Human Papillomavirus (HPV)

What is the HPV virus?

Human papilloma virus (HPV) is a prominent etiological agent of cutaneous and mucosal epithelial disorders, including genital warts and cervical cancer. This viral pathogen is widely distributed and can be transmitted via unprotected sexual intercourse with an infected individual, affecting both genders. Recent evidence has highlighted cervical cancer as the most prevalent neoplasm among women, serving as a leading cause of mortality in young females.

The incidence of this malignancy varies across different regions of the world, with a relatively higher burden observed in Western societies. Conversely, developing nations often confront a higher burden of cervical cancer, representing up to a quarter of all malignancies affecting women. The World Health Organization (WHO) has reported a concerning estimate of approximately 500,000 new cases of HPV infection worldwide annually, with a staggering 250,000 resulting in fatality. Moreover, recent investigations have indicated a greater vulnerability of women to this infection, with twice the susceptibility compared to their male counterparts.

HPV virus types

Until now, more than 120 strains of Human Papillomavirus (HPV) have been identified and their genetic sequences have been elucidated. From a phylogenetic standpoint, this viral pathogen can be categorized into two distinct groups, namely α-HPV and β-HPV, which are respectively linked to mucosal and cutaneous infections. Human papillomavirus types are classified into two groups based on the degree of risk they pose: low risk and high risk. The former is associated with the development of benign warts, whereas the latter is implicated in the formation of malignant tumors such as cervical, anal and head and neck cancers. Among the high-risk strains, we find strains 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Notably, strains 16 and 18 are responsible for many malignancies attributed to this virus. On the other hand, the low-risk HPV strains, which include strains 6, 11, 42, 43, and 44, play a role in the development of squamous intraepithelial lesions (SILs).

Symptoms of HPV infection

In the majority of cases, HPV infection does not manifest with any discernible symptoms and is eventually cleared by the immune system. However, in certain individuals, it can give rise to various clinical manifestations. Some of these include:

Feeling of burning or itching in the genital area
Appearance of genital warts or other types of warts in other parts of the body

In general, Warts represent the most prevalent symptom of HPV infection and can appear weeks, months or even years after initial infection. These growths typically resemble cauliflower-like masses that emerge on the skin, although other variants with a flat appearance may also be observed. In cases where high-risk HPV strains are involved, symptoms do not become apparent until they progress to the point of causing cancer. Cervical cancer, head and neck cancer, anal cancer and laryngeal cancer are among the malignancies associated with HPV.

HPV virus structure

1) Components and Physical Characteristics of Viruses

In general, the HPV virus is characterized as a small (approximately 55 nm) and non-enveloped viral entity with a circular double-stranded DNA genome spanning around 8 kb in length. The genetic material of this virus is intimately associated with cellular histones and resides within a proteinaceous capsid composed of 72 pentamer capsomers. This capsid encompasses two structural proteins, namely L1 (55 kDa, representing 80% of cellular proteins) and L2 (70 kDa), both of which are encoded by the viral genome. The construction of virus-like particles (VLPs) can be achieved by the expression of L1 alone or in conjunction with L2 in either mammalian or non-mammalian expression systems.

2) Genome, Proteins and Cell Cycle of the HPV Virus

The genome of the HPV virus consists of approximately 8 open reading frames (ORFs), all of which are transcribed from the same DNA strand. These ORFs can be subdivided into three distinct functional regions, namely:

Region E: encompassing a variety of genes responsible for early viral functions. Among these genes,

E1/E2 encode proteins involved in the regulation of E6 and E7 gene functions, serving as factors that identify the initiation site of transcription.

The E4 gene encodes a protein with a relatively unexplored function, likely implicated in viral dissemination from infected cells.

Hydrophobic proteins, encoded by the E5 gene, play a role in the immortalization of cells.

The E6 gene encodes proteins that hinder the activity of negative cell cycle regulators, while concurrently inhibiting the P53 gene, which is integral to apoptosis induction.

The E7 gene encodes proteins that interact with products of the Rb tumor suppressor gene, ultimately leading to its inactivation.

Region L: encompassing the L1/L2 genes responsible for late viral functions, contributing to the comprehensive assembly of viral components.
LCR region: t is a long control region (Long Control Region) located between E and L regions and does not code any protein.

Viral E proteins are transcribed from the early promoter, such as P97 in the case of HPV 31, while L proteins are primarily transcribed from the late promoter, P742 in the case of HPV 31. Human papilloma viruses have a strong affinity for epithelial cells and have a profound impact on the epithelial component of the skin in various regions such as the oral cavity and genital area. The life cycle of this virus is closely intertwined with the differentiation process of infected epithelial cells. The stability of the HPV virus genome in basal cells is dependent on several genes, including E1, E2, E6 and E7. Under normal circumstances, when basal cells divide, the daughter cells detach from the basement membrane and enter the suprabasal compartment, exiting the cell cycle and initiating the final differentiation program.

