**3. Identification of miRNAs encoded by avian viruses**

The majority of viral miRNAs had initially been identified by a protocol involving size fractionation of small RNA, ligation of linkers, reverse transcription, concatamerization, cloning followed by Sanger sequencing [7]. The bioinformatics approaches to identify miRNA-encoding loci in viral genome based on commonalities in the predicated secondary structures of pre-miRNAs have also been developed. Exploring the small RNAs libraries with a higher degree of reliability and unprecedented depth has become possible with the advent of massively parallel sequencing technologies. We and others have reported the identification of miRNAs encoded by a number of avian herpesviruses. These include 14 miRNAs (26 mature sequences) encoded by MDV-1 [24, 25], 18 miRNAs (36 mature sequences) encoded by MDV-2 [26, 27], 17 miRNAs (28 mature sequences) encoded by HVT [27, 28], 7 miRNAs (10 mature sequences) encoded by ILTV [27, 29], 24 miRNAs (33 mature sequences) encoded by DEV [30], and 1 miRNA (2 mature sequences) encoded by ALV-J [11] (**Figure 1**).

#### **3.1 MDV-1 miRNAs**

*Non-Coding RNAs*

the miRNA pathway.

control, epigenetic control of viral/host gene expression, and restricted gene expression [5, 6]. The miRNAs, with the small size along with the ability for specific repression of multiple transcript targets, make them ideal tools for herpesviruses to reshape the gene expression to favor viral replication in an infected cell. Hence, it is of no surprise that herpesviruses encode 91% of virus-encoded miR-NAs identified so far. The number of miRNAs encoded by different herpesviruses varies, i.e., 5 miRNAs in Bovine herpesvirus 5 and 70 in Rhesus lymphocryptovirus (miRBase V22.1). In addition to herpesviruses, some of other virus families also encode miRNAs [7, 8] including miRNAs encoded by the human Torque Teno Virus [9]. Although retroviruses have not been widely documented to exploit the miRNA pathway [10], demonstration of E (XSR) miRNA encoded by avian leukosis virus subgroup J (ALV-J) using the canonical miRNA biogenesis pathway [11], and a conserved cluster of RNA polymerase III (pol III)-transcribed miRNAs from the bovine leukemia virus (BLV) genome [12, 13] showed that retroviruses also exploit

**2. miRNA-encoding avian viruses and associated diseases**

Avian herpesviruses are a major group of pathogens affecting different species of poultry. All of the pathogenic avian herpesviruses belong to the subfamily *Alphaherpesvirinae*. These include infectious laryngotracheitis virus (ILTV, Gallid herpesvirus) in the *Iltovirus* genus, pathogenic Marek's disease virus-1 (MDV-1, Gallid herpesvirus 2) in *Mardivirus* genus, and unassigned duck enteritis virus (DEV). ILTV induces laryngotracheitis, a contagious respiratory tract infection that results in severe losses in egg production and high mortality in infected poultry flocks. Although the safety of vaccine strains has been questionable, live attenuated vaccines are used to control this disease [14]. *Mardivirus* genus is classified into three closely related, but distinct groups including the pathogenic Marek's disease virus-1 (MDV-1, Gallid herpesvirus 2), attenuated Marek's disease virus-2 (MDV-2, Gallid herpesvirus 3), and the antigenically related herpesvirus of turkey (HVT, Meleagrid herpesvirus 1). Based on their pathogenicity, MDV-1 is further grouped into different pathotypes named as virulent (vMDV), very virulent (vvMDV), and very virulent plus (vv + MDV) [12]. Marek's disease (MD) is an immunosuppressive, neurological, and oncogenic disease caused by highly contagious MDV-1. MD that is widespread in the poultry population around the world causes estimated economic losses of US\$ 2000 million annually [15]. Although, controlled by the use of vaccines such as nonpathogenic HVT and MDV-2 vaccines or live attenuated MDV-1 strains [16], there is concern on the continued evolution of the virus toward greater virulence, challenging the sustainability of Marek's disease vaccination strategy [17, 18]. DEV induces acute disease in waterfowl species with high

Avian leukosis viruses (ALVs) are members of *Alpharetrovirus* genus of the *Retroviridae* family. ALVs cause neoplastic diseases and other reproduction problems with enormous economic losses in the global poultry industry. According to their mode of transmission, the ALVs are classified as either endogenous (ALV-E) or exogenous viruses. Based on viral envelope interference, host range, and crossneutralization patterns, exogenous ALVs from chickens have been further divided into different subgroups (A, B, C, D, and J) [21]. ALV-J was first described in the UK in the late 1980s [22] and has been primarily associated with myeloid leukosis in meat-type chickens. ALV-J caused more serious damage than all other subgroups worldwide. ALV-induced disease particularly by ALV-J is still widespread in poultry

population in China and causes huge economic losses [23].

