**14. Long intergenic noncoding RNAs (lincRNAs) and their association with DENV-host interaction**

Considering as another important class of regulatory RNAs, lincRNAs (sometimes known as dark matter) have various biological functions including genomic imprinting and cell differentiation specifically in host-pathogen interaction [100]. A growing number of evidences also reflect their possible role in gene regulation either epigenetics or nonepigenetics. However, their role in immune cell differentiation and activation is poorly understood, but the recent discoveries show their potential role in defense system as well as rapid responses to various stimuli and stress factors. Some studies also predict their active role to enhance viral replication or decrease antiviral immunity. It was also depicted that some lincRNAs also interact with other noncoding RNAs like miRNAs. The *Ae. aegypti*-linked lincRNAs are shorter in length (approximately 3000 nucleotide bases) than their protein-coding genes. Similarly, their GC content was also lower (mean:40.1%) than their protein-coding gene sequences (mean:47.8%). However, the AT enrichment or lower GC content is a typical characteristic of lincRNAs and congruent with other lincRNAs in other species [100].

Etebari et al. identified lincRNAs in dengue fever vector *Ae. aegypti* and demonstrated their potential role in host antiviral defense [100]. They evaluated lincRNA's expression in DENV serotype 2 (DENV-2) and *Wolbachia*-infected and noninfected adult mosquitoes as well as in Aa20 cells. The findings revealed the increased number of host lincRNAs under the circumstances of DENV-2 infection, some of which inhibit viral replication in mosquito cells. Furthermore, the silencing of only one lincRNA\_1317 by RNA interference enhanced the viral replication in host cells, which clearly indicates their possible role in host antiviral defense. The lincRNA\_1317 suppression was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). It was found that the lincRNA\_1317 expression was increased substantially upon the progression of infection, indicating the possible role of this lincRNA in antiviral defense. The findings might be consistent as the highly overexpressed lincRNA\_1317 expression (2.33 fold) was reported in *Wolbachia-*infected mosquitoes as compared to noninfected [100].

technical issues such as library preparation. In addition to RNAi, piRNAs (piwi RNAs) have been reported in mosquitoes and knockdown of piRNAs-related specific proteins indicates their endogenous antiviral activity instead of well-established exogenous RNAi activities in mosquitoes. Similarly, virus-specific piRNAs with typical A10 bias in sense RNA and U1 bias in antisense RNA have been reported to be produce by Bunya viruses and alphaviruses. However, the piRNAs expressed by dengue virus and cell fusing agent virus have only A10 bias in sense RNA. Interestingly, the PCV-specific small RNAs do not show either A10 or U1 bias features. In this scenario, it is uncertain that either they are piRNAs or just viral degradation products. This situation is very similar seen in other flavivirus-specific piRNAs, which map to very small number of sequences in the genome where the presence of one copy of genome indicates more specific targeting than random RNA degradation. The production of virus small RNAs and piRNAs through vDNA synthesis is also documented in mosquitoes or in their derived cell lines. It is worth mentioning to note the contribution of vDNA in the production of small RNAs of virus and piRNAs in mosquitoes, although insect-specific

viruses like PCV establish persistent infection in their hosts [99].

**association with DENV-host interaction**

104 Current Topics in Tropical Emerging Diseases and Travel Medicine

congruent with other lincRNAs in other species [100].

**14. Long intergenic noncoding RNAs (lincRNAs) and their** 

Considering as another important class of regulatory RNAs, lincRNAs (sometimes known as dark matter) have various biological functions including genomic imprinting and cell differentiation specifically in host-pathogen interaction [100]. A growing number of evidences also reflect their possible role in gene regulation either epigenetics or nonepigenetics. However, their role in immune cell differentiation and activation is poorly understood, but the recent discoveries show their potential role in defense system as well as rapid responses to various stimuli and stress factors. Some studies also predict their active role to enhance viral replication or decrease antiviral immunity. It was also depicted that some lincRNAs also interact with other noncoding RNAs like miRNAs. The *Ae. aegypti*-linked lincRNAs are shorter in length (approximately 3000 nucleotide bases) than their protein-coding genes. Similarly, their GC content was also lower (mean:40.1%) than their protein-coding gene sequences (mean:47.8%). However, the AT enrichment or lower GC content is a typical characteristic of lincRNAs and

Etebari et al. identified lincRNAs in dengue fever vector *Ae. aegypti* and demonstrated their potential role in host antiviral defense [100]. They evaluated lincRNA's expression in DENV serotype 2 (DENV-2) and *Wolbachia*-infected and noninfected adult mosquitoes as well as in Aa20 cells. The findings revealed the increased number of host lincRNAs under the circumstances of DENV-2 infection, some of which inhibit viral replication in mosquito cells. Furthermore, the silencing of only one lincRNA\_1317 by RNA interference enhanced the viral replication in host cells, which clearly indicates their possible role in host antiviral defense. The lincRNA\_1317 suppression was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). It was found that the lincRNA\_1317 expression was increased substantially upon the progression of infection, indicating the possible role of this The authors also described lincRNAs potential involvement in mosquito-pathogen interaction by determining its association with host-endogenous small RNAs and its direct interaction with DENV-2 infection. lincRNA\_1317 was not found to be located in any of the known piRNA clusters; however, no differences were found in the mapping pattern and mapped read length distribution when reads from DENV-infected and noninfected small RNA libraries were mapped to lincRNA\_1317. Gene silencing like role of piRNAs on lincRNA\_1317 transcriptome was also speculated. However, a little information is available about piRNAmediated lincRNA although some recent studies predict piRNA-mediated degradation of lincRNAs in mouse's late spermatocytes [100].

The researchers also tested the hypothesis that *Ae. aegypti* lincRNA\_1317 response to microbial challenge could be due to cross regulation between miRNAs and the lincRNAs. For this purpose, the normalized minimum free energy (mfe) of hybridization for each *Ae. aegypti* miRNA and lincRNA\_1317 was calculated by using RNAhybrid core script. The basic objective was to identify *Ae. aegypti* miRNA recognition elements on lincRNA\_1317. The results were quite interesting as binding sites enrichment for a few miRNAs with more than two recognition elements were detected on lincRNA\_1317 (e.g., more than four recognition sites for miR-278-5p and miR-252-3p were predicted on lincRNA\_1317). Furthermore, some hot spots for miRNA recognition sites on lincRNA\_1317 were also identified, which may facilitate multiple miRNAs to bind the same regions. microRNAs contain the capability to shake lincRNA stability by targeting their transcripts similar to targeted mRNAs. Similarly, lincRNAs possessing multiple recognition sites might act as a competitive inhibitor of miRNA by eliminating them to bind their genuine targets by sequestration. The mfe values of hybridization for miRNA-lincRNA recognition sites could be a strong predictor of a binding event between two; however, further investigations and trials are still needed to validate this interface [100].