However, in keratinocytes and cervical epithelial cells infected with HPV, the suprabasal cells are unable to exit the cell cycle and instead continue to replicate DNA. Moreover, the E5 oncoprotein significantly contributes to this characteristic in both HPV16 and HPV31 strains. Within the suprabasal compartment, the cells undergo amplification of viral genome replication and expression of capsid genes. E6 and E7 proteins are the two main proteins involved in the carcinogenesis pathway of the HPV virus. These proteins lead to the inactivation and disruption of the host cell cycle by binding to tumor suppressor genes. Currently, the presence of this virus can be detected using various methods, including Real-Time PCR and the utilization of fluorescent dyes.

HPV related diseases

Today, there exists knowledge regarding the association between the HPV virus and a wide array of diseases, encompassing genital and oral warts, cervical, anal and head and neck cancer, all of which, in the majority of instances, exhibit a benign nature. It is evident that individuals from diverse backgrounds are impacted by this virus. Particularly, low-risk strains such as types 6 and 11, induce the occurrence of genital warts in patients which manifest in various regions such as the vagina, cervix, groin, anus, penis, lips and mouth. These warts manifest as hypertrophied areas of skin filled with keratin. Within a span of a few years, most low-risk strains are effectively eliminated by the body’s immune system. The subsequent table enumerates several diseases and their respective HPV viruses with which they are associated.

Most frequently associated HPV types	Disease
HPV 2, 4, 7; occasionally other types in immunosuppressed (e.g. HPV 75–77)		Common warts
HPV 3, 10, occasionally HPV 26–29 and 41		Flat plane warts
HPV 1, 2, 4		Plantar warts
HPV 3, 10	Plane warts	Epidermodysplasia verruciformis
HPV 5, 8; less commonly 9,12,14,15, 17, 19,20, 21–25, 36–39, 47,49	Pityriasis-like plaques
HPV 5, 8, less commonly 14,17,20 and 47	Squamous cell carcinomas of sun exposed skin
HPV 6, 11, 40, 42, 43, 44, 54, 61, 72, 81, 89	External warts	Anogenital warts
HPV 6	Buschke–Lowenstein tumour
HPV 16, 55	Bowenoid papulosis
HPV 16,18, 31, 33, 45, 51, 52	Group 1: Carcinogenic to humans	Anogenital cancers and precancers
HPV 68	Group 2A: Probably carcinogenic to humans
HPV 26, 53, 64, 65, 66, 67, 69, 70, 73, 82	Group 2B: Possibly carcinogenic to humans
HPV 2,6,7,11,16,18,32,57	Oral papillomas	Oral lesions
HPV 6,11	Laryngeal papillomas
HPV 13, 32	Focal hyperplasia (Heck’s disease)
HPV 16 predominantly,18	Oropharyngeal carcinoma

Cervical cancer ranks as the second most prevalent form of cancer in women in the United States, following skin cancer and breast cancer. However, in developing nations, cervical cancer is the most common cancer in women accounting for approximately 25% of all cancer cases. Almost all instances of cervical cancer are caused by high-risk strains of the HPV virus. According to statistics from the World Health Organization (WHO), the optimal methods for preventing this disease are screening and vaccination. Cervical cancer typically originates at the junction of squamous cells in the ectocervix and columnar and glandular cells in the endocervical canal. When an individual becomes infected with HPV, metaplastic changes occur in this area, thereby increasing the risk of cervical cancer in the future.

Approximately 80% to 90% of cervical cancers are classified as squamous cell carcinomas (SCC), while 10-20% of cases are adenocarcinomas, a percentage that has risen in recent years. In certain instances, both types of cells undergo cancerous changes, known as Aden squamous or mixed carcinomas. In the early stages, SCC may not exhibit any specific symptoms, but as the disease progresses, abnormal vaginal secretions, pelvic pain, bleeding, and pain during intercourse may occur. According to statistics, HPV 16 is responsible for 50% of cervical cancer cases, while HPV 16 and HPV 18 together account for over 70% of cases. Prior to confirming the presence of malignancy in the cervical region, a type of neoplasia known as Cervical intraepithelial neoplasia (CIN) is observed. CIN is a precancerous condition in which abnormal cells develop on the surface of the cervix. Cervical dysplasia is categorized into three distinct groups based on the likelihood of these cells becoming cancerous. These groups are as follows:

CIN 1: In this group, the epithelial layer is affected by abnormal cells to the extent of one third. In such instances, the likelihood of these abnormal cells progressing to cancer is infrequent, and in certain cases, they will be eradicated.
CIN 2: During this phase, the epithelial layer is influenced by abnormal cells ranging from one-third to two-thirds.
CIN 3: At this stage, abnormal cells have affected more than two-thirds of the epithelial layer. In both CIN 2 and CIN 3 stages, individuals will necessitate monitoring of the disease and reception of appropriate therapeutic interventions to preclude cancer.