**30**

mortality [19, 20].

High throughput sequencing of small RNA libraries from highly virulent RB1B strain infected chicken embryo fibroblast (CEF) has led to the identification of the first MDV-1-encoded miRNAs in 2006 [24]. Subsequently, more MDV-1 miRNAs were discovered by analyzing small RNA library of MSB-1, a MDV-1 cell line established from an MDV-induced spleen lymphoma [25, 31]. A total of 14 precursor sequences producing 26 mature miRNAs have been identified from the MDV-1 genome (www.miRbase.org; **Figure 1A**). The MDV-1 miRNAs are clustered in three separate genomic loci: cluster 1 (Meq-cluster), cluster 2 (Mid-cluster) flank the Meq oncogene, and cluster 3 (LAT-cluster) lies in the region encoding the latency-associated transcripts (LATs) [24, 25, 31] (**Figure 1A**). All three MDV-1 miRNA clusters are in the repeat regions of the viral genome. Although differing in virulence, the miRNA sequences are highly conserved among 23 different MDV-1 strains [31, 32]. Despite this, the expression level of Meq-cluster miRNAs is higher in vv+ MDVinduced lymphomas than vvMDV strain-induced lymphomas. One polymorphism in the miRNA promoter region has been proposed to be responsible for this differential expression. On the other hand, there is no difference on the level of the LATcluster miRNAs expression [31, 33], implying that miRNAs from Meq-cluster may play a more significant role in MD oncogenesis. Indeed, the significantly decreased oncogenicity of the virus with the deletion of the Meq-cluster miRNAs has proved this hypothesis [34, 35]. mdv1-miR-M4-5p, a functional ortholog of gga-miR-155 and a member of Meq-cluster miRNA, is the most highly expressed viral miRNA in tumors, representing over 70% of MDV miRNA sequencing reads [31]. Similar to the role of gga-miR-155 in lymphoid malignancies, mdv1-miR-M4-5p was shown to play a key role in MDV-1-induced oncogenesis [34, 35]. The promoter prmiRM9M4, corresponding to the 1300-bp immediately upstream from the first Meq-cluster miRNA mdv1-miR-M9, has been shown to drive the transcription of both Meqcluster and Mid-cluster miRNAs with two distinct transcriptional models during different infection phases [33]. Indeed, this promoter has been shown to be active during MDV-1 latency by both DNA hypomethylation and active histone marks [36], confirming its transcriptional activity. In contrast, the transcription of LATclustered miRNAs is driven by a p53-dependent promoter, which contains at least two 60-bp tandem repeats harboring a p53-response element but has no consensus core promoter element [37].

Studies on correlation of miRNA expression signature with MDVtransformation have been undertaken. Microarray analysis has been carried out to determine the global miRNA expression profiles in seven distinct MDVtransformed cell lines [38]. Compared to uninfected splenocytes and CD4+ T-cells or non-MDV avian viral transformed cell lines, these profiles revealed that the expression of a set of host miRNAs is altered in the MDV-transformed cell lines. The downregulated host miRNAs in the MDV-transformed cell lines include miR-26a, miR-150, miR-223, miR-155, and miR-451. The differential expression of these host miRNAs may contribute to MDV pathogenesis, considering the link of several mammalian homologs of these miRNAs with cancer development [39]. For example, miR-26a with tumor suppressor function is downregulated in a number of avian lymphoma cells. miR-26a has been shown to regulate interleukin 2 (IL-2) expression [40]. It is well known that IL-2 regulates T-cell proliferation, suggesting that downregulation of miR-26a and the upregulation of IL-2 expression could be a conserved mechanism in avian viral transformation. Taken together, analysis of the functional targets of the differentially expressed miRNAs would contribute toward better understanding of the molecular pathways of MD oncogenicity.

