Diagnosis and Treatment

#### **Chapter 8**

## Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique

*Douglas Millar and John Melki*

#### **Abstract**

We have developed a novel strategy to simplify microbial nucleic acids termed 3base™. This technology uses the chemical sodium bisulphite to reduce the genome from adenine, cytosine, guanine, and thymine or uracil, in the case of RNA containing viruses, to adenine, guanine and thymine thus reducing genome complexity. The method has been applied to the detection of high-risk human papilloma virus (HPV), gastrointestinal pathogens, alphaviruses, flaviviruses, dengue and more recently coronaviruses. Currently, there are very few real-time RT-PCR based assays that can detect the presence of all members of these viral families using conventional approaches. This strategy allows the design of assays that are capable of pan-family detection. The pan-viral assays provide a sensitive and specific method to screen and thereafter speciate viral families in clinical samples. The assays have proven to perform well using clinical samples and additionally during an outbreak of dengue fever that occurred in 2016/17 on the islands of Vanuatu. The 3base™ assays can be used to detect positive clinical samples containing any viral family generally in less than 3 hours making them ideally suited to viral surveillance and perhaps the discovery of emerging viruses in families without prior sequence knowledge of the pathogen.

**Keywords:** human papilloma virus, gastrointestinal pathogens, flavivirus, alphavirus, dengue, coronavirus, simplification, RT-PCR

#### **1. Introduction**

Many viruses are members of large families in which the individual viruses can be diverse at the molecular level. For example, SARS-CoV-2 belongs to the family *Coronaviridae* that contains 4 distinct genera namely the Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Many other viruses such as *Flaviviridae* and *Togaviridae* again contain many individual viruses in their designated family (see **Table 1** for examples).

Due to the genomic heterogenicity of viral families virtually all molecular diagnostic tests target individual viruses for disease diagnosis. However, even this can be challenging as viruses such as Influenza A contains many different strains based on the composition of their haemagglutinin and neuramidase genes. This chapter


#### **Table 1.**

*Examples of the diversity contained within a number of different viral families.*

describes a novel genomic simplification technique that enables the use of pan-family primers and probes to detect the presence of viral pathogens such as high-risk HPV, gastrointestinal pathogens, flavivirus, alphavirus, dengue and coronaviruses in clinical samples.

#### **2. 3base™ a novel RNA simplification method**

In order to simplify and improve the detection of viral families in clinical samples, we have developed an assay that is able to detect the presence of any high-risk HPV, gastrointestinal pathogen, flavivirus, alphavirus, dengue or coronavirus virus using a single primer and probe set for each type. These assays are based on the use of the chemical sodium bisulphite to reduce the complexity of genomes from 4 to 3 bases by deaminating cytosine to an uracil intermediate. The deamination reaction of cytosine to uracil was first described in 1970 by Hayatsu [1, 2] and has been studied in detail since. The first step of the reaction involves the sulphonation of cytosine to cytosine sulphonate followed by deamination to an uracil sulphonate intermediate and subsequently the removal of the sulphate adduct to uracil, traditionally by the use of strong alkali (**Figure 1**).

*Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

Sulphonated uracils are unable to be copied by DNA polymerases [3] due to steric hindrance as a result of the presence of the sulphate group at the C6 position. This causes distortions of DNA geometry and reduced stacking interactions [4]. Therefore, this adduct has to be removed if the resulting template is to be copied by a polymerase or reverse transcriptase enzyme. The bisulphite reaction was further refined in 1992 by Frommer and her colleagues [5] and used to differentiate cytosine from 5-methylcytosine in mammalian DNA as 5-methyl-cytosine is resistant to the deamination reaction. Since the publication of the genomic sequencing method, it has become the gold standard for studying the presence of methylated cytosine residues in the human genome (**Figure 2**).

However, this method resulted in up to 96% degradation of the DNA template [6] and would completely destroy RNA due to the need to desulphonate the uracil adduct with strong alkali. We have subsequently refined the method so that the degradation of DNA and RNA has been eliminated allowing "simplification" of both microbial DNA and RNA.

The 3base™ protocol deaminates all cytosine residues in nucleic acid to uracil, which are subsequently copied as thymine by a polymerase (**Figure 3**) or reverse transcriptase enzyme [5]. After simplification individual species become more similar in base composition resulting in reduced complexity of primer and probe sets for panfamily identification. The resulting primer and probe sets have fewer mismatches to the original sequences thus allowing binding of these to regions of nucleic acid that were previously heterogeneous in nature. The use of the simplification method does not result in a loss of specificity as it is still possible to design individual primer sets that can detect the viral species responsible for disease.

**Figure 1.** *Shows the reaction of cytosine with sodium bisulphite.*

#### **Figure 2.**

*Schematic representation of published papers using the bisulphite method with arrows representing the publication of the description of the cytosine deamination method and the bisulphite sequencing protocol.*


**Figure 3.** *Shows the simplification process where cytosine residues are converted to uracil.*

#### **3. Viral surveillance**

New viruses will continue to appear due to evolutionary pressure, climate change and the demise of natural habitats as a result of human intervention. The Zika virus epidemic that began in 2016 demonstrates that a flavivirus originally thought to be relatively benign can emerge as a significant public health threat within a relatively short space of time [7]. SARS-CoV-2 emerged in 2019 and subsequently gave rise to a global pandemic which to date has resulted in over 620,000,000 confirmed cases and over 6,500,000 deaths [8]. SARS-CoV-2 more than likely emerged from an animal reservoir, and it is highly likely that other coronavirus threats will emerge in the future likely by the same route. While it is impossible to predict the rise of a particular virus in the human population it is almost certain that in the future new viral threats will emerge which will more than likely result in widespread morbidity and mortality.

As a result of the recent SARS-CoV-2 pandemic it is likely that governments will in the future invest in a more extensive network of testing equipment, stockpile reagents and enable easier regulatory protocols. While this could reduce the time required for testing, a critical phase exists of when a new pathogen becomes infectious to the general population, and when reliable diagnostic tests are generally available. A strategy that may allow for less-restricted screening for novel pathogens during this period is the use of pan-family assays: molecular diagnostic tests which target a family of viruses rather than a single species [9].

The use of species-specific PCR is unlikely to pick up new strains of a virus and this was demonstrated in the case of SARS-CoV-2, which was only detected on Next Generation Sequencing (NGS) and not by conventional PCR using species-specific primers and probes [10]. Interestingly, we had already developed a pan-coronavirus 3base™ assay that on publication of the complete genome of SARS-CoV-2 [11] would have picked up this variant without prior knowledge of the viral genomic sequence. The pan-family PCR approach is thus perhaps a simpler and more cost-effective alternative to NGS for viral surveillance?

It has been postulated that one of the more obscure viruses in the *Flaviviridae* family such as Spondweni virus (SPOV), Usutu virus (USUV), Ilheus virus (ILHV), Rocio virus (ROCV), Wesselsbron virus (WSLV) or tick-borne flaviviruses may be the next pathogen to emerge into the human population [7]. The use of the panflavivirus 3base™ assay would be the ideal tool to screen for emerging flaviviruses entering the population without the expense and labour costs of screening each and every sample for all of the individual flavivirus species that are currently known.

#### **4. Pan viral diagnosis**

There are several methods available for molecular pan-viral diagnosis (see **Table 2**). Perhaps the first was the use of arrays fabricated with large numbers of oligonucleotides probes specific for individual pathogens. Hybridisation of a clinical sample to such arrays was then be used to detect the presence of viral genomes in infected individuals [39]. However, using this approach prior sequence knowledge of the pathogens are required to design the specific oligonucleotides to be arrayed, and as stated previously emerging pathogens are highly likely to contain divergent nucleic acid sequences. Another approach for the detection of novel pathogens is the use of Next Generation Sequencing (NGS). Unlike the array approach no prior knowledge of an emerging viral sequence is required as all nucleic acids in the sample can be


#### **Table 2.**

*Examples of the pan-family approach applied to molecular diagnostics.*

sequenced then assembled by alignment with established genomes to produce a best match. Recently the costs associated with NGS have reduced dramatically from when the technology was in its infancy thus it is now possible to apply this technique to viral discovery [40]. However, the use of viral arrays and NGS is still more costly, labour intensive and less sensitive compared to the more routine technique of RT-PCR which can generate clinically meaningful data in around 1 h.

