**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 mortality [19, 20].

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].

**31**

core promoter element [37].

Studies on correlation of miRNA expression signature with MDVtransformation have been undertaken. Microarray analysis has been carried

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

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

and 1 miRNA (2 mature sequences) encoded by ALV-J [11] (**Figure 1**).

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],

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

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

**3.1 MDV-1 miRNAs**