#### **3.2 MDV-2 miRNAs**

MSB-1 is a MDV-transformed lymphoblastoid cell line, which is co-infected with both BC-1 strain of MDV-1 and HPRS24 strain of MDV-2 [41]. About 17 novel MDV-2 miRNAs were identified by analyzing small RNA library from MSB-1 [26]. Of these, 16 are expressed in the same direction and clustered in a 4.2-kb long repeat region that encodes R-LORF2 to R-LORF5, suggesting that they may be derived from a common primary transcript. The single miRNA outside the cluster was located within the C-terminal region of the ICP4 homolog in the short repeat region. Despite lacking sequence homology, the relative genomic positions of miRNA clusters in MDV-1 and MDV-2 are conserved by locating within the repeat regions of the viral genome. Subsequently, an additional miRNA mdv2-miR-M32 has been reported by high throughput sequencing of small RNAs from MSB-1 cells [27]. Uniquely, all 18 MDV-2 encoded miRNA precursors give rise to 2 mature forms resulting in 36 mature MDV-2 miRNAs, representing both strands of the duplex (www.miRbase.org; **Figure 1A**). Two separate studies on sequencing of MSB-1 small RNA library showed that MDV-2 miRNAs accounted for 10 and 13% of the sequencing reads, respectively [25, 27].

#### **3.3 HVT miRNAs**

HVT-encoded miRNAs were identified from HVT-infected CEF using both traditional cloning and sequencing of a small RNA library [28] and high throughput sequencing technology [27]. About 17 precursor miRNAs producing 28 mature miRNAs (www.miRbase.org; **Figure 1A**) have been identified in the HVT genome. Except for miR-H11, which is located within an intron of UL21 in the UL region, all other HVT miRNAs are located in the long-repeat regions, demonstrating some degree of positional conservation with MDV-1 and MDV-2. Interestingly, 10 HVT miRNAs were located in a region containing two tandem repeats, and small sequence variations were observed following multiple sequence alignment of the miRNA precursors, suggesting evolution by duplication. This represents the first example of evolution of virus-encoded miRNAs by duplication.

#### **3.4 ILTV-miRNAs**

A total of seven ILTV precursor miRNAs producing 10 mature miRNAs from ILTV-infected chicken embryo kidney (CEK) cells and leghorn male hepatoma

**33**

*Role of Virus-Encoded microRNAs in Avian Viral Diseases*

(LMH) cell line have been identified by deep sequencing of small RNA populations [27, 29] (www.miRbase.org; **Figure 1B**). Four of the miRNAs (iltv-miR-I1-I4), which are not associated with any annotated ORFs were located at the extreme terminus of the ILTV genome. miR-I5 and miR-I6, the two most highly expressed miRNAs, are located in the repeat regions within ICP4. The iltv-miR-I7 was mapped in the replication origin (oriL) of the palindrome stem loop sequence. Out of seven

*DOI: http://dx.doi.org/10.5772/intechopen.89688*

*Role of Virus-Encoded microRNAs in Avian Viral Diseases DOI: http://dx.doi.org/10.5772/intechopen.89688*

(LMH) cell line have been identified by deep sequencing of small RNA populations [27, 29] (www.miRbase.org; **Figure 1B**). Four of the miRNAs (iltv-miR-I1-I4), which are not associated with any annotated ORFs were located at the extreme terminus of the ILTV genome. miR-I5 and miR-I6, the two most highly expressed miRNAs, are located in the repeat regions within ICP4. The iltv-miR-I7 was mapped in the replication origin (oriL) of the palindrome stem loop sequence. Out of seven

*Non-Coding RNAs*

**3.2 MDV-2 miRNAs**

**3.3 HVT miRNAs**

**3.4 ILTV-miRNAs**

out to determine the global miRNA expression profiles in seven distinct MDVtransformed cell lines [38]. Compared to uninfected splenocytes and CD4+ T-cells or non-MDV avian viral transformed cell lines, these profiles revealed that the expression of a set of host miRNAs is altered in the MDV-transformed cell lines. The downregulated host miRNAs in the MDV-transformed cell lines include miR-26a, miR-150, miR-223, miR-155, and miR-451. The differential expression of these host miRNAs may contribute to MDV pathogenesis, considering the link of several mammalian homologs of these miRNAs with cancer development [39]. For example, miR-26a with tumor suppressor function is downregulated in a number of avian lymphoma cells. miR-26a has been shown to regulate interleukin 2 (IL-2) expression [40]. It is well known that IL-2 regulates T-cell proliferation, suggesting that downregulation of miR-26a and the upregulation of IL-2 expression could be a conserved mechanism in avian viral transformation. Taken together, analysis of the functional targets of the differentially expressed miRNAs would contribute toward

better understanding of the molecular pathways of MD oncogenicity.