Many viruses that infect humans cause non-specific symptoms such as headache, fever, arthralgia, myalgia, and lethargy making initial diagnosis based on clinical symptoms challenging. This is especially true of respiratory viruses thus pan-family diagnosis can reduce the number of primer and probe sets that are require for molecular syndromic testing. **Table 3** shows that if the pan-family approach was used for respiratory viruses screening the number of individual reactions that would be required to identify the infectious agent is reduced from 20 to 7 reducing costs and the labour involved. After identifying the family responsible for infection individual typing primers could then be used to detect the exact species if required. Likewise,

*Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*


**Table 3.**

*Shows that using the pan-family screening approach the number of individual reactions required for a comprehensive respiratory screen is reduced from 20 to just 7.*

infection with arboviruses manifest in similar symptoms thus the use of the panfamily screen can provide a rapid diagnosis of the family involved without the need to perform multiple individual PCR reaction to determine the cause of infection. After determination of the species responsible for infection again samples can then be typed using species-specific PCR if required.

#### **5. Human papilloma virus (HPV)**

The family *Papillomaviridae* contains a group of double stranded DNA viruses containing a circular genome of approximately 8000 base pairs [41, 42] that were first described to be associated with skin warts in 1907 [43]. The family papillomavirus contains over 100 individual members many of which cause no symptoms with the vast majority (90%) resolving after 2 years [44]. HPV can infect many different sites in the body including the skin, throat, tonsils, mouth, cervix, vulva, vagina, penis, and anus.

It was first postulated in 1976 that HPV could be associated with the development of cervical cancer [45]. Genital HPV infection can be caused by at least 50 individual


#### **Table 4.**

*HPV viruses classified according to the risk of cervical cancer development.*

viruses that can be split into four classes as shown in **Table 4** [46]. The high-risk types of HPV have been shown to be associated with the development of cervical cancers [47–49].

Traditional methods for the diagnosis of cervical cancer have relied heavily on cytology in which cells of the cervix are observed under the microscope for the presence of cancerous or precancerous lesions. This test, known as the Papanicolaou (Pap) test was invented in the 1920s by Georgios Papanikolaou and Aurel Babeș and subsequently named after Papanikolaou. A simpler version of the test was discovered by Anna Marion Hilliard in 1957. The use of the Pap test when used in combination with molecular methods has been shown to increase the sensitivity in which precancerous lesions can be detected in the cervix [50].

#### **5.1 Molecular detection of HPV**

There are a number of molecular methods that can be used to detect the presence of HPV in clinical samples [51–55]. One common primer pair, the MY set, was first described in 1989 [56] detects a common region of the viral L1 gene that is found in all HPV types. Improvements on these primers generated the GP set that are able to detect more strains of the virus [57]. However, these primer sets are unable to differentiate the presence of high-risk HPV from low risk therefore amplicons must be sequenced or hybridised to oligonucleotide arrays to determine the strain of the virus responsible for infection.

One of the earliest FDA approved molecular tests for HPV was the hcII HPV test (Digene Corporation, USA). This test used oligonucleotide probes that were specific for each of the high and low risk viruses. The method was based on capture of specific HPV sequences present in the clinical sample coupled with a chemiluminescent readout. However, it has been demonstrated that this assay could generate both false positive and negative results [58].

#### *5.1.1 3base™ detection of high-risk HPV types*

To produce an assay capable of detecting specifically the high-risk viruses we aligned the sequences of the complete genomes of the high-risk HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 along with the low-risk types 6, 11, 43 and 44 to serve as reference for non-target HPV strains. Using this approach, we were able to design a nested PCR assay that was specific for high-risk HPV types only. The primers were tested extensively on a large number of previously typed ThinPrep® liquid-based cytology samples to ensure that the assay was specific for the high-risk-types.

#### *5.1.2 3base™ high-risk HPV clinical trial*

A total of 834 ThinPrep® samples were tested using the 3base™ simplification method and compared to the reference hcII method. Discordant samples were the

#### *Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

amplified using a reference method (MY09/MY11 and the GP5+/GP6+ primer sets) and the amplicons subsequently sequenced to identify the strain of virus present. As can be seen from **Table 5** both methods demonstrated a similar sensitivity which was not statistically significant (*p* = 0.398). However, the specificity of the 3base™ was significantly higher than the hcII method (*p* = 0.001) and as would be expected the PPV for the 3base™ test was also significantly higher [59].


**Table 5.**

*The results generated in an independent clinical trial comparing the 3base™ to a commercially available assay for the detection of HPV in cervical samples.*

#### **6. Gastrointestinal disease**

Gastrointestinal (GI) diseases occur globally and are a major cause of morbidity and mortality. In developed countries the mortality due to GI disease is relatively low compared to that of developing countries [60]. It has been estimated that in developing countries up to 2 million children under the age of five die from GI infections annually [61].

There are many viral, bacterial, and protozoan agents that are responsible for gastroenteritis in humans. Perhaps the most notable of these are the viral agents that include Norovirus, Rotavirus, Sapovirus, Astrovirus and the Adenoviral group. One of the most common causes of gastroenteritis is Norovirus which is responsible for outbreaks of disease especially in children with an estimated 685 million cases and around 200,000 deaths occurring annually worldwide [62].

The viruses that cause gastroenteritis are a diverse group of pathogens with many genotypes and genogroups responsible for disease (see **Table 6** below).

There are also a wide range of bacterial species that are responsible for gastrointestinal illness with the most common agents that of *Campylobacter* spp. and *Salmonella*. The CDC estimated that in the USA that 43% of bacterial gastrointestinal cases are caused by *Salmonella* spp. followed by *Campylobacter* spp. representing a further 33% [66]. Other notable causes of GI disease are *Shigella* spp., *Yersinia enterocolitica*, *Clostridium difficile* and pathogenic strains of *Escherichia coli*. Different bacterial agents also show distinct geographical distributions with species such as *Vibrio cholera* and *Shigella* spp. more common in developing countries [67].

Protozoan species also contribute to the burden of GI diseases with notable agents such as *Giardia* spp., *Cryptosporidium* spp. and *Enteramoeba histolytica* the most


#### **Table 6.**

*The diversity of viral agents responsible for GI disease.*

common causes. Other agents such as *Dientamoeba fragilis* and *Blastocystis hominis* have also been implicated in the aetiology of gastroenteritis [68–70].

The symptoms of gastrointestinal disease can include diarrhoea, vomiting, abdominal pain, fever, general lack of energy, and dehydration. These symptoms are shared between the many organisms that cause symptoms thus traditionally disease was diagnosed by a combination of culture, microscopy and EIA for bacteria, protozoan and viral disease respectively. These techniques are laborious and in some cases such as conventional culture can take up to 4–5 days to yield positive results [71].

To simplify the detection of gastrointestinal pathogens and streamline the process we sought to use 3base™ technology to not only detect the complex viral causes of gastroenteritis but also the individual bacterial and protozoan agents responsible for gastroenteritis [72].

#### **6.1 Viral pathogens**

Sequences for all genotypes of Norovirus, Rotavirus, Astrovirus, Sapovirus and Adenovirus were downloaded from the NCBI nucleotide database and aligned using the free web-based alignment tool Dialign (https://dialign.gobics.de). Regions were then chosen to produce primer and probe sets to amplify each viral group. After initial optimisations the best sets were used to screen a bank of archived clinical samples with the results are shown in **Table 7**.

As can be seen from **Table 7** the assay was able to detect the presence of all viral targets. In addition, the assay was validated independently yielding similar results.

#### **6.2 Bacterial and protozoan pathogens**

Although bacterial and protozoan causes of gastroenteritis are not as complex as the viral targets it was important that the assay was able to detect these pathogens as gastroenteritis is a syndromic disease. **Tables 8** and **9** demonstrate the ability of the 3base™ method to detect organisms at the species level.

As can be seen from the data the 3base™ assay does not suffer from a loss of specificity when primer and probe sets are designed to detect organisms at the species level. The syndromic multiplex PCR assay is thus a useful tool for the detection of viral, bacterial, and protozoan causes of gastroenteritis without the need for time consuming and labourious conventional methods. In addition, testing can be centralised with a turnaround time of less than 4 h.

