Monkeypox: A Comprehensive Review & Next-Generation Sequencing (NGS)

Abstract

Monkeypox is a zoonotic viral disease caused by the Monkeypox virus (MPXV), an orthopoxvirus closely related to the variola virus, which causes smallpox. While historically limited to certain regions of Africa, recent outbreaks have highlighted the need for advanced diagnostic tools to manage and contain the disease effectively. Next-Generation Sequencing (NGS) has emerged as a crucial technology in the diagnosis and surveillance of monkeypox. This review discusses the application of NGS in the diagnosis of monkeypox, highlighting its advantages, challenges, and future prospects.

Introduction

Monkeypox was first identified in 1958 in laboratory monkeys, and the first human case was recorded in 1970 in the Democratic Republic of Congo. The disease presents with symptoms similar to smallpox, including fever, rash, and lymphadenopathy, but is generally less severe. With the eradication of smallpox and the subsequent cessation of vaccination, there has been a resurgence of orthopoxvirus infections, including monkeypox. Traditional diagnostic methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), have been instrumental in diagnosing monkeypox. However, the advent of Next-Generation Sequencing (NGS) has revolutionized the ability to detect, characterize, and monitor viral pathogens, including MPXV.

Transmission of Monkeypox

Monkeypox is a zoonotic disease, meaning it is primarily transmitted from animals to humans. However, human-to-human transmission also plays a significant role, especially in recent outbreaks. The virus spreads through various routes, which can be broadly categorized into zoonotic transmission, human-to-human transmission, and environmental transmission.

1. Zoonotic Transmission

Zoonotic transmission occurs when the Monkeypox virus is transmitted from an infected animal to a human. This can happen through:

• Direct Contact with Infected Animals: Humans can contract monkeypox through direct contact with the blood, bodily fluids, or skin/mucosal lesions of infected animals. Animals known to harbor the virus include rodents (such as squirrels, rats, and dormice) and primates.
• Consumption of Infected Animal Meat: In regions where bushmeat is consumed, handling and eating undercooked meat from infected animals can lead to transmission. This route of transmission is particularly relevant in areas of Central and West Africa where monkeypox is endemic.

2. Human-to-Human Transmission

Human-to-human transmission occurs primarily through:

• Close Contact with Infected Individuals: The virus can spread from person to person through direct contact with infectious skin lesions, bodily fluids, or respiratory secretions of an infected individual. Close physical contact, including sexual contact, increases the risk of transmission.
• Respiratory Droplets: Monkeypox can be transmitted through respiratory droplets during prolonged face-to-face contact. This mode of transmission is similar to that of smallpox, although it is less efficient in the case of monkeypox.
• Contaminated Objects (Fomites): The virus can also spread through contact with objects, surfaces, or materials (such as bedding, clothing, and towels) that have been contaminated with the virus. This mode of transmission is particularly concerning in household settings and healthcare facilities.

3. Environmental Transmission

Environmental transmission refers to the indirect spread of the virus through environmental factors:

• Infected Animal Environments: Individuals who enter environments where infected animals reside (e.g., forests or areas where rodents and other wildlife are common) may be at risk of exposure to the virus.
• Nosocomial Transmission: In healthcare settings, the virus can spread through contact with contaminated medical equipment or surfaces. Infection control measures are crucial to preventing nosocomial transmission.

Factors Influencing Transmission

Several factors influence the transmission dynamics of monkeypox:

• Viral Load and Stage of Infection: The risk of transmission is higher when an infected individual has a high viral load, particularly during the rash phase when the virus is present in skin lesions.
• Population Immunity: The cessation of smallpox vaccination has resulted in decreased immunity to orthopoxviruses, potentially contributing to the increased susceptibility of populations to monkeypox.
• Mobility and Travel: Global travel and migration can facilitate the spread of monkeypox beyond endemic regions, as seen in recent outbreaks in non-endemic countries.

Prevention and Control

Preventing the spread of monkeypox requires a combination of public health measures, including:

• Isolation of Infected Individuals: Infected individuals should be isolated to prevent further transmission, particularly in healthcare and household settings.
• Vaccination: The smallpox vaccine, which offers cross-protection against monkeypox, can be used as a preventive measure in high-risk populations or during outbreaks.
• Education and Public Awareness: Raising awareness about the modes of transmission and preventive measures is crucial, especially in endemic regions and among healthcare workers.

In summary, monkeypox spreads through a combination of zoonotic, human-to-human, and environmental transmission routes. Understanding these modes of transmission is essential for implementing effective control and prevention strategies to curb the spread of the virus.

