Although ILTV causes less mortality than the highly pathogenic avian influenza virus and Newcastle disease, its impact on avian productivity has caused significant economic losses to the poultry industry worldwide . However, no scientific data on ILTV surveillance in poultry farms in Myanmar was available until now. In this study, we investigated the presence of ILTV in Myanmar among 20 poultry farms in Myanmar using PCR targeting the TK gene and we detected ILTV in six farms which were located in southern Myanmar.
Molecular characterization of ILTV is required to differentiate between field and vaccine strains [15, 17, 18]. ICP4 is responsible for regulation of gene expression early in infection  and has been proposed as a potential differentiation marker due to differences in this gene in the wild-type and vaccine strains . The sequences from the isolates in Myanmar in the present study had a 12 bp-deletion at positions 259–270 in the ICP4 gene fragment 1; this deletion is typically not present in the TCO vaccine strains. In addition, the nucleotide sequences of ICP4 gene fragment 2 in the isolates showed distinct differences from TCO vaccine strain sequences. According to the local veterinarians from Myanmar poultry farms, TCO vaccine strain is used to prevent the incidence of ILT in poultry farms that we visited. Therefore, the isolates detected in the present study appear to be field strains.
Glycoprotein B encoded by UL27 gene is one of the major proteins in ILTV, playing a fundamental role in virus attachment to target cells and cell entry . According to our data, the point mutation at position 1931 in the gB gene was found in most virulent and vaccine strains (including TCO and CEO strains). Gracía et al. also reported that the codon at position 1931 in the gB gene from most field strains coded for cytosine whereas the one from most vaccine strains coded for thymine . Therefore, the SNP at position 1931 in the gB gene could act as a good differentiation marker for field and vaccine strains [9, 22]. In contrast, the isolate from Farm Ya-5 showed similarity to the vaccine strains as well as a few field isolates.
gJ protein is a major viral antigen and plays an important role during egress of ITLV . Craig et al.  compared seven different partial fragments of some ILTV genes (TK, gD, gG, gB, gC, gJ, and ICP4), and indicated that the gJ sequence was the most informative segment to discriminate between field and vaccine strains. Furthermore, five distinct haplotypes were defined according to the specific changes in select nucleotide positions of the gJ gene. Sequence analysis in the present study showed that haplotype 2 was the predominant type circulating in Myanmar (data not shown).
Sequencing analysis of the gG gene has also been used to characterize ILTV isolates . By comparing the partial sequence of gG genes with those of other reference strains, a non-synonymous substitution (Glu-to-Asp) at position 34 was identified in the gG gene of field isolates from this study. To our knowledge, no other studies have reported this mutation in the gG gene of either field or vaccine strains. Further investigation of ILTV strains circulating in the other regions of Myanmar is therefore necessary. Furthermore, since ILTV gG is a known virulence factor that can bind chemokines with high affinity and inhibit leukocyte chemotaxis [25, 26], the biological significance of this amino acid substitution (Glu 34 Asp) in the gG gene requires further investigation to determine whether it impacts on the pathogenicity of ILTV.
In the present study, ILTV was mainly detected in the Yangon farms (southern area of the country). All the Yangon samples were collected in May, which is the wet season in Myanmar. In contrast, the Mandalay and Pyin Oo Lwin samples were collected in February, which is the dry season, and almost all were negative for ILTV. The climate during the dry season is much warmer than during the wet season in Myanmar. Since ILTV is a temperature-sensitive virus that cannot resist high environmental temperatures, it is possible that ILTV transmission may be limited during the dry season, thus partially explaining why most positive samples were detected from Yangon farms and very few from Mandalay and Pyin Oo Lwin farms. Therefore, future studies should ensure that sampling is conducted during similar seasons to ensure accurate representation of the circulating ILTV strains in Myanmar.
Phylogenetic analyses of the ICP4 and gB genes indicated that the Myanmar ILTV isolates were closely related to ILTV reference strains including Asian strains, especially three Korean field isolates, which most likely originated from the Serva vaccine strain . According to the phylogenetic analysis comparing the gB and gG gene sequences obtained in this study and those previously published in Genbank, five Myanmar isolates clustered into separate branches belonging to the CEO vaccine and TCO vaccine strains. In contrast, phylogenetic analysis using the gJ and ICP4 gene sequences revealed that these isolates clustered together with CEO vaccine. In a previous study by Oldoni et al , three isolates could only be differentiated from the CEO vaccine by the analysis of glycoprotein M gene. Meanwhile, molecular techniques have identified live-attenuated vaccines as one of the main causes of ILTV outbreaks worldwide . CEO vaccine has been banned in Argentina for more than 10 years due to its associated reversion to virulence . Shehata et al.  also isolated three highly pathogenic CEO-like field strains and suggested that CEO vaccine strains could increase in virulence after bird-to-bird passages causing severe outbreaks in susceptible birds. It is more likely that the ILTV isolates circulating in poultry farms in Myanmar originated from CEO-like viruses. However, such a hypothesis requires further periodical surveillance using larger sample sizes and sequence analysis based on additional ILTV genomic regions.