#### *Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*


#### **Table 7.**

*Results generated using the 3base™ on stool samples with viral gastroenteritis.*


#### **Table 8.**

*Detection of bacterial causes of gastroenteritis.*


#### **Table 9.**

*Detection of protozoan causes of gastroenteritis.*

#### **7. Coronaviridae**

The Coronavirus family members are sub classified as alpha, beta, gamma and deltacoronaviruses [73, 74]. Alphaconoronaviruses contain least 10 known species including human coronavirus (hCoV) 229E that causes the common cold, many bat, feline, canine coronaviruses, and the porcine transmissible gastroenteritis coronavirus. The Betaconoronaviruses contain members such as SARS-CoV-1,

MERS-CoV, human coronavirus 0C43 and now SARS-CoV-2. The Gammacoronaviruses genera contain avian, duck coronavirus and the infectious bronchitis virus. Finally, the deltacoronaviruses members include HKU11, HKU12, HK13 that cause the common cold. **Figure 4** illustrates a phylogenetic tree showing the relatedness of various coronavirus strains. Most of the members of the coronavirus family exhibit a zoonotic lifecycle, that in rare occasions result in a spill over event to the human population.

A number of notable human coronaviruses have emerged in the last two decades which can result in severe respiratory disease. The severe acute respiratory syndrome (SARS) originated as a mystery illness in Guangdong, China in 2002 and resulted in an epidemic that killed 10% of the 8000 people it infected [75]. The etiological agent was subsequently identified as the severe acute respiratory syndrome coronavirus (SARS, now renamed SARS-CoV-1). This was the fifth hCoV to be identified and is thought to have originated as an animal virus from an unknown animal reservoir. The disease was characterised by flu-like symptoms, high fevers exceeding 38°C, myalgia, dry non-productive cough, difficult breathing, and an infiltrate seen on chest radiography.

Ten years later in 2012, a sixth hCoV was isolated from a patient presenting with severe respiratory illness in Jeddah, South Arabia [76]. The etiological agent was later designated Middle East respiratory syndrome coronavirus (MERS-CoV). MERS-CoV has been detected in more than 27 countries across the Middle East, Europe, North Africa, and Asia. There has been a total of 2040 MERS-CoV laboratory confirmed cases, with 712 deaths (34%) making this the most lethal coronavirus to date.

Another novel coronavirus (SARS-CoV-2) emerged into the human population in December 2019 in Wuhan, China, and has subsequently become the deadliest coronavirus to emerge in the human population in the past two decades [77], bringing the number of hCoV to seven. The disease (Covid-19) is believed to have been contracted from an animal virus that crossed over into the human population, more than likely from bats. The virus has spread globally and infected over 620,000,000 people resulting in over 6,500,000 deaths [8] which although far more than the MERS-CoV epidemic represents only a 1% case fatality rate compared to 34% for MERS-CoV. Such a large-scale spread is a result of efficient human-human transmission as the virus evolves to improve its ability to infect its human host.

The severity of Covid-19 and the rapid spread of the virus is a wakeup call to rethink diagnostic approaches, especially for the coronavirus family that has many members maintained by a variety of animal reservoirs such as bats, birds, pangolins, and snakes [78–80]. Covid-19 is an example of what can happen if a spill over event involves a virus well attuned to human-human transmission. The severity of coronavirus disease and the potential for new emerging viruses calls for rapid diagnostic tests which can quickly and accurately detect these viruses in clinical samples and animal hosts. The pan-family molecular approach could be an ideal method to screen for coronaviruses in general and detect novel strains as they emerge.

#### **7.1 Design of the pan-coronavirus assay**

Whole genomic sequences of SARS-CoV-1, MERS, HKU-1, NL63, 229E and OC43 were downloaded from the data base and aligned using the Geneious Prime™ software to generate optimal regions for the design of 3base™ primers and probes. These were them tested using synthetic constructs to determine assay sensitivity and specificity (see **Table 10**).

*Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

**Figure 4.** *Shows a phylogenetic tree generated using whole genomes of various coronaviruses using the Geneious prime™ tree building software.*

After initial assessment and assay validation including cross-reactivity studies the pan-coronavirus component was then multiplexed with the SARS-CoV-2 E and N genes for clinical studies using cultured SARS-CoV-2 virus. **Table 11** shows that the triplex assay could detect low levels of SARS-CoV-2 virus.

The clinical performance of the assay was established using 1, 662 patient samples sourced from a local hospital in 2020 when the virus was still relatively rare in Australia. Twenty-five samples were found to be positive for SARS-CoV-2 by both the pan-coronavirus and gene specific assays. In addition, a further 45 samples were positive using the pan-coronavirus assay and negative with the SARS-CoV-2 specific primer and probe sets. These samples were then tested with a confirmatory assay that detected the presence of seasonal coronaviruses. This assay detected 37 samples as either NL63, 229E, OC43 or HKU-1. Of the eight samples that were negative by the confirmatory assay, five were available for sequencing using the pan-coronavirus amplicons. On sequencing the results showed that these samples contained a novel HKU-1 variant not targeted in the confirmatory test.


#### **Table 10.**

*Sensitivity of the pan-coronavirus assay tested on synthetic construct.*


#### **Table 11.**

*Shows the results using the pan-coronavirus triplex assay on cultured viral samples.*

#### **8. Current pan-flavivirus/alphavirus assays**

A PubMed.*gov* search was performed using the keywords pan-flavivirus real time PCR (RT-PCR), pan-alphavirus RT-PCR and pan-dengue RT-PCR to determine the number of assays that employed a pan-family approach. Although this is not a definitive search the results give an idea of what is possible at present using conventional real-time PCR. From 1996 to 2022 a total of 1, 182 paper were found that used realtime PCR to detect the presence of flaviviruses in general. Of these only 2 papers used the pan-flavivirus detection approach. Similarly, from 2004 to 2022 a total of 294 papers mentioned RT-PCR for the detection of alphavirus with only 1 using a panspecies approach with this assay using multiple primers due to target sequence degeneracy. With dengue virus from 2001 to 2022 a total of 782 papers were published that mentioned dengue virus and real-time PCR with 32 using pan-dengue RT-PCR primers and probes.

As the dengue virus family contains only 4 members it makes sense that this was the target to which most pan-family assays were designed. The flavivirus and

#### *Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

alphavirus virus families are much more complex and contain 54 and 32 members respectively and are much underrepresented with pan-family tests compared to dengue. The reduction in the number of assays able to detect pan-flavivirus and panalphavirus is presumably due to sequence divergency of the individual members making the selection of suitable primers and probes for pan-family identification using conventional RNA more challenging.

This is where the use of the chemical simplification step can make the selection of regions to design primers and probes easier (see **Table 12**). As can be seen before the genomic simplification process the consensus sequence for a pan-alphavirus primer would contain a total of 576 individual primers to produce sequences that were a perfect match for all targets. However, after the simplification process the primer pool would be reduced to just 27 representing a major reduction in genomic complexity.

#### **8.1 Flavivirus/alphavirus and dengue**

The *Flaviviridae* family of viruses contain many individual members that result in a heavy toll in terms of morbidity and mortality globally on an annual basis. Notable members include dengue which has been estimated to cause over 400 million


#### **Table 12.**

*Genomic simplification of alphavirus sequences reduces the number of primer variations from 576 to just 27.*

infections yearly with 100 million cases in 2010 [81]. Other species include Zika which caused epidemics between 2014 and 2017, yellow fever virus which is endemic in Africa and South America, Japanese encephalitis virus and West Nile virus which has been associated with sporadic outbreaks in the USA. Flaviviruses infections range from asymptomatic to life threatening conditions such as hemorrhagic fevers. Flaviviruses are characterised by a positive sense single stranded RNA genome that ranges in size from 10 to 11 Kb. The genome consists of 8 non-structural and 3 structural proteins [82].

Alphaviruses are members of the *Togaviridae* group of viruses with genomes of around 11–12 Kb that like flaviviruses contain a single stranded positive sense genome [83]. Alphaviruses infect a wide range of birds, fish and mammals including humans. Probably the best-known alphaviruses are chikungunya, Barmah Forest virus and O'nyong'nyong virus. Both flavi- and alphaviruses are arboviruses and are most commonly transmitted to the human population via a bite from an infected mosquito or tick.

The global distribution of flaviviruses and alphaviruses can be overlapping or unique with some viruses specific for certain geographical locations (**Figure 5**). Epidemics of flavivirus and alphavirus occur on an annual basis with different degrees of severity thus rapid and specific molecular diagnostic approaches are required to aid in patient management.

#### **8.2 Design of 3base™ primers and probes**

To determine if the pan-flavivirus, pan-alphavirus and pan-dengue simplification method could be used in screening and outbreak management we designed 3base™ assays for each family of pathogens.

