed J, Mounch M. An endpoint assay to evaluate the 50% tissue sytopatic effect. J Gen Virol. 1938; 5: 25-9.

Virus genome detection - After MPXV isolation, the virus genome was detected to confirm viral growth in cell culture. Total viral DNA was extracted using ReliaPrep Viral TNA Miniprep (Promega®), according to the manufacturer’s instructions. An MPXV generic real-time PCR (qPCR) test was performed according to the Centre for Disease Control and Prevention (CDC) protocol, using primers and probes already described.4343. Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. Real-time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA. J Virol Methods. 2010; 169(1): 223-7.,4848. CDC - Centers for Disease Control and Prevention. Test procedure: monkeypox virus generic real-time PCR test 2022 [updated 2022 06 June 2022; cited 2023 03 February 2023]; Available from: https://www.cdc.gov/poxvirus/monkeypox/pdf/pcr-diagnostic-protocol-508.pdf.
https://www.cdc.gov/poxvirus/monkeypox/p...
The reactions were performed using a GoTaq® Probe qPCR RT-qPCR System (Promega, Madison, WS, USA) in a QIAquant 96 5plex Real-Time PCR System (Qiagen®).

Ultrastructural analysis - MPXV infected and non-infected Vero cell monolayers (control/mock) were incubated with 200 µL of trypsin-EDTA 0.25% (Life Technologies, Grand Island, NY, USA) per well and collected after six days post-infection. Then, FBS (200 µL/well) was added, and cells were fixated in 2.5% glutaraldehyde in sodium cacodylate buffer (0.2 M, pH 7.2), post-fixated in 1% osmium tetroxide in water, dehydrated in acetone, embedded in epoxy resin, and polymerised at 60ºC for three days.4949. Vieira DFB, Barth-Schatzmayr OM, Schatzmayr HG. Modelo animal experimental para o estudo da patogênese dos vírus dengue sorotipos 1 e 2. 1st ed. Rio de Janeiro: Fiocruz/FAPERJ/Interciência; 2010. 82 pp.,5050. Barreto-Vieira DF, da Silva MAN, Garcia CC, Miranda MD, Matos AR, Caetano BC, et al. Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells. Mem Inst Oswaldo Cruz. 2021; 116: e200443.,5151. Barth OM, Silva MAN, Barreto-Vieira DF. Low impact to fixed cell processing aiming transmission electron microscopy. Mem Inst Oswaldo Cruz. 2016; 111(6): 411-3. Ultrathin sections (50-70 nm) were obtained from the resin blocks. The sections were picked up using copper grids (uncoated 300 mesh grids) and observed using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope. No staining was performed.

RESULTS AND DISCUSSION

The first MPXV isolation and identification was performed in 1959 when monkeys were shipped from Singapore to Denmark. However, only in the 1970’s the first human infection case was confirmed in a child from the Democratic Republic of Congo, suspected to have smallpox.5252. Moore MJ, Rathish B, Zahra F. Mpox (Monkeypox). Treasure Island: StatPearls; 2023.,5353. Breman JG, Kalisa R, Steniowski MV, Zanotto E, Gromyko AI, Arita I. Human monkeypox, 1970-79. Bull World Health Organ. 1980; 58(2): 165-82. Herein, we analysed the MPXV isolation and growth starting from a positive sample from the Brazilian outbreak.

