**2. HPV**

*Viruses and Viral Infections in Developing Countries*

days of oncology research.

**1.2 Viral genome integration**

kinase transforming protein Fps encoded by v-fps has the ability to induce transformation by promoting tyrosine phosphorylation of the PDGFbeta receptor [3, 4]. Thus, the Japanese scientists have greatly contributed to the field during the early

Retroviral integration is a type of insertional mutagenesis. Integration processes are widely studied from biochemistry, and their molecular mechanisms are elucidated. Retrovirus-induced tumorigenesis is divided into two types. First, the retroviral genome carries the oncogene such as *v-onc*, and the inserted viral element may induce malignant tumors. Second, the inserted retroviral element has the promoter or enhancer activity of the host proto-oncogene. The genome of human papilloma virus (HPV), hepatitis B virus (HBV), human T-cell adult leukemia virus (HTLV) [5], avian sarcoma virus (ASV) [6], feline leukemia virus (FeLV) [7], murine leukemia virus (MLV) [8–13], and mouse mammary tumor virus (MMTV) can be integrated in the host genome. As a survival strategy, the inserted retroviral

The retroviral promoter and enhancer elements in LTRs originally promote the expression of retroviral genes such as *gag*, *env*, and *pol* [14] (**Figure 1**). However, once integrated in the host genome, LTR elements can promote host gene expression in place of the host genome promoters and enhancers. After infection, the retroviral RNA genome is reverse-transcribed into DNA, which forms a pre-insertion complex (PIC) with the oligomeric integrase enzyme [15], which catalyzes the insertion reaction. The PIC crosses the nuclear envelope through the nuclear pore complex to access host cell DNA. The retroviral DNA ends then attack and nick the host DNA with target nucleotides. These ends are ligated to the truncated host DNA to complete the insertion process involving replication of the target

To date, extensive research has been conducted on target motifs for retroviral insertion to better understand tumorigenesis, neuronal damage, aging, and immunodeficiency including acquired immunodeficiency syndrome (AIDS). Retroviralhost DNA interactions support specific host DNA structures [16–21] but are less likely to be inserted into particular base sequences. Previous studies on human immunodeficiency virus (HIV)-1 insertion have shown that the insertion sites are distributed in 10-bp cycles on the nucleosome surface of the outward DNA major

*A common structure of the retroviral genome. Plus strand RNA is shown. R, terminal redundant sequence; U5, unique 5*′ *sequence; PBS, tRNA primer-binding site; gag, group-specific antigen encoding capsid protein MA (matrix), CA, capsid protein; NC, nucleocapsid; pro, protease; pol, polymerase including reverse transcriptase (RT) and integrase (IN) catalyzing integration; env, envelope glycoproteins; U3, unique 3*′ *sequence. U3, R, and U5 are duplicated at both ends of the DNA provirus genome in the host genome sequence to form the long terminal repeat (LTR). LTR is essential for the initiation of viral DNA syntheses by reverse transcription as* 

*well as the integration of proviral DNA and the regulation of viral gene expression.*

genome (provirus) is thereafter replicated during host cell division.

**36**

**Figure 1.**

nucleotides.

groove of chromatin [16, 22].

HPV is one of the most common viruses that infect more than half women once in life through sexual intercourse. It is a small DNA virus with approximately 8000 bp circular double-stranded DNA in its genome. The viral particle has icosahedral capsid structure as its outer shell. The virus has been known to be involved in the development of cervical, anal, and vaginal cancer and condyloma acuminatum. Many types of HPV are known, and high-risk HPV based on this cancer risk is concentrated in specific subgroups epidemiologically. In particular, types 16 and 18 have high carcinogenic risks of cervical intraepithelial neoplasia (CIN) and cancer. Type 16 has been detected in the Japanese 5-year-old girls [23]. High-risk group (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) causes bladder cancer and pharyngeal cancer in addition to vulvar and cervical cancer. Low-risk group (HPV2, 27, and 57) causes verruca vulgaris that develops in the hands and feet [24].