#### *8.2.1 3base™ pan-flavivirus/pan-dengue assays*

The complete genomes of the following flaviviruses were analysed using Geneious software to determine the optimal regions for 3base™ primers and probes; Karshi virus (AY863002), Powassan virus (EU670438), Kyasanur forest disease virus

#### **Figure 5.**

*Shows the global distribution of a number of important arboviruses (this map was prepared using information in Socha et al. [84] using the free web based MapChart software).*

*Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

(AY323490), Langat virus (NC\_003690), Omsk hemorrhagic fever virus (AB507800), Tick-borne encephalitis virus (KU761572), Yellow fever virus (MF423374), Sepik virus (DQ859063), Wesselsbron virus (JN226796), Dengue 4 (EU854296), Dengue 2 (AF038402), Dengue 3 (AB189125), Dengue 1 (AB189120), Zika (KU820899), Saint Louis encephalitis virus (MN233312), West Nile virus (KT57320), Kunjin virus (KX394405), Japanese encephalitis virus (AF080251), Usutu virus (AY453411) and Murray Valley encephalitis virus (AF161266).

#### *8.2.2 3base™ pan-alphavirus assay*

The complete genomes of the following alphaviruses were analysed using Geneious software to determine the optimal regions for 3base™ primers and probes; Barmah Forest virus (NC\_001786), Ndumu virus (NC-01659), Chikungunya virus (NC\_004162), O'nyong-nyong virus (NC\_001512), Middelburg virus (NC\_024887), Mayaro virus (NC\_003417), Ross River virus (NC\_001544), Semliki forest virus (NC\_003215), Una virus (NC\_043403), Aura virus (NC\_003900), Rio Negro virus (NC\_038674), Mucambo virus (NC\_038672), Everglades virus (NC\_038671), Venezuelan equine encephalitis virus (NC\_001449), Eastern equine encephalitis virus (NC\_003899) and Western equine encephalitis virus (NC\_003908).

#### **8.3 Assay performance**

Numerous primer/probe sets were designed for the pan-flavivirus, pan-dengue and pan alphavirus assays and sets then wet tested to determine optimal sensitivity and specificity. After initial screening the best performing sets were tested using individual synthetic oligonucleotoides specific for each virus. The pan-flavivirus assay was able to detect the presence of DENV-1, DENV-2, DENV-3, DENV-4, TBEV, WNV, YZV and Zika virus with a lower limit of detection (LLOD) of 12.5 copies/PCR for all species tested.

Likewise, the pan-alphavirus assay was able to detect the presence BFV, CHIKV, EEEV, MVE, NV, RRV, VEEV and WEEV with a sensitivity of 10 copies/PCR for VEEV, RRV, NV, BFV and MV, 25 copies/PCR for CHIKV and EEEV and 50 copies/ PCR for WEEV.

To assess potential cross reactivity with other viruses after the 3base™ simplification process a large number of RNA and DNA samples were obtained from a number of human viruses. No cross reactivity was observed with any component of the assays using a wide range of both DNA and RNA containing human pathogens.

Molecular quality assurance panels obtained from QCMD for dengue, Zika virus and chikungunya from 2016 to 2018 demonstrated that the pan-flavivirus/panalphavirus/pan-dengue assays were in 100% concordance with the expected results. These results indicate that the simplification assays are performing well, if not better than other molecular assays used worldwide.

#### **8.4 Vanuatu 2016/2017 dengue outbreak**

T0 date traditional methods such as Enzyme Immuno Assays (EIAs) have been the method used for the detection of both flavi- and alphaviruses. It has been shows that dengue EIAs show and high degree of cross reactivity with Zika virus and likewise

Zika EIAs cross react with dengue [85, 86]. Unlike molecular approaches conventional EIAs are unable to differentiate the individual dengue serotypes and in addition are generally less sensitive than molecular assays. However, unlike the RNA simplification approach there are very few RT-PCR assays can target all members of complex groups such as flavivirus or alphavirus using a single primer and probe set.

There have been numerous outbreaks of arboviruses in the South pacific regions. From 2012 to 2014 it was estimated that at least 28 outbreaks of disease have occurred which were attributed mainly to dengue virus but notable outbreaks as a result of chikungunya and Zika virus were also recorded [87]. These outbreaks cause severe stress on both the public health system and on the islands economy which for the most part are tourist driven.

During 2016/2017 an outbreak of dengue fever occurred on the islands of Vanuatu [88]. Vanuatu consists of a group of over 80 islands that are located in the South Pacific region the largest of which is Efate home to over 86, 000 residents. The population on the rest of the islands range from as many as 46,000 to as low as a few hundred. From the 12th to 24th March 2017, we tested both archived and fresh samples obtained from Port Villa central hospital, Efate, to determine if the 3base™ pan-flavivirus, pan alphavirus and pan-dengue assays were useful in an outbreak situation. We included a dengue 2 specific primer and probe set since this was the genotype responsible for the outbreak. Samples were extracted using a small footprint automated extraction platform along with a small portable PCR machine weighing less than 2 kg.

Over the study period we tested 187 serum sample for the presence of dengue (see **Table 13**). One hundred and sixteen samples tested positive for the presence of panflavivirus, pan-dengue and the specific dengue 2 assay representing a positivity rate of 62%. Seven samples were inconclusive as only signals were obtained with the pandengue component of the assay which could be explained by a very low viral load in these particular samples.

When we plotted the dengue positivity from December to March (see **Figure 6**) we found that the number of positive dengue cases peaked in the month of January followed by a marked decline in positivity in February. Routine testing of patients with dengue like symptoms using the pan-family assays commenced in the middle of March and we found that the number of cases began to increase again at this time. As molecular methods are more sensitive than the conventional EIA assays the rise could be attributed to increased sensitivity of the pan-family assays [80].


#### **Table 13.**

*Results of clinical sample obtained during the Vanuatu outbreak.*

*Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

**Figure 6.**

*Shows the weekly positive results from December to the 24th March 2017.*

In addition, when we looked at the distribution of dengue cases across the islands, we found a statistically significant concentration of infection on the islands of Emae (*p* < 0.00001), Tongoa (*p* < 0.00001) and Ambae (*p* < 0.00001) compared to the regional average, which was calculated to be 0.416 per 1000 people, suggesting that these islands may possibly harbour animal reservoirs infected with the dengue virus.

#### **9. Conclusion**

In summary it has been shown that the pan-family screening approach is a sensitive and specific method for the detection of viral families that contain a large number of diverse pathogens. Viruses will continue to emerge from animal and avian hosts in the future and at present there are very few assays that can detect complex viral families. Coronaviruses are a good example of a family of viruses that have adapted well to human-to-human transmission. In just 20 years three significant pathogens, SARS-CoV-1, MERS-CoV and SARS-CoV-2, have emerged from zoonotic hosts and resulted in two epidemics and one global pandemic which has infected more than 620,000,000 people. It is likely that in the near future a new coronavirus variant will emerge and spill over into the human population resulting in significant morbidity and mortality.

Individual flavivirus and alphaviruses have to date shown different global distribution patterns. Yellow fever is predominately found in African and South America with JEV mainly confined to Asia. It has been suggested that new flaviviruses will continue to emerge or re-emerge into the human population which may cause more serious infections than previously realised as was the case with the recent Zika virus epidemics. Climate change [88] will challenge the current distribution of these

viruses globally as was demonstrated recently with JEV which for the first time was found in Victoria and New South Wales, Australia [89, 90]. The pan-family assays have been tested using insect vectors to screen for flavi- and alphaviruses and preliminary results look promising (John Waitumbi, personal communication) opening the potential of these assays to be used to screen arbovirus vectors for the presence of novel or re-emerging pathogens. One advantage of the current pan-flavivirus/panalphavirus/pan-dengue screening test is that the assays can be used in any region worldwide to quickly detect the presence of an unknown arboviral infection and with the boundaries to infection expanding their use is even more urgent.

It would be possible to design unique primer and probe sets that covered the major families of viruses that are pathogenic to the human population. These assays could be multiplexed to produce screening panels that could be used in front line hospitals or sentinel laboratories to screen animal, bats, birds, or vectors such as mosquitoes at regular intervals for emerging viruses. If a sample is positive using the pan-family assay but negative using species specific primers the sample could then be quickly screened by NGS to determine if a novel virus is present. In this way we would be forewarned to the presence of an emerging viral threat.

This simplifies and reduces the costs of broad screening approaches in disease outbreaks or during pathogen surveillance in humans, animal or vectors and importantly has the possibility to identify emerging pathogens without prior sequence knowledge.