MSB-1 is a MDV-transformed lymphoblastoid cell line, which is co-infected with both BC-1 strain of MDV-1 and HPRS24 strain of MDV-2 [41]. About 17 novel MDV-2 miRNAs were identified by analyzing small RNA library from MSB-1 [26]. Of these, 16 are expressed in the same direction and clustered in a 4.2-kb long repeat region that encodes R-LORF2 to R-LORF5, suggesting that they may be derived from a common primary transcript. The single miRNA outside the cluster was located within the C-terminal region of the ICP4 homolog in the short repeat region. Despite lacking sequence homology, the relative genomic positions of miRNA clusters in MDV-1 and MDV-2 are conserved by locating within the repeat regions of the viral genome. Subsequently, an additional miRNA mdv2-miR-M32 has been reported by high throughput sequencing of small RNAs from MSB-1 cells [27]. Uniquely, all 18 MDV-2 encoded miRNA precursors give rise to 2 mature forms resulting in 36 mature MDV-2 miRNAs, representing both strands of the duplex (www.miRbase.org; **Figure 1A**). Two separate studies on sequencing of MSB-1 small RNA library showed that MDV-2 miRNAs accounted for 10 and 13% of the sequencing reads, respectively [25, 27].

HVT-encoded miRNAs were identified from HVT-infected CEF using both traditional cloning and sequencing of a small RNA library [28] and high throughput sequencing technology [27]. About 17 precursor miRNAs producing 28 mature miRNAs (www.miRbase.org; **Figure 1A**) have been identified in the HVT genome. Except for miR-H11, which is located within an intron of UL21 in the UL region, all other HVT miRNAs are located in the long-repeat regions, demonstrating some degree of positional conservation with MDV-1 and MDV-2. Interestingly, 10 HVT miRNAs were located in a region containing two tandem repeats, and small sequence variations were observed following multiple sequence alignment of the miRNA precursors, suggesting evolution by duplication. This represents the first

A total of seven ILTV precursor miRNAs producing 10 mature miRNAs from ILTV-infected chicken embryo kidney (CEK) cells and leghorn male hepatoma

example of evolution of virus-encoded miRNAs by duplication.

**32**

#### **Figure 1.**

*Diagrammatic representation of the viral genomes showing the positions of miRNAs of MDV1, MDV2, HVT (A) and ILTV, DEV and ALV-J (B). Position and orientation of selected transcripts are shown. The genome size (base pairs) of viruses is shown on the top-left corner. Adapted from Yao et al., [92].*

miRNAs, only three (iltv-miR-I3, -I5, -I6) have been confirmed by Northern hybridizations although the expression was confirmed for all by the end point PCR using small RNA libraries generated from ILTV-infected CEK.

**35**

*Role of Virus-Encoded microRNAs in Avian Viral Diseases*

About 33 mature miRNAs derived from 24 pre-miRNA sequences within the DEV genome have been identified by deep sequencing of small RNAs from DEV vaccine strain-infected CEF cultures [30] (www.miRbase.org; **Figure 1B**). Compared to the location of most Mardivirus-encoded miRNAs which are at the repeat regions, genomic positions of DEV-encoded miRNAs are unique. Five out of six miRNA clusters from both the coding and non-coding regions of the 158,091-bp viral genome were encoded within the unique long region. Similar to miRNA-offset RNAs, two miRNA precursors, DEV miR-D18 and miR-D19, overlapped with each other although only the dev-miR-D18-3p was functional in reporter assays. In addition, 12 putative DEV miRNAs have been reported by computational approach [42], none of these miRNAs overlapped with the 24 DEV miRNAs described above [30]. In a recent report, 39 mature viral miRNAs have been identified from a Chinese virulent strain of DEV infected duck embryo fibroblast cells [43]. Of those, only 13 miRNA sequences and 22 "seed sequences" of miRNA were identical with the previously reported DEV-miRNAs encoded by vaccine strain [30]. About eight additional

viral miRNAs were detected and confirmed by stem-loop RT-qPCR [43].