Viral replication in the MPXV-infected Vero cell culture was monitored daily under optical microscopy for six days. Vero cells without virus infection were used as control (Fig. 1A). After three days post-inoculation, the onset of CPE was observed in the cell monolayer infected with a 1:10 sample dilution (Fig. 1B). The CPE observed was typical as of other poxviruses, characterised by rounding of cells, plaque formation, and detachment of cells from the plastic well.5454. Ali HMS, Nimir AH, Khalafalla AI. Growth characteristic of Camel pox and vaccinia viruses in embryonated eggs and cell culture. Trop Anim Health Prod. 2009; 41(3): 393-6.,5555. Mangana-Vougiouka O, Markoulatos P, Koptopoulos G, Nomikou K, Bakandritsos N, Papadopoulos P. Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel immunoprecipitation assay. Mol Cell Probes. 2000; 14(5): 305-10.,5656. Mosadeghhesari M, Oryan A, Zibaee S, Varshovi HR. Molecular investigation and cultivation of camelpox virus in Iran. Arch Virol. 2014; 159(11): 3005-11.,5757. Samour JH, Kaaden OR, Wernery U, Bailey TA. An epornitic of avian pox in houbara bustards (Chlamydotis undulata macqueenii). Zentralbl Veterinarmed B. 1996; 43(5): 287-92. Viral growth was maintained for another three days, when about 80% of the cell monolayer was disrupted (Fig. 1C-D). In agreement with our results, previous studies have shown MPXV isolation from clinical samples in monkey kidney cell lines (Vero E6, RMK, BGM, MA-104, LLCMK-2, OMK and BSC-40) with the onset of CPE occurring two- and three-days post infection.5858. Hutin YJ, Williams RJ, Malfait P, Pebody R, Loparev VN, Ropp SL, et al. Outbreak of human monkeypox, Democratic Republic of Congo, 1996 to 1997. Emerg Infect Dis. 2001; 7(3): 434-8.,5959. Hughes CM, Liu L, Davidson WB, Radford KW, Wilkins K, Monroe B, et al. A tale of two viruses: coinfections of monkeypox and varicella zoster virus in the Democratic Republic of Congo. Am J Trop Med Hyg. 2020; 104(2): 604-11.,6060. Noe S, Zange S, Seilmaier M, Antwerpen MH, Fenzl T, Schneider J, et al. Clinical and virological features of first human monkeypox cases in Germany. Infection. 2023; 51(1): 265-70.,6161. Ma A, Langer J, Hanson KE, Bradley BT. Characterization of the cytopathic effects of monkeypox virus isolated from clinical specimens and differentiation from common viral exanthems. J Clin Microbiol. 2022; 60(12): e0133622. Furthermore, human lung cell line types (A549 and MRC-5), also used for MPXV isolation, showed the emergence of CPE in the same period, regardless of the cell type.5858. Hutin YJ, Williams RJ, Malfait P, Pebody R, Loparev VN, Ropp SL, et al. Outbreak of human monkeypox, Democratic Republic of Congo, 1996 to 1997. Emerg Infect Dis. 2001; 7(3): 434-8.,5959. Hughes CM, Liu L, Davidson WB, Radford KW, Wilkins K, Monroe B, et al. A tale of two viruses: coinfections of monkeypox and varicella zoster virus in the Democratic Republic of Congo. Am J Trop Med Hyg. 2020; 104(2): 604-11.,6060. Noe S, Zange S, Seilmaier M, Antwerpen MH, Fenzl T, Schneider J, et al. Clinical and virological features of first human monkeypox cases in Germany. Infection. 2023; 51(1): 265-70.,6161. Ma A, Langer J, Hanson KE, Bradley BT. Characterization of the cytopathic effects of monkeypox virus isolated from clinical specimens and differentiation from common viral exanthems. J Clin Microbiol. 2022; 60(12): e0133622.

Fig. 1:
Monkeypox (Mpox) cytophatic effect (CPE) at three- and six-days post-infection. Images of Vero cells without virus inoculation (A) and inoculated with a 1:10 diluted positive sample were obtained at three days post-infection at 100x (B), and six days post-infection at 100x (C); and 200x (D). Images were obtained through an inverted microscope, EVOS XL Core Imaging System (AMEX1200, Thermo Fisher Scientific).

The supernatant of the Vero cell monolayers was harvested after six days of infection. The viral growth obtained from the 1:10 dilution was titrated by TCID50/mL and the viral genome detected by qPCR. The virus title was 104.7 TCID50/mL with a cycle threshold (Ct) equal to 30. The cell monolayers infected with the two dilutions (1:10 and 1:100) were pooled for ultrastructural analysis.

Ultrastructural analysis of Vero cells on the sixth-day post-infection revealed cellular changes associated with MPXV infection, including electron-dense ribosomes, mitochondrial swelling and vacuolation, numerous vesicles, pyknotic nuclei, and nuclei presenting altered chromatin profile (Fig. 2B-D). Such changes were not observed in uninfected cells (control) (Fig. 2A). Virus particles were observed in the cytosol with a rectangular morphology and presenting lateral bodies and a central core (Fig. 3A-D) as has been observed by Bayer-Garner in ultrastructural studies by transmission electron microscopy with cutaneous biopsy specimens from patients.6262. Bayer-Garner IB. Monkeypox virus: histologic, immunohistochemical and electron-microscopic findings. J Cutan Pathol. 2005; 32(1): 28-34.,6363. Rodríguez-Cuadrado FJ, Nájera L, Suárez D, Silvestre G, García-Fresnadillo D, Roustan G, et al. Clinical, histopathologic, immunohistochemical, and electron microscopic findings in cutaneous monkeypox: a multicenter retrospective case series in Spain. J Am Acad Dermatol. 2023; 88(4): 856-63. In agreement with previous reports the virion size and morphologic characteristics observed were similar to other virus particles of the Poxviridae family (200 by 300 nanometres).6464. Kim JW, Lee M, Shin H, Choi CH, Choi MM, Yi H, et al. Isolation and identification of monkeypox virus MPXV-ROK-P1-2022 from the first case in the Republic of Korea. Osong Public Health Res Perspect. 2022; 13(4): 308-11.,6565. Reed KD, Melski JW, Graham MB, Regnery RL, Sotir MJ, Wegner MV, et al. The detection of monkeypox in humans in the Western Hemisphere. N Engl J Med. 2004; 350(4): 342-50.,6666. Muller M, Ingold-Heppner B, Stocker H, Heppner FL, Dittmayer C, Laue M. Electron microscopy images of monkeypox virus infection in 24-year-old man. Lancet. 2022; 400(10363): 1618.,6767. Witt ASA, Trindade GS, Souza FG, Serafim MSM, da Costa AVB, Silva MVF, et al. Ultrastructural analysis of monkeypox virus replication in Vero cells. J Med Virol. 2023; 95(2): e28536.