#### **Acknowledgements**

We would like to thank all the staff past and present at Genetic Signature. In particular we would like to thank the late Dr. Geoff Grigg for conversation and suggestions which without his help would not have allowed development of this technology. We would also like to appreciate the support of staff at the Prince of Wales Hospital, Sydney, especially Professor William Rawlinson for advice and suggestions. In addition, we would like to thank the staff of the Vila Central Hospital for their support and kindness during the Vanuatu study period. I especially thank Crystal Garae, George Junior Pakoa and Kalkie Sero. Finally we would like to acknowledge Chris Abbott and Phill Isaacs for their continuing support of Genetic Signatures.

#### **Conflict of interest**

DM and JR are paid employees of Genetic Signature the inventors of 3base™ technology.

*Diagnosis of Viral Families Using a Nucleic Acid Simplification Technique DOI: http://dx.doi.org/10.5772/intechopen.109632*

### **Author details**

Douglas Millar\* and John Melki Genetic Signatures, Sydney, Australia

\*Address all correspondence to: doug.millar@geneticsignatures.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 9** Imaging in Dengue Fever

*Rolando Reyna*

#### **Abstract**

Dengue is a viral disease caused by a flavivirus transmitted by Aedes aegypti mosquitoes in tropical regions but has spread to regions of Europe, subtropical regions, and South America. The clinic is varied, so imaging methods are important before having a positive confirmatory test. Clinically, dengue is a disease that increases vascular permeability with loss of plasma and albumin, causing polyserotis. The most accessible imaging methods in the emergency room are chest radiography and abdominal ultrasound. Chest radiography shows that the most frequent finding is pleural effusion. Abdominal ultrasound has several findings, including thickened gallbladder wall, ascites, and hepatic and splenomegaly. The thickened gallbladder wall is an indicator of disease severity since the more severe the thickening, the more severe the clinical picture. The patient's platelet count is also related to the ultrasound findings, since the lower the platelet count, the more severe is the thickened gallbladder wall. The differential diagnosis of dengue should include other febrile states such as influenza, Zika, Chikungunya, and COVID-19.

**Keywords:** dengue, abdominal ultrasound, pleural effusion, gallbladder wall thickening, ascites

#### **1. Introduction**

Dengue is the most important arboviral infection affecting humans and presents a major challenge for public health services worldwide.

Most infections are asymptomatic or result in only a brief systemic viral illness; a small proportion of patients develop potentially fatal complications.

Although dengue fever disease is mild in most cases and does not progress to severe disease, it can cause many cases in an epidemic form, resulting in overcrowding of health services. Therefore, the ability to recognize cases that progress to severe disease is important.

The World Health Organization classifies dengue into two main categories: dengue with or without warning signs and severe dengue. The secondary classification of dengue with or without warning signs is designed to assist health care professionals in selecting patients for hospital admission for close observation and to minimize the risk of progression to the more severe form of dengue.

The differential diagnosis should be made with febrile states (especially if it is in time of dengue epidemic), such as influenza, Zika, Chikungunya, Hanta Virus (in regions with endemic cases of hanta), and COVID-19.

#### **2. Imaging methods**

The most frequently used imaging methods in dengue are chest radiography and abdominal ultrasound, especially in emergency rooms.

The initial evaluation of a patient with dengue is with chest X-ray, and according to the clinical picture and its evolution, other diagnostic methods are requested [1, 2].

Dengue consists of a significant increase in vascular permeability, with loss of plasma and albumin from the intravascular space, causing polyserositis.

Abdominal ultrasound is a widely available imaging technique to study abdominal pain and acute febrile processes. It allows to assess with a high degree of certainty the abdominal findings related to dengue fever, which are thickening of the gallbladder wall, ascites, hepatomegaly and splenomegaly, pericardial effusion, and pleural effusion [2–4].

In chest radiography, pleural effusion is the most frequent finding, which can be unilateral or bilateral, of variable quantity and mainly on the right side. In cases of severe dengue, it may demonstrate the presence of vascular congestion or lead to acute respiratory distress syndrome [4, 5].

#### **3. Imaging findings related to dengue fever**

#### **3.1 Gallbladder wall thickening**

It is one of the most frequent findings, but it is non-specific since it is found in other viral infections, cholecystitis, liver cirrhosis, and portal hypertension. There are different forms of gallbladder wall thickening that can be observed in ultrasound. These can be lamellar or layered, diffuse, and reticular thickening. Of these forms of thickening, the diffuse thickening is the most frequent form. Lamellar and reticular thickening are observed more frequently in children or young adults. Reticular thickening is more frequent in patients with severe dengue. This type of thickening is usually located at the bottom of the gallbladder **Figures 1** and **2** [4, 5].

#### **3.2 Ascites**

Ascites develops with the pathophysiological process of polyserositis, correlating with the severity of the disease. Ascites is detected on physical examination when it exceeds 1000 cc in volume, while ultrasound can demonstrate the existence of scant amounts of peritoneal fluid (approx. 100 cc). Its appearance is usually anechoic and may be of variable quantity. **Figures 3** and **4** [5, 6].

#### **3.3 Pleural effusion**

As in ascites, pleural effusion is part of the process of polyserositis, resulting in plasma leakage into the pleural cavity. It is generally an infrequent finding being right or bilateral. Pleural effusion in dengue is one of the markers of severity, but it is mild and self-limiting without the need for intervention. The type of pleural effusion is exudative. **Figures 5** and **6** [4–7].

**Figure 1.** *Abdominal ultrasound axial section. Diffuse gallbladder wall thickening is observed diffusely, (white arrow).*

#### **Figure 3.**

*Abdominal ultrasound. The presence of free fluid around the right kidney is observed at the level of Morrison's fossa (White arrow).*

**Figure 4.** *Free fluid in the pelvic excavation. UB: urinary bladder, (White arrow).*

**Figure 5.** *Chest X-ray. Right costal diaphragmatic angle obliteration due to pleural effusion (arrow).*

#### **3.4 Hepatomegaly and splenomegaly**

Both hepatomegaly and splenomegaly's growth is homogeneous, without focal lesions. In some cases, the liver may present steatosis. Liver growth may be present in up to 30% of cases of dengue fever. Splenomegaly may be present in 14% of cases. **Figure 7** [7].

#### **3.5 Pericardial effusion**

It may occur in severe cases after the fifth or seventh day of illness in up to 28% of cases. Its sonographic characteristic is a simple anechoic effusion [7, 8].

There may be a combination of sonographic findings in a patient with a diagnosis of dengue. We can find gallbladder wall thickening with ascites and pleural effusion at any age.

### **4. Platelet count and imaging findings**

Several hematological parameters have been considered as potential predictors, most commonly the platelet count.

The severity of the course of the disease, which is directly linked to the platelet count, can also be assessed by sonography.

#### **Figure 7.** *Abdominal Ultrasound. Hepatic cross-sectional view. Shows mild liver enlargement with homogeneous liver parenchyma.*

*Imaging in Dengue Fever DOI: http://dx.doi.org/10.5772/intechopen.109858*

In patients whose platelet counts are less than 40,000, the most frequent findings are gallbladder wall thickening, ascites, and pleural effusion. With platelet counts between 40,000 and 80,000, the most frequent findings are gallbladder wall thickening and pleural effusion. With platelet counts greater than 80,000, pleural effusion is more frequent followed by gallbladder wall thickening [7].

#### **5. Conclusion**

In the clinical context of a patient with suspected dengue fever, findings of gallbladder wall thickening, ascites, pleural effusion, and hepato-splenomegaly strongly favor the diagnosis of dengue fever. An abdominal ultrasound examination can effectively recognize these and guide the clinician to initiate prompt treatment without waiting for serologic results. Ultrasound can also estimate the severity of the disease. The degree of thrombocytopenia shows a direct relationship with abnormal ultrasound findings.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Rolando Reyna Saint Thomas Hospital, Panama City, Republic of Panama

\*Address all correspondence to: rolando0572@gmmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Castrillón ME, Iturrieta N, Cativelli S, Padilla F. Hallazgos ultrasonográficos en pacientes con Dengue. Revisión de la literatura. Revista argentina de radiología. 2010;**75**:71-76

[2] Sophie Y, Bridget W. Predicting outcome from dengue. BMC Medicine. 2014;**12**:147

[3] Basawaraj NG, Dasan TA, Patil SS, Deepashri B. Sonography in the diagnosis and assessment of dengue fever. International Journal of Research in Medical Sciences. 2015;**3**(11):3131-3136

[4] Venkata Sai PM, Krishnan R. Role of ultrasound in dengue fever. British Journal of Radiology. 2005;**78**:416-418