One of the distinct features of HPRS-103 sequence, the ALV-J prototype virus, is the presence of E (also called XSR) element, a distinct hairpin stem-loop structure in the 3′ untranslated region. Indeed, the expression of miRNA from this hairpin-like structure of E element has been demonstrated in ALV-J-infected/ transformed cells [11]. E (XSR) miRNA, a small RNA population encoded from within the E (XSR) element, was identified using deep sequencing approach on ALV-J-transformed cell line IAH30 [11] (**Figure 1B**). E (XSR) miRNA is suggested to play a major role in ALV-J pathogenesis and neoplastic transformation as it accounted for a quarter of the sequences of the IAH30 "miRNAome." Although the E element per se is not absolutely essential for ALV-J induced tumor, the E element does contribute to the oncogenicity in certain genetic lines of chicken when the oncogenicity of viruses derived from the parental and E element deleted HPRS-103 viruses was compared [44]. The hypothesis of polymorphisms, particularly those in any potential E (XSR) miRNA target sites could account for differential susceptibility phenotypes among these lines can be investigated by comparison of the genomes

Two classes of virus-encoded miRNAs are grouped: viral specific miRNAs and orthologs of host miRNAs. Similar to some viral regulatory proteins, several viral miRNAs have evolved to mimic host effectors. The "seed" region of a miRNA (~nucleotides 2–8 at the 5′ end) plays a key role in directing RISC to its mRNA targets. Binding with perfect seed complementary to the target transcript takes ~60% of regulation by a particular miRNA [45]. Some of viral miRNAs share seed sequences with host miRNAs and at least three viruses: BLV, MDV-1, and Kaposi's Sarcoma-associated Herpesvirus (KSHV) have been shown to regulate transcripts via the same target docking sites as their counterpart host miRNAs [46–48]. Such viral miRNAs could potentially regulate hundreds of transcripts having target sites for a particular host miRNA. Such regulatory networks could affect specific func-

*DOI: http://dx.doi.org/10.5772/intechopen.89688*

**3.5 DEV miRNAs**

**3.6 ALV-J miRNAs**

of these lines.

**4. Viral orthologs of host miRNAs**

tions such as inhibiting apoptosis.

#### **3.5 DEV miRNAs**

*Non-Coding RNAs*

**34**

**Figure 1.**

miRNAs, only three (iltv-miR-I3, -I5, -I6) have been confirmed by Northern hybridizations although the expression was confirmed for all by the end point PCR using

*Diagrammatic representation of the viral genomes showing the positions of miRNAs of MDV1, MDV2, HVT (A) and ILTV, DEV and ALV-J (B). Position and orientation of selected transcripts are shown. The genome* 

small RNA libraries generated from ILTV-infected CEK.

*size (base pairs) of viruses is shown on the top-left corner. Adapted from Yao et al., [92].*

About 33 mature miRNAs derived from 24 pre-miRNA sequences within the DEV genome have been identified by deep sequencing of small RNAs from DEV vaccine strain-infected CEF cultures [30] (www.miRbase.org; **Figure 1B**). Compared to the location of most Mardivirus-encoded miRNAs which are at the repeat regions, genomic positions of DEV-encoded miRNAs are unique. Five out of six miRNA clusters from both the coding and non-coding regions of the 158,091-bp viral genome were encoded within the unique long region. Similar to miRNA-offset RNAs, two miRNA precursors, DEV miR-D18 and miR-D19, overlapped with each other although only the dev-miR-D18-3p was functional in reporter assays. In addition, 12 putative DEV miRNAs have been reported by computational approach [42], none of these miRNAs overlapped with the 24 DEV miRNAs described above [30]. In a recent report, 39 mature viral miRNAs have been identified from a Chinese virulent strain of DEV infected duck embryo fibroblast cells [43]. Of those, only 13 miRNA sequences and 22 "seed sequences" of miRNA were identical with the previously reported DEV-miRNAs encoded by vaccine strain [30]. About eight additional viral miRNAs were detected and confirmed by stem-loop RT-qPCR [43].

### **3.6 ALV-J miRNAs**

One of the distinct features of HPRS-103 sequence, the ALV-J prototype virus, is the presence of E (also called XSR) element, a distinct hairpin stem-loop structure in the 3′ untranslated region. Indeed, the expression of miRNA from this hairpin-like structure of E element has been demonstrated in ALV-J-infected/ transformed cells [11]. E (XSR) miRNA, a small RNA population encoded from within the E (XSR) element, was identified using deep sequencing approach on ALV-J-transformed cell line IAH30 [11] (**Figure 1B**). E (XSR) miRNA is suggested to play a major role in ALV-J pathogenesis and neoplastic transformation as it accounted for a quarter of the sequences of the IAH30 "miRNAome." Although the E element per se is not absolutely essential for ALV-J induced tumor, the E element does contribute to the oncogenicity in certain genetic lines of chicken when the oncogenicity of viruses derived from the parental and E element deleted HPRS-103 viruses was compared [44]. The hypothesis of polymorphisms, particularly those in any potential E (XSR) miRNA target sites could account for differential susceptibility phenotypes among these lines can be investigated by comparison of the genomes of these lines.