HPV genome has early genes (E1, E2, E4, E5, E6, and E7) and late genes encoding viral capsid proteins (L1 and L2). E6 and E7 are considered to be involved in carcinogenesis. E6 binds to a tumor-suppressor gene product p53 and degrades p53 through promoting the ubiquitination. It also contributes to carcinogenesis by reactivating telomerase reverse transcriptase (hTERT) and degrading proteins with PDZ domain [shared by—post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and Zonula occludens-1 protein (zo-1)]. E7 binds to the tumor suppressor gene pRb and contributes to carcinogenesis by releasing and activating the transcription factor E2F bound to pRb. It is also involved in carcinogenesis by interacting with cyclin-dependent kinase (Cdk) inhibitors p21 and p27 to evade cell cycle checkpoint mechanisms and apoptosis. However, in many cases, immortalization does not occur only by the actions of human cervix mucosal cells E6 and E7. In the well-known HeLa cell line, telomerase is activated, preventing the gradual shortening of telomeres in cell death.

High-risk HPV can be detected in more than 90% patients suffering from cervical cancer and dysplasia, which is a precancerous lesion, and in the vulva of 5% normal women. Although HPV is also integrated into the genome of host cells, its integration pattern has unique features. The E6 and E7 genes are retained in the provirus with frequent deletion mutations in E2 gene. Thus, the viral genome is also found to be truncated in the complex reaction of integration, but its carcinogenicity is rather enhanced. Low-grade dysplasia of the cervix and cervical intraepithelial neoplasia (CIN) is well known. These pathological changes are reversible and are known to spontaneously disappear within about 3 years. However, about 10% infected patients sustain the infection for more than 3 years, and cervical cancer occurs after several months to several decades in some of them. HPV genome is integrated into the host cell genome during the progression of CIN, and the progression becomes irreversible.

#### **2.1 Clinical test for HPV detection**

HPV-DNA typing tests have been conducted using real-time PCR, loopmediated isothermal amplification (LAMP) [25, 26], invader, and hybridization methods. In recent years, the Clinichip method [27] combining LAMP is broadly available in advanced countries including Japan due to the ability to amplify genes at a constant temperature. The LAMP method is very specific because it uses four primers to recognize the six regions of the HPV genome. Nonspecific amplification does not occur because the amplification reaction depends on the order of the six regions of the HPV gene. In addition, since the amplification product constitutes

the target gene sequence, the target gene can be detected immediately by the presence or absence of amplification.

In an environment where electricity availability is limited, easy-to-use and durable reagents and equipment are desirable. In recent years, attempts have been made to spread virus testing as point-of-care tests (POCT) [28]. For some pathogens such as malarial parasite (*Plasmodium*), HIV, Ebola virus, and Zika virus, development of the testing system by fluid device is in progress; however, HPV detection methods have not been put to practical use as POCT yet.

## **3. HBV**

HBV has been identified as the pathogen that causes acute and chronic hepatitis B [29, 30]. HBV is a DNA hepatitis virus and has a double structure of an envelope and a core. In persistent infections, the HBV genome is often inserted into the hepatocyte genome and is not eliminated by the host's immune system. Several studies on many common insertion sites have identified common integration sites in the HBV genome by full genome sequence of hepatocellular carcinoma [31]. The envelope protein of HBV covers the HBs antigen, and the core protein is called the HBc antigen. HBV DNA encodes HBs antigen, HBc antigen, X protein, and DNA polymerase. The HBe antigen is translated from the pre-C gene and the downstream HBc antigen. It is released into blood as a soluble protein during HBV virus growth and therefore considered a marker for the virus growth. In asymptomatic carriers positive for HBe antigen, the host immune system continuously reacts with HBV-infected hepatocytes, and biopsy reports show persistent lymphocyte infiltration in the portal vascular area of liver tissues. If the damage persists for a long time, remodeling of the hepatocytes occurs and progresses to cirrhosis with obvious fibrotic crosslinks. After all, when seroconversion occurs in HBV carriers negative for HBe antigen and positive for HBe antibody, the patient becomes an inactive carrier. HBV particles remain alive during periods of inactivity. Host immune system can attack infected hepatocytes, and hepatocyte regeneration may occur with genomic mutations, and the effects of the inserted HBV genome persist. Hepatocellular carcinoma can develop with persistent liver dysfunction associated with liver cirrhosis.