[5] Sachar S, Goyal S, Sachar S. Role of ultrasonography ("Honeycomb Sign") in early detection of dengue hemorrhagic fever. Archives of Clinical and Experimental Surgery. 2013;**2**(1):38-42

[6] Oliveira GA, Machado RC, Horvat JV, et al. Transient reticular gallbladder wall thickening in severe dengue fever: A reliable sign of plasma leakage. Pediatric Radiology. 2010;**40**:720-724

[7] Vedaraju KS, Vijay Kumar KR, Vijayaraghavachari TV. Role of ultrasound in the assessment of dengue fever. International Journal of Scientific Study. 2016;**3**:59-62

[8] Shabbir M, Ameen F, Roshan N, Israr M. Nature and clinical course of pleural effusion in dengue fever. Internal and Emergency Medicine. 2018;**1**(1):1006

#### **Chapter 10**

## Dengue Virus Gene-Silencing Techniques: A Current Assessment

*Samir Casseb, Karla Melo, Carolina Santos and Edna Franco*

#### **Abstract**

Infection with the Dengue virus (DENV) has become a global threat, affecting approximately 100 nations. There is not a recognized antiviral treatment for dengue at the moment. Therefore, it is crucial to create therapeutic approaches to treat this fatal condition. A critical and successful method of silencing genes, RNA interference breaks down targeted RNA according to its sequence. Over the past ten years, a number of studies have been carried out to determine how well siRNA works to prevent dengue virus replication. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most effective and widely used tools for RNA and DNA manipulation in numerous organisms. In our review, we describe and discuss the use of these technologies to comprehend and treat DENV-related infections.

**Keywords:** dengue, CRISPR, RNAi, genetic engineering, siRNA

#### **1. Introduction**

Dengue is the most dangerous virus spread by mosquitoes, and any of the four DENV serotypes (DENV-1 to DENV-4) can cause it. A DENV infection can cause a broad spectrum of clinical symptoms, ranging from a mild flu-like condition known as dengue fever (DF) to the potentially fatal dengue shock syndrome (DSS) [1].

Approximately half of the global population is at risk for Dengue fever, and the mosquito-borne virus is the leading cause of death in certain Latin American and Asian nations. Nevertheless, despite the rapid increase in cases and decades of drug development efforts, there is no specific treatment and only one vaccine with a limited application [2].

Symptoms of DF include fever, nausea, vomiting, rash, and aches and pains; however, in DSS, severe hemorrhage and shock can develop, and if left untreated, the fatality rate can reach 20%. Previously, the World Health Organization (WHO) classified dengue disease states as undifferentiated fever, dengue fever, and dengue hemorrhagic fever (DHF) [3].

The categorization of DHF was revised into four levels of severity, with grades III and IV being classified as DSS. However, in 2009, the WHO updated its case categorization method, discontinuing the previous categories of probable Dengue, Dengue with unexpected symptoms, Dengue with warning symptoms, and severe Dengue. Currently, the focus is on understanding DENV's biology, epidemiology, and transmission characteristics, including circulating serotypes and genotypes, DENVspecific immune responses, illness etiology, improved diagnostic tools, therapies, and vaccine development [2, 3].

There is no antiviral treatment for dengue fever, and the only approved vaccine, Dengvaxia from Sanofi, can be dangerous. Dengvaxia can reduce the severity of Dengue fever in previously infected individuals. However, Dengvaxia may increase the risk of severe Dengue in uninfected individuals [4].

Experts say that the development of vaccines and antivirals has been slowed down by poorly coordinated clinical trials, problems with animal models and lab tests, and a complicated and constantly changing virus. Experts say that manufacturers could make progress on this disease if they simplified the endpoints for symptoms in clinical trials and used less common designs like platform trials and human challenge studies [5].

In this way, scientists worldwide have been working hard to find treatments and ways to avoid getting sick. In the search for treatments to stop the spread of DENV, new technologies like RNA interference (RNAi) and CRISPR have become more popular.

#### **2. Dengue treatment technologies**

As previously stated, diverse dengue treatment technologies are currently being developed. Our text will elaborate on RNAi and CRISPR, two technologies that are getting more and more interesting in this field.

#### **2.1 RNAi**

Post-transcriptional gene silencing (PTGS) is observed in many species, including plants, fungi, and animals. RNA interference (RNAi), an ancient defense mechanism, is the common denominator [6].

When put into cells, long dsRNA can efficiently and precisely lead to the degradation of cognate mRNAs in a way that depends on the gene. This powerful technology has been used to change how genes are expressed, determine how signals are sent, and determine what genes do on a whole-genome scale [7].

Researchers worldwide have used RNA interference (RNAi) for basic research. They are currently making drugs based on RNAi to prevent and treat viral infections, tumors, and metabolic disorders in humans [8].

Although there have been significant improvements in the treatment of viral diseases, current medications and vaccines are still limited by a variety of issues, including toxicity, complexity, cost, and resistance. Eukaryotic get a defense mechanism called RNAi that helps them avoid getting infected by viruses [9].

Viral mRNA is sent to cellular enzymes to be broken down, which can stop the production of crucial viral proteins. Human cells can now be protected from viruses that cause disease thanks to new technology [10].

#### *2.1.1 Machinery of RNAi*

Through biochemical and genetic research, scientists have discovered how dsRNA causes the breakdown of target messenger RNA at the molecular level. RNA interference involves the initiation and effector steps [9].

#### *Dengue Virus Gene-Silencing Techniques: A Current Assessment DOI: http://dx.doi.org/10.5772/intechopen.110421*

Dicer, a member of the RNase III family of ATP-dependent ribonucleases, binds to long dsRNA (introduced directly or via a transgene or virus) with high affinity and cleaves it into small interfering RNA (siRNA) duplexes. An N-terminal DEXHbox RNA helicase domain, a domain with an unknown function (DUF283), a PAZ domain, two RIII domains, and a dsRNA-binding domain are all common features of dicer enzymes (dsRBD). In order to create siRNAs or microRNAs (miRNAs), the dicer can cut stem-loop precursors from dsRNA [11].

siRNAs are dsRNA duplexes with 21–23 nucleotides, 2-nt 3′ overhangs, a 5′-monophosphate, and a 3′-hydroxyl group. During the "effector" (RISC) step, siRNA duplexes are incorporated into the RNA-induced silencing complex (RISC). The phosphorylation of the 5′-terminus of siRNA is required for entry into RISC. A helicase domain of RISC binds to one end of the duplex and unwinds it ATPdependently [12].

The thermodynamic stability of the initial few base pairs of siRNA can affect the proportion of RISC containing antisense or sense siRNA strands. Dicer with R2D2 (Dcr-2-associated protein) binds siRNA and assists with its loading onto RISC. The active RISC then identifies the homologous transcript via base-pairing interactions and cleaves the mRNA between the 10th and 11th nucleotides of the 5′ end of the siRNAs [13, 14].

Animals make these short RNA species using Dicer to cut long (70 nt) endogenous precursors with an imperfect hairpin RNA structure into short RNA species. Mature miRNAs stop translation by partially matching their bases to the 5′ or 3′ ends of mRNAs. A miRNA that is completely complementary to its target mRNA (endogenous siRNA) can cause the target mRNA to be broken down [15, 16].

Furthermore, it is likely that many other proteins, such as eukaryotic translation initiation factor 2C2 (eIF2C2) and Argonaute proteins, work in both pathways. Argonaute proteins are the essential RISC components. With two distinct domains, PAZ and PIWI, they are evolutionarily conserved. The PIWI domain is exclusive to Argonautes, whereas the PAZ domain is shared with proteins 21 from the Dicer family [13, 17].

#### *2.1.2 Silencing mechanisms of RNAi*

The mRNA targets multiple siRNA sequences, and long dsRNA effectively stops the gene from being expressed. Virus-infected cells always produce dsRNA, but viruses can evade a severe cellular response. The dsRNA binds to dsRNA-binding proteins (dsRBPs), which have been shown to stop RNA interference (RNAi) and block the effects of interferon (IFN). Recent research has shown that 21-nucleotide siRNAs cannot cause mammalian cells to make interferon. Since siRNAs can stop viruses from spreading, more and more scientists are becoming interested in this field [16].

It has been shown that siRNA molecules can stop a virus from spreading by sending viral mRNA to be broken down. Compared to other conventional medications, siRNA has numerous advantages. Because sequence-specific target mRNA and complementary siRNA make it much easier and more flexible to choose target sites, siRNAs can stop mRNA from doing its job by going after different parts of the target mRNA for a given mRNA molecule. Second, to silence a gene, a substoichiometric amount of siRNA is enough to reduce homologous mRNA by a lot within 24 hours [18].