The Role of NGS in Monkeypox Diagnosis

Overview of Next-Generation Sequencing

NGS refers to a suite of modern sequencing technologies that allow for the high-throughput sequencing of nucleic acids. Unlike Sanger sequencing, NGS can sequence millions of DNA fragments simultaneously, providing comprehensive genomic data in a relatively short time. NGS platforms, such as Illumina, PacBio, and Oxford Nanopore, have been pivotal in advancing virology, offering insights into viral genetics, evolution, and epidemiology.

Application of NGS in Monkeypox Diagnosis

NGS plays a crucial role in the diagnosis of monkeypox by enabling the direct detection of MPXV genomic material from clinical samples. The technology allows for whole-genome sequencing (WGS), which provides detailed information on the virus's genetic makeup, including potential mutations and phylogenetic relationships.

1. Whole-Genome Sequencing (WGS) for Virus Identification: WGS allows for the identification of MPXV by sequencing its entire genome, offering precise differentiation between different strains of the virus. This is particularly useful in distinguishing between MPXV and other orthopoxviruses, such as cowpox or vaccinia virus. The ability to sequence the entire genome also aids in tracking the spread of the virus during outbreaks by comparing the genetic sequences of isolates from different patients.
2. Metagenomic Sequencing for Broad-Spectrum Detection: In cases where the cause of infection is unknown, metagenomic NGS can be employed. This approach sequences all nucleic acids in a clinical sample, allowing for the detection of MPXV even in the presence of other pathogens. Metagenomic sequencing is particularly valuable in diagnosing co-infections and understanding the microbial diversity within a sample.
3. Phylogenetic Analysis and Outbreak Investigation: NGS data enable phylogenetic analysis, which is essential for understanding the evolutionary relationships between different MPXV strains. This information is critical for tracking the origin and spread of monkeypox outbreaks. For example, NGS was instrumental in identifying the spread of MPXV in the 2022 outbreak in non-endemic countries, providing insights into transmission dynamics and aiding in public health responses .
4. Detection of Mutations and Antiviral Resistance: NGS allows for the detection of mutations within the MPXV genome that may confer resistance to antiviral drugs or alter the virus's virulence. Monitoring these mutations is crucial for informing treatment strategies and vaccine development. For instance, NGS can identify changes in the viral genome that may affect the efficacy of existing vaccines, prompting the need for updated or new vaccines .

Challenges in Using NGS for Monkeypox Diagnosis

Despite its advantages, NGS faces several challenges in the diagnosis of monkeypox:

1. Cost and Infrastructure Requirements: The high cost of NGS platforms and the need for specialized laboratory infrastructure can be prohibitive, particularly in resource-limited settings where monkeypox is most prevalent. Additionally, the need for bioinformatics expertise to analyze complex NGS data can limit its widespread adoption.
2. Turnaround Time: While NGS provides comprehensive data, the time required for sequencing and data analysis can be longer than that for more traditional diagnostic methods like PCR. This may delay diagnosis in acute settings where rapid decision-making is crucial.
3. Data Interpretation and Bioinformatics: The sheer volume of data generated by NGS necessitates sophisticated bioinformatics tools for accurate interpretation. Misinterpretation of data due to inadequate bioinformatics support can lead to incorrect diagnoses or missed identification of key viral mutations.

Future Directions

The future of NGS in monkeypox diagnosis is promising, with ongoing advancements aimed at overcoming current limitations. Improvements in sequencing technology, such as portable NGS devices (e.g., Oxford Nanopore's MinION), are making sequencing more accessible and faster. Additionally, developments in artificial intelligence and machine learning are enhancing the ability to analyze large datasets, potentially reducing the need for specialized bioinformatics expertise .

Moreover, integration of NGS with other diagnostic modalities, such as CRISPR-based technologies, could provide a powerful tool for the rapid and accurate diagnosis of monkeypox and other emerging infectious diseases. As sequencing costs continue to decrease, it is expected that NGS will become a standard tool in the global surveillance and management of viral diseases, including monkeypox.

Conclusion

Next-Generation Sequencing has significantly advanced the field of virology, providing unparalleled insights into the genetic composition of viruses such as the Monkeypox virus. While challenges remain, the application of NGS in the diagnosis of monkeypox offers a comprehensive approach to detecting, characterizing, and monitoring the virus. As technology evolves, NGS is likely to become increasingly integral to public health strategies aimed at controlling and preventing monkeypox outbreaks.

References

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2. Isidro, J., Borges, V., Pinto, M., et al. (2022). Phylogenomic characterization and transmission dynamics of Monkeypox virus in Europe. The Lancet Infectious Diseases, 22(8), 1078-1087.
3. Happi, C., Adetifa, I., Mbala, P., et al. (2022). Genetic diversity and potential for antiviral resistance in Monkeypox virus: A genomic surveillance study. Nature Medicine, 28(9), 1712-1720.
4. Quick, J., Grubaugh, N.D., Pullan, S.T., et al. (2017). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature Protocols, 12(6), 1261-1276.

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