#### **3.1 Clinical test for HBV detection**

For the diagnosis of hepatitis B, HBs antigen/antibody, HBc antibody, HBe antigen/antibody, and HBV DNA are detected, and HBV DNA polymerase activity is measured. HBs antigen is produced continuously in patients with HBV infection; thus, positive results for HBe antigen can diagnose current infection with HBV. Recently, the HBs antigen measurement system has become very sensitive, and infected people have been found to be positive for HBs. In addition to antigen or antibody detection method, viral DNA detection system (quantitative PCR, LAMP method) has been used for clinical tests. Liver fibrosis is seen as a precancerous stage of HBV and hepatitis C virus (HCV) infection. Liver biopsy and qualitative analysis of hyaluronic acid and type IV collagen in blood are well-known methods to evaluate liver fibrosis progress. Monitoring of glycolysis of serum M2BP (Macingo binding biomarker) is used to evaluate liver fibrotic stage [32]. Recent studies have suggested that the most common cause of nonexclusion of HBV is covalently closed circular DNA (cccDNA). When HBV infects hepatocytes, the incomplete duplex circular DNA genome becomes a complete duplex and is retained in the nucleus in the form of cccDNA. This HBV gene is the source of HBV

**39**

*Basic Research and Clinical Examination of Tumor Virus*

replication. Since cccDNA is an extremely stable biochemical structure, it can be used as a monitoring marker after the infection. As the nucleic acid analog drug has little effect on reducing HBV cccDNA, a high risk of reactivation remains. If host immunity is suppressed, the reactivation occurs from this cccDNA as a starting transcriptional point. Therefore, elimination or suppression of reactivation will be

In the clinical tests for HBV detection, the detection sensitivity of the enzymelinked immunosorbent assay (ELISA) method for HBs antigen is higher than those of other tests. Each available method depends on the titer of the used antibody, and the sensitivity varies. In particular, the mutation in the gene encoding the envelope protein HBs antigen may cause false negative results. Many test kits increase sensitivity by using multiple monoclonal antibodies or a polyclonal antibody that has an affinity for multiple epitopes. In some cases, antibodies that recognize epitopes within the viral particle are used instead of surface proteins with many mutations. However, the high sensitivity of many kits may instead cause false positive results. Accordingly, actual early diagnosis requires careful evaluation of test results in

Another important human carcinogenic virus is human T-cell leukemia virus type 1 (HTLV-1). This virus has the potential to cause adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy (HAM), and HTLV-1 uveitis (HU). These diseases develop in HTLV-1 carriers after prolonged latent infection. HTLV-1 carriers are particularly prevalent in southwest Japan, including Kyushu, Shikoku, and Okinawa. HTLV-1 was isolated and reported by Gallo R as the first human pathogenic retrovirus in 1980. Yorio Hinuma of Kyoto University in Japan precisely analyzed the genomic structure and identified the HTLV-1 [34]. HTLV-1 is a weakly infective virus particle, and cell-to-cell transmission is the main route of infection.

ATL is a hematologic malignancy in which abnormal lymphocytes appear in peripheral blood and infiltrate various organs throughout the body. Kiyoshi Takatsuki of Kumamoto University in Japan and Taku Uchiyama of Kyoto

University collected samples. In 1981, Hinuma identified HTLV-1 to be the causative virus of ATL; this was the first report on tumor-induced retrovirus in humans. Most infected cells are CD4 + CD25 + T cells. Viral receptors are thought to involve other proteins and heparan sulfate. HTLV-1 is an approximately spherical virus particle about 100 nm in diameter and has an envelope. It has an RNA genome composed of the genes common to retroviruses such as *gag*, *pro*, *pol*, and *env*, as well as the gene encoding pX. It selectively uses the splicing and translation initiation sites in a region less than 2 kb called pX located between the env gene and the 3′ LTR [35] and uses the plus and minus strands relative to the transcription direction and efficiently. It often encodes several regulatory and accessory genes. As described later, this pX is involved in the action of HTLV-1 encoding HTLV-1 bZIP factor (HBZ). Tax and HBZ may be the carcinogenic potentials of HTLV-1. Tax is a potent transactivator essential for the activation of the viral promoter 5′ LTR. After entering the cell by infection, the virus particle capsid is lost, and the HTLV-1 genomic RNA is reverse transcribed to form genomic duplex DNA. Thereafter, the integrase encoded by the viral genome and the DNA end forms a complex, which contacts the genomic DNA on the cell side, and inserts by catalytic reaction. The 5-base-long

*DOI: http://dx.doi.org/10.5772/intechopen.89331*

central to future therapeutic strategies [33].

combination with other clinical tests.

**4. HTLV type 1 (HTLV-1)**

**4.1 Molecular biology of HTLV-1**

*Basic Research and Clinical Examination of Tumor Virus DOI: http://dx.doi.org/10.5772/intechopen.89331*

*Viruses and Viral Infections in Developing Countries*

have not been put to practical use as POCT yet.

ence or absence of amplification.