Third, siRNAs can cause cognate mRNA to break down in the cells of different species. Scientists are working on siRNA delivery systems that will make it easier for siRNA to get into the cells of almost all organs. Fourthly, siRNAs appear to have no negative effect on cell control mechanisms. The length of the siRNA and how similar it is to the target region of the cognate transcription make sure that only the desired transcript will be destroyed. siRNAs lacking suitable targets appear to be inactive within cells. The best thing about RNAi as a way to fight viruses is that it is very specific and does not have any bad side effects.

Fifthly, siRNAs can effectively silence genes. Using plasmid and viral vectors, siRNAs can exhibit their long-lasting biological effects. The siRNAs made in vivo or in vitro and then put into cultured cells or animals may silence messenger RNA (mRNA) molecules based on their sequence. Since proof-of-concept studies showed that siRNAs work, they have become a popular alternative therapy [19].

#### *2.1.3 RNAi and DENV*

RNA interference is an exciting field of functional genomics that can silence viral genes. This virus-fighting system, found in many eukaryotes, could be used to treat flavivirus infections in hosts. However, RNA interference against flaviviruses has received scant research [20, 21].

RNAi has been utilized against multiple human pathogens, such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, poliovirus, influenza virus A, and DENV. In the cytoplasm, the ssRNA genomes of these viruses are visible and could be used as RNAi targets. Between viral RNA uncoating and viral replication, this occurs [22].

Certain mosquitoes are capable vectors of arthropod-borne viruses (arboviruses), while others are not. It has been established that Aedes species possess a Rnai pathway. The first piece of evidence is that recombinant Sindbis viruses expressing an RNA fragment from a genetically unrelated dengue-2 virus (DENV-2) inhibit DENV-2 replication in *Aedes aegypti* mosquitoes in a manner analogous to how plants shut down genes [6].

The second evidence is that the replication of the homologous virus is stopped when dsRNA or siRNA made from the arbovirus genome is put into C6/36 (*Aedes albopictus*) cells. The third evidence is that virus-resistant C6/36 cells were made from DENV-2-specific hairpin RNA copied from a plasmid. These things show that RNA interference is present in *Aedes* species, just like in plants and other animals [23].

Both innate and adaptive immune responses highly influence the DENV infection, but little is known about the innate immune response of the mosquito vector *A. aegypti* to arbovirus infection. DENV-2 does not completely evade RNA interference, as silencing the expression of dcr2, r2d2, or ago2 genes increases virus replication in vectors and shortens the extrinsic incubation period for viral transmission. Sánchez-Vargas and his team showed that RNA interference is a key factor in controlling mosquito infections [24].

Dendritic cells (DC) infected with AAV-siRNA demonstrated a dose-dependent reduction in dengue infection. DCs treated with AAV-siRNA were also protected from dengue-induced apoptosis. Thus, AAV-mediated siRNA delivery can reduce dengue infection and replication in humans. Through extensive siRNA screening, more than 100 proteins of host factors involved in DENV replication have been identified. In drug design, these host factors serve as drug targets. Host factors (proteases, glucosidases, other) have yet to be identified via siRNA screening. Also, these studies could not find genes for natural immunity that protect against DENV infection. The biggest problem is getting siRNA to patients; a good way to do that has yet to be found [25].

#### *Dengue Virus Gene-Silencing Techniques: A Current Assessment DOI: http://dx.doi.org/10.5772/intechopen.110421*

The fact that DENV-2-derived siRNA was found in RNA extracts from the midguts of Carb77 and that the resistance phenotype was lost when the RNAi pathway was blocked [26] showed that an RNAi response caused DENV-2 resistance. C6/36 cells transfected with siRNA against the dengue PreM gene were then attacked by the DENV1 virus [25].

After seven days, the number of transfected cells that were still alive increased by 2.26 times, while the amount of virus RNA dropped by 97.54 percent. This finding provides evidence that siRNA inhibits dengue replication effectively [27]. Mukherjee et al. [28] showed that DENV can replicate in Drosophila S2 cells and that the RNAi pathway controls DENV replication. The downregulation of HSP60 in infected cells reduced viral load, RNA copy number, and IFN concentration [29].

High levels of HSP60 in infected cells make it easier for viruses to multiply and could be a target for treating dengue infection. RNAi, plasmid transfection, and inducible vectors can temporarily turn off genes' effects. siRNA is extremely specific for target RNA. Therefore, siRNA is important for discovering and understanding gene function [29, 30].

Using siRNA to silence the attachment receptor and clathrin-mediated endocytosis, it is possible to lower the amount of virus in like this using siRNA to stop the attachment receptor and clathrin-mediated endocytosis, the amount of virus in the body can be lowered. Thus, preventing the progression of dengue fever to more severe forms (DHF/DSS) [31].

Importantly DENV infection identified key cellular genes involved in endocytosis and cytoskeletal dynamics. siRNA targeting genes involved in clathrin-mediated endocytosis prevented DENV entry into Huh7 cells [32]. Villegas-Rosales et al. [26] recently found that three siRNAs that target NS4B and NS5 sequences can silence four DENV genome serotypes.

Combining siRNA and endogenous RNAi processing machinery can prevent severe dengue infection. DC-3 siRNA is a new way to fight against different serotypes of Dengue, so it can help develop new treatment plans [33].

Korrapati et al. [34] used a human adenovirus type 5 vector that could not replicate to target conserved viral genome sites with short-hairpin RNA. This shorthairpin RNA grows into the corresponding siRNA and stops all four dengue serotypes from making antigens and more viruses.

These studies and their clear results show that RNA interference prevents DENV from replicating in cell cultures and animal models [35].

#### **2.2 CRISPR**

This adaptive immune response protects bacteria and archaea from bacteriophages and plasmids. CRISPR-Cas immunity is mediated by crRNA and an endonuclease Cas that targets genetic elements. The mode of action includes three distinct phases: acquisition, expression, and interference. In the acquisition step, foreign nucleic acids are added in a specific order as new CRISPR spacers to a CRISPR array made up of repeat sequences. This creates a memory of the genetic elements outside the cell [36–38].

The CRISPR locus is turned into a pre-CRISPR RNA transcript (pre-crRNA) during the expression step. This pre-crRNA is then changed into a mature crRNA that has some CRISPR spacer sequences joined to some CRISPR repeats. A transactivating RNA (tracrRNA) is also made by the CRISPR locus. Its repeat regions match those of the crRNA transcripts. In addition to the CRISPR array, the CRISPR locus can code for one or more Cas nucleases, such as Cas9. During the interference phase, the repeat region sequences that match each other bind to make a hybrid of crRNA and tracrRNA. This RNA hybrid tells the Cas nuclease to go after complementary DNA sequences. This allows invading genetic elements to be found and cut out [39, 40].

Most CRISPR effector proteins depend on a protospacer-adjacent motif (PAM) in the targeted nucleic acid, like NGG for Cas9. The PAM is essential for self-DNA recognition, cleavage, and differentiation from non-self DNA [41].

For Cas9, perfect complementarity will cause the endonuclease to change shape, leading to a structure that can cut DNA. The protein and RNA parts of Streptococcus pyogenes's class 2 CRISPR system have been changed to work in eukaryotic cells, like human cells [42].

Mammalian cells send Cas9 to the nucleus by joining it to a nuclear localization signal (NLS) that works best with human codons. To make single-guide RNAs (sgRNAs) for editing the genome that looks like the natural crRNA–tracrRNA hybrid, crRNA-like sequences are fused to a partial tracrRNA using a synthetic stem-loop [43].

#### *2.2.1 Gain-of-function approaches*

Strategies that use the ectopic overexpression of genes have helped find cell surface receptors needed for viruses to get into cells and host factors that stop viruses from getting into cells. An infection-resistant cell line is often transduced with a complementary DNA library (cDNA library) made from an infection-permissive cell type to find entry receptors. In a cDNA library made from hepatocellular carcinoma cells and a non-permissive cell line, claudin 1 (CLDN1) and occludin (OCLN) were found to be HCV entry receptors [44, 45].

In addition to identifying receptors, an independent expression screen revealed that SEC14-like protein 2 (SEC14L2), a cytosolic lipid-binding protein, promotes the replication of clinical strains of HCV20. Also, proteins important for the immune system's natural defenses against DNA and RNA viruses were found using a library of about 380 interferon-stimulated genes (ISGs) [46–48].