Fig. 2:
Monkeypox virus (MPXV) particles inside Vero cells six days post-infection. MPXV infected cell (A) with MPXV particles (white arrow) in cytosol (B-C), microscope images show pyknotic nuclei (*) (C), nuclei presenting an altered chromatin profile (N) and mitochondria alteration (M) (A, B, D), and numerous vesicles (V) (C-D). Images were obtained using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope (TEM).

Fig. 3:
Vero cells six days post-infection (A-D) with Monkeypox virus (MPXV) particles (arrow) inside the cytoplasm (C). Virus particles presenting 391 rectangular shapes with lateral bodies and a central core. Virus particles measuring between 250 and 300 nanometers were observed. 392 nucleus (N). images were obtained using a Hitachi HT 7800 (Hitachi, Tokyo, Japan) transmission electron microscope (TEM).

While nucleic acid amplification testes (NAATs) have largely replaced the practice of viral culture, knowing viral growth in cell models remains an important alternative to the differential diagnosis of poxvirus, even with many advances in molecular biology, still presents specific and limited targets. Thus, a wide range of pathogens not covered by common NAATs can be detected through viral culture methods.6161. Ma A, Langer J, Hanson KE, Bradley BT. Characterization of the cytopathic effects of monkeypox virus isolated from clinical specimens and differentiation from common viral exanthems. J Clin Microbiol. 2022; 60(12): e0133622.,6868. Martin RM, Burke K, Verma D, Xie H, Langer J, Schlaberg R, et al. Contact transmission of vaccinia to an infant diagnosed by viral culture and metagenomic sequencing. Open Forum Infect Dis. 2020; 7(4): ofaa111. Furthermore, the viral variability or genetic mutations that may contribute to false NAATs negative results are less impacted by culture growth.6969. Ogilvie M. Molecular techniques should not now replace cell culture in diagnostic virology laboratories. Rev Med Virol. 2001; 11(6): 351-4. For different viruses infections, in situations of prolonged viral shedding, culture assays are important to determining virus infectivity and phenotypic antiviral susceptibility.7070. Souza TM, Salluh JI, Bozza FA, Mesquita M, Soares M, Motta FC, et al. H1N1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis. PLoS One. 2010; 5(11): e14158.,7171. AlGhounaim M, Xiao Y, Caya C, Papenburg J. Diagnostic yield and clinical impact of routine cell culture for respiratory viruses among children with a negative multiplex RT-PCR result. J Clin Virol. 2017; 94: 107-9.,7272. Mileto D, Foschi A, Mancon A, Merli S, Staurenghi F, Pezzati L, et al. A case of extremely prolonged viral shedding: could cell cultures be a diagnostic tool to drive COVID-19 patient discharge? Int J Infect Dis. 2021; 104: 631-3. In addition, understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties. Beside this, the use of in vitro models diminishes the use of animal models.4141. Rosa RB, de Castro EF, da Silva MV, Ferreira DCP, Jardim ACG, Santos IA, et al. In vitro and in vivo models for monkeypox. iScience. 2023; 26(1): 105702. Moreover, the appearance of MPXV CPEs has been poorly characterised from the standpoint of the practicing clinical microbiologist.6161. Ma A, Langer J, Hanson KE, Bradley BT. Characterization of the cytopathic effects of monkeypox virus isolated from clinical specimens and differentiation from common viral exanthems. J Clin Microbiol. 2022; 60(12): e0133622. Thus, the evaluation of MPXV viral growth, starting from clinical samples, and the study of cellular structures modulation by viral infection is relevant.

ACKNOWLEDGEMENTS

To the Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz (INI-Fiocruz) for the collection of clinical sample; Instituto Oswaldo Cruz-Fiocruz (IOC) for providing us with its technology and facilities during the course of the research; to Arthur da Costa Rasinhas; the Plataforma de Microscopia Eletrônica Rudolf Barth/HPP (IOC-Fiocruz); the BSL3 Platform (Pavilhão Leônidas Deane, IOC, Fiocruz); and the CENABIO (Federal University of Rio de Janeiro).

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Publication Dates

  • Publication in this collection
    28 Aug 2023
  • Date of issue
    2023

History

  • Received
    22 May 2023
  • Accepted
    17 July 2023
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