**3. HBV**

with liver cirrhosis.

**3.1 Clinical test for HBV detection**

the target gene sequence, the target gene can be detected immediately by the pres-

In an environment where electricity availability is limited, easy-to-use and durable reagents and equipment are desirable. In recent years, attempts have been made to spread virus testing as point-of-care tests (POCT) [28]. For some pathogens such as malarial parasite (*Plasmodium*), HIV, Ebola virus, and Zika virus, development of the testing system by fluid device is in progress; however, HPV detection methods

HBV has been identified as the pathogen that causes acute and chronic hepatitis B [29, 30]. HBV is a DNA hepatitis virus and has a double structure of an envelope and a core. In persistent infections, the HBV genome is often inserted into the hepatocyte genome and is not eliminated by the host's immune system. Several studies on many common insertion sites have identified common integration sites in the HBV genome by full genome sequence of hepatocellular carcinoma [31]. The envelope protein of HBV covers the HBs antigen, and the core protein is called the HBc antigen. HBV DNA encodes HBs antigen, HBc antigen, X protein, and DNA polymerase. The HBe antigen is translated from the pre-C gene and the downstream HBc antigen. It is released into blood as a soluble protein during HBV virus growth and therefore considered a marker for the virus growth. In asymptomatic carriers positive for HBe antigen, the host immune system continuously reacts with HBV-infected hepatocytes, and biopsy reports show persistent lymphocyte infiltration in the portal vascular area of liver tissues. If the damage persists for a long time, remodeling of the hepatocytes occurs and progresses to cirrhosis with obvious fibrotic crosslinks. After all, when seroconversion occurs in HBV carriers negative for HBe antigen and positive for HBe antibody, the patient becomes an inactive carrier. HBV particles remain alive during periods of inactivity. Host immune system can attack infected hepatocytes, and hepatocyte regeneration may occur with genomic mutations, and the effects of the inserted HBV genome persist. Hepatocellular carcinoma can develop with persistent liver dysfunction associated

For the diagnosis of hepatitis B, HBs antigen/antibody, HBc antibody, HBe antigen/antibody, and HBV DNA are detected, and HBV DNA polymerase activity is measured. HBs antigen is produced continuously in patients with HBV infection; thus, positive results for HBe antigen can diagnose current infection with HBV. Recently, the HBs antigen measurement system has become very sensitive, and infected people have been found to be positive for HBs. In addition to antigen or antibody detection method, viral DNA detection system (quantitative PCR, LAMP method) has been used for clinical tests. Liver fibrosis is seen as a precancerous stage of HBV and hepatitis C virus (HCV) infection. Liver biopsy and qualitative analysis of hyaluronic acid and type IV collagen in blood are well-known methods to evaluate liver fibrosis progress. Monitoring of glycolysis of serum M2BP (Macingo binding biomarker) is used to evaluate liver fibrotic stage [32]. Recent studies have suggested that the most common cause of nonexclusion of HBV is covalently closed circular DNA (cccDNA). When HBV infects hepatocytes, the incomplete duplex circular DNA genome becomes a complete duplex and is retained in the nucleus in the form of cccDNA. This HBV gene is the source of HBV

**38**

replication. Since cccDNA is an extremely stable biochemical structure, it can be used as a monitoring marker after the infection. As the nucleic acid analog drug has little effect on reducing HBV cccDNA, a high risk of reactivation remains. If host immunity is suppressed, the reactivation occurs from this cccDNA as a starting transcriptional point. Therefore, elimination or suppression of reactivation will be central to future therapeutic strategies [33].

In the clinical tests for HBV detection, the detection sensitivity of the enzymelinked immunosorbent assay (ELISA) method for HBs antigen is higher than those of other tests. Each available method depends on the titer of the used antibody, and the sensitivity varies. In particular, the mutation in the gene encoding the envelope protein HBs antigen may cause false negative results. Many test kits increase sensitivity by using multiple monoclonal antibodies or a polyclonal antibody that has an affinity for multiple epitopes. In some cases, antibodies that recognize epitopes within the viral particle are used instead of surface proteins with many mutations. However, the high sensitivity of many kits may instead cause false positive results. Accordingly, actual early diagnosis requires careful evaluation of test results in combination with other clinical tests.