In addition to these screens, full cDNA libraries with all annotated ORFs from humans have been cloned into lentiviral expression vectors. This has led to the creation of an expression vector library, which will likely make the gain of function screens more useful for studying the interactions between a host and a pathogen [49, 50].

#### *2.2.2 Function loss genetic analyses*

Screens for loss of function rely on the stable knockdown or knockout of genes. Initial RNA interference-based approaches have yielded valuable insights into virushost relationships [50].

In contrast to RNAi, which only stops some genes from being expressed, recent technological advances have made it possible to stop all genes from being expressed. One way is to use insertion mutagenesis to change genes in haploid cell lines in culture. This is called "haploid genetic screening." Retroviral gene traps with a splice acceptor site, for example, can become part of the host genome and cause truncated mRNA transcripts to be made. Completely turning off the expression of a gene can have big effects on viral replication and help figure out which parts of the host are most

#### *Dengue Virus Gene-Silencing Techniques: A Current Assessment DOI: http://dx.doi.org/10.5772/intechopen.110421*

important for viral infection. Using insertion mutagenesis in haploid cells, researchers have found the essential receptors for many viruses, like Ebola and Lassa [51, 52].

As receptors, both viruses utilize abundant lysosomal proteins. The interaction between the Ebola virus glycoprotein and its receptor Niemann–Pick C1 protein (NPC1) is set off by cathepsin cleavage. In contrast, the interaction between the Lassa virus glycoprotein and its receptor lysosome-associated membrane glycoprotein 1 (LAMP1) is set off by acidification of the endosome. Subsequent structural studies determined the viral glycoprotein and NPC1 binding interface. During the 2013–2016 Ebola epidemic, several mutations occurred in the host-binding site of the viral glycoprotein [53–56].

These changes made the virus more infectious in cells from primates but not in cells from rodents. This implies that they aided the virus's adaptation and spread in humans. Haplotypic genetic screens helped find a cellular phospholipase that lets viruses get around an antiviral restriction mechanism that works against many *picornaviruses* [57].

Recently, a haploid screen found a protein-based receptor that allows multiple different serotypes of adeno-associated virus (AAV) to enter cells. This may change how AAV is used as a vector for gene therapy. Loss of function screens is a good way to find out which host factors are necessary for viral replication, as shown by these and other studies [58].

#### *2.2.3 Insights from CRISPR-CAS screens*

CRISPR-Cas screens have a great potential for identifying host factors essential for viral pathogenesis, which could lead to developing new antivirals. CRISPR-Cas screens have been used to study several viruses [59].

CRISPR-Cas screens could find host factors essential for viral pathogenesis, which could lead to developing new antivirals [60].

Using CRISPR-Cas screens, the cotranslational and posttranslational insertion of several membrane-spanning hydrophobic helices and polyprotein cleavage by a viral protease and several host proteases into the mature viral proteins have been studied. Even though these processes are known, not enough is known about the involved host proteins [61, 62].

Using DENV, different CRISPR-Cas screens have each found several ER proteins needed for the virus to spread. A lot of these proteins are needed for the endoplasmic reticulum (ER) to do its important job of making membrane and secretory proteins [63].

The identified proteins have been implicated specifically in N-linked glycosylation, ERAD, and signal peptide insertion and processing. Notably, these proteins were identified in duplicate screens conducted in the same lab as well as independent screens conducted in separate labs using distinct cell lines and virus strains. There was also substantial overlap between the results of haploid genetic testing. This technology's remarkable reproducibility is a major advantage [64].

Furthermore, CRISPR-Cas technology is a reliable way to test candidate genes and figure out how gene knockouts affect a virus copies itself. Gene knockouts differ from knockdown methods like RNA interference (RNAi) because they are permanent and do not lead to different levels of depletion. This lets people use quantitative tests for virus replication, like quantitative PCR, immunostaining, or plaque assays, to compare genes accurately. When the most enriched host factors were taken out of the screens, flavivirus replication dropped by 100–10,000,000. This shows that pooled sgRNA screens could be used to find host factors needed for virus replication [65].

CRISPR-Cas knockout cells can also be used to understand the molecular basis of knockout phenotypes and find out which stage of a virus's life cycle the host factor is involved. For instance, it was discovered that the OST complex is required for viral RNA synthesis but not for viral entry and translation [63].

The OST complex glycosylates newly synthesized proteins via N-linked glycosylation. In mammalian cells, there are two different OST multiprotein complexes. Each comprises a catalytic subunit (one of two paralogs, STT3A or STT3B) and accessory subunits [66].

DENV replication needs both isoforms, as either knocking out STT3A or STT3B stopped DENV replication completely. Other *flaviviruses* that are spread by mosquitoes, like ZIKV, only use the STT3A isoform for viral RNA replication. This strongly suggests that the virus and the host interact differently. Inactive mutant proteins were able to bring DENV replication back to the knockout cells. This proves that the OST complex plays a role in DENV replication that was not expected. Multiple viral proteins that are not structural but are part of the RNA synthesis complex at ER61 were found to bind to the OST complex. This suggests that the OST complex is a framework to help create a DENV RNA replication complex that works [67, 68].

SEC61A1 and SEC63, which form the translocon channel in the ER membrane; the translocon-associated protein (TRAP) complex, which stimulates cotranslational translocation of polypeptides into the ER73; and the signal peptidase complex, which cuts signal peptides in the ER lumen are also essential for flavivirus replication. Multiple *flaviviruses* exhibited severe polyprotein cleavage deficiencies when a subset of signal peptidase complex subunits (SPCSs) was absent. Particularly, the cleavage of the structural proteins prM and E from the polyprotein was impaired, resulting in significant defects in virus particle release [69, 70].

Other host factors essential for flavivirus replication include SEC61A1 and SEC63, which form the translocon channel in the ER membrane; the translocon-associated protein (TRAP) complex, which stimulates cotranslational translocation of polypeptides into the ER73; and the signal peptidase complex, which cuts signal peptides in the ER lumen. Multiple flaviviruses exhibited severe polyprotein cleavage deficiencies when a subset of signal peptidase complex subunits (SPCSs) was absent. In particular, separating the structural proteins prM and E from the polyprotein was hard for the virus particles to get out of the cell [71].

It is important to know that genetic screenings of WNV and DENV have not found a specific receptor for viruses to enter host cells. This is not the case with many other viruses, such as Ebola. This is probably because there is more than one way for a virus to get into a cell. If a virus receptor is knocked out, the cell is still open to infection in a different way. Indeed, numerous DENV receptors have been identified. Nevertheless, CRISPR-Cas screens have contributed to our understanding of flavivirus biology by revealing the central role of ER complexes in flavivirus infection promotion [51, 71].

#### *2.2.4 CRISPR-CAS antiviral strategies*

The CRISPR-Cas technology could be used to prevent and treat diseases by going after viruses and the things that spread them. Vector control has been used to stop the spread of viruses carried by vectors, like ZIKV, DENV, and yellow fever [72].

Using CRISPR-Cas tools, scientists have made gene drives that could reduce the number of mosquitoes. CRISPR-Cas technology could also be used to treat HIV, HBV, HCV, and the herpes simplex virus, which do not go away on their own. HBV

*Dengue Virus Gene-Silencing Techniques: A Current Assessment DOI: http://dx.doi.org/10.5772/intechopen.110421*

covalently closed circular DNA (cccDNA), a sign of a persistent HBV infection, has been successfully targeted in cell cultures and animal models [73–75].

Additionally, CRISPR-Cas screens can be utilized to determine the mechanism of action of antivirals. For example, CRISPR-Cas and short hairpin RNA (shRNA) screens were used to determine how the antiviral drug GSK983 works. This drug may stop a wide range of RNA and DNA viruses. By stopping the enzyme dihydroorotate dehydrogenase from making pyrimidine in cells and lowering the number of nucleotides inside cells, which are needed for viral nucleic acid synthesis, GSK983 was found to stop viruses from spreading [76, 77].

#### **3. Conclusions**

Technologies like CRISPR and RNAi have become important ways to learn more about how viruses, like DENV, cause infections.

In addition, it is noteworthy that both CRISPR and RNAi have emerged as viable alternatives for treating viral infections and managing *aedes* vectors.

The new information we get from these technologies will be significant for a better understanding of how viruses replicate and interact with their hosts.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Samir Casseb\*, Karla Melo, Carolina Santos and Edna Franco Instituto Evandro Chagas, Universidade Federal do Pará, Ananindeua, Brazil

\*Address all correspondence to: samircasseb@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 5