Methods of detecting HPV infection

Today, diverse techniques facilitate the identification of HPV virus in individuals, which will be elucidated below:

1) Pap smear

One of the prevailing approaches for prompt identification of cervical cancer is to perform a Pap smear or Pap test. This technique involves acquiring samples from the uterus and cervix employing a brush. It is recommended that married women above the age of 21 undergo this examination once every three years.

2) Colposcopy and biopsy:

Colposcopy serve as one of the conventional methodologies for examining uterine cells through employment of a specialized microscope termed as a colposcope. This examination is commonly advised for individuals with abnormal Pap smear outcomes. During this examination, if the physician observes abnormal cells, he or she will procure a specimen or biopsy from them. In the subsequent step, the biopsy is transferred to the laboratory for more comprehensive evaluations.

3) HPV molecular test

Today, for molecular detection of HPV a variety of methodologies are employed, among which Real-Time PCR stands out. This particular approach is regarded as one of the most precise and dependable techniques within the realm of diagnosis, as it facilitates the identification of distinct genotypes of the virus subsequent to DNA extraction from a pap smear sample or any genital tissue. The advantages of this method encompass the ability to ascertain the viral load, the requirement for minute quantities of the initial sample and the determination of the particular strain of HPV present in an individual.

Individuals who undergo a pap smear test in conjunction with an HPV test (co-testing) are advised to repeat this examination every five years so as to promptly diagnose and treat any potential infection. Real-Time PCR, also referred to as quantitative PCR or qPCR, is a form of Multiplex PCR that enables the user to monitor and observe the progression of the reaction. Noteworthy benefits of this method encompass its rapidity, accuracy and high sensitivity, which has made it a key tool in the diagnosis of diseases. An important issue regarding Real-Time PCR is that the amount of primary DNA sample in the sample determines the number of amplicons in the PCR reaction.

An important application of this technique lies in the realm of examining gene expression, where mRNA is initially transformed into cDNA by Reverse Transcriptase enzyme, subsequently serving as a template for the real-time PCR reaction through the aid of labeled probes and primers.

These probes are designed in such a manner that the amount of light emitted from them signifies the progression of the reaction stages and product production. Within the Real-Time PCR technique, a term known as Ct or threshold cycle exists, which denotes the initial cycle in which the amount of fluorescent light emitted surpasses the initial phase. The outcomes of Real-Time PCR are displayed in the form of graphs simultaneously with the advancement of the reaction process and these graphs illustrate the four primary phases of the reaction. These distinct phases consist of:

The first phase or Linear ground phase: During this stage, the fragments are rapidly synthesized, albeit with a signal that is not sufficiently robust to be detected by the device.
The second phase or early exponential phase: In this phase, exponential amplification takes place and the device registers the signals.
The third phase or Log-linear phase: This phase is characterized by a gradual reduction in the rate of the Real-Time PCR reaction, accompanied by a decline in the efficiency of the reaction materials.
The fourth phase or Plateau phase: In this final phase, no alterations in the level of fluorescent light received by the device are detected, thereby indicating the depletion of compounds within the reaction.

Real-Time PCR fluorescent dyes

In the Real-Time PCR technique, it is customary to employ two groups of fluorescent dyes. The first group comprises of fluorescent dyes that have an affinity for double-stranded DNA, such as SYBR Green and EvaGreen, which exhibit an affinity for each DNA strand. Both strands become bound non-specifically in the reaction medium. On the other hand, the second group consists of fluorescent dyes that are attached to specific oligonucleotide sequences called probes. Consequently, these dyes can only attach to specific sequences, including hydrolytic probes like TaqMan probes and beacons.

The Trita® r-HPV Detection Kit product utilizes the Real-Time PCR method in conjunction with four different fluorescent dyes (FAM, Cy5, HEX, and ROX) across four distinct color channels to detect 14 high-risk HPV strains. The genotypes that are examined by this kit encompass types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.

HPV virus treatment

Today, individuals afflicted with skin warts have several treatment alternatives at their disposal, such as the surgical removal of the lesions, cryotherapy (the freezing of infected tissue), immune-stimulating or modulatory drugs and laser. The most efficient way to prevent HPV virus infection is to receive vaccination against it. Consequently, it is recommended that both boys and girls receive HPV vaccination between the ages of 11 and 12 in order to prevent infection by the most prevalent high-risk and low-risk strains. The treatment of anogenital and oropharyngeal warts remains feasible as long as the patient possesses sufficient immunity. In the event that an individual is diagnosed with cervical cancer, the commencement of chemotherapy and radiation therapy shall be determined by the doctor’s opinion.