Molecular characterisation of varicella zoster virus genotypes in Sri Lanka.

Papers Abstract Introduction Genotyping of wild type of varicella zoster virus (VZV) in Sri Lanka would help to distinguish the VZV wild type infection from varicella vaccine associated infections.


Introduction
Varicella zoster virus (VZV) causes chicken pox (varicella) during primary infection, which is usually a benign self-limiting illness in children.However, it can cause severe disease and even death in adults, neonates, pregnant women and in the immunosuppressed [1].As common to all alpha herpes viruses it then becomes latent in the dorsal root sensory ganglia.The virus may reactivate later in life to cause shingles (herpes zoster) [2].
VZV is a double stranded DNA virus and only one serotype exists.The VZV genome is highly conserved with 72 ORFs [2].The genomic variation between VZV strains is limited to about 0.05% to 0.06% and consists almost entirely of single nucleotide polymorphisms (SNPs) Ceylon Medical Journal 2013; 58: 153-6 dispersed across the genome [3].On the basis of analysis of SNP at least 3-4 geographically distinct genotypes have been described.By analysis of selected SNPs in ORFs 1, 21, 50 and 54, Barrett-Muir et al have identified four genotypes; namely, A (Africa/Asia), B and C (Europe and America) and J (Far East) [4].Loparev et al have shown that by the analysis of a short region in ORF22, three major genotypes can be distinguished: E (European), J (Japanese), and M (mosaic).J strains are most common in Japan and E strains are most common in temperate latitudes [5].The M strains were commonest in Africa, India, China and Central America [5,6].
The epidemiology of VZV infections is remarkably different in tropical and temperate climates.In the tropics, infection mainly occurs among young adults resulting in significant morbidity, higher hospital admission rates and mortality [7].The seroprevalance rates of VZV among 5 year olds in India was 29% while in Sri Lanka it was 10% [8,9].In Sri Lanka, only 50% of those living in the rural areas had chickenpox even at the age of 60 years [9].Determining the genotype of wild type VZV in Sri Lanka could possibly help us to determine if the genotypic differences could contribute to differences in disease transmission.In addition, genotyping the wild type strain would also enable us to differentiate VZV infection due to the wild type virus from the vaccine associated infection.RFLP analysis of the PCR products of VZV ORF 38, 54 and 62, has been used to distinguish the wild type strain for the Oka virus (VZV vaccine strain) [6,10].All Japanese strains (Oka vaccine strain) are either Pst I+ BglI + or PstI -BglI + , while most isolates in the United States, Germany and the United Kingdom are PstI + BgI - [10].PstI + BglI + strains have been shown to be prevalent in countries with low adult immunity to varicella, such as India and also among Asian immigrants in European countries [11].

Methods
Thirty one isolates were obtained from either blister fluid or blood from 28 patients with chickenpox and 3 patients with herpes zoster.Informed written consent

Papers
was obtained from all study participants and the study was approved by the Ethical Review Committee of the University of Sri Jayawardenapura.Viral DNA was extracted using QIAmp Blood Kit (QIAGEN) according to the manufacturer's instructions and the live attenuated varicella vaccine.PCR was performed as described in previous study, using primers for ORF 38, 54 and 62 [62].
Restriction Endonuclease digestion of PCR products were carried out with 1-3 μl of PCR product, 1-3μl of Restriction Endonuclease (BglI, PstI or SmaI), 4 μl of Restriction Enzyme buffer (10×, Promega) and 4 μl of Acetylated Bovine Serum Albumin (10μg/μl).The final reaction volume was adjusted to 20μl with DNase/RNasefree water.Reactions were incubated at 37°C for BglI and PstI or 25°C for SmaI overnight.BglI and PstI digested products were separated by gel electrophoresis on 3% agarose at 80 V for 150 minutes and 100 minutes respectively.SmaI digested products were electrophoresed on 4% agarose gel at 80 V for 2.5 hours.

Results
In order to characterize the genotype of the wild type VZV strain in Sri Lanka, we analysed 31 VZV isolates from patients with chicken pox or herpes zoster by RFLP of DNA fragments of open reading frames (ORFs) 38, 54, and 62.We found that except for one strain, all other VZV isolates from clinical samples had the genotype characteristic of the wild type VZV strain PstI + BglI + SmaI -.None of the isolates had the American or the European VZV profile (PstI + BglI -) but were similar to the virus isolates from Africa and Asia (PstI + BglI + ).

Restriction fragment length polymorphism (RFLP) analysis of ORF 38
After PCR amplification with the primers for ORF 38, a 647 bp fragment was obtained as expected.Subsequent PstI digestion of the PCR products yielded two fragments of 357 bp and 290 bp in all except one isolate (Figure1).In contrast, the 647 bp fragments were obtained from one isolate and the Oka vaccine strain.These results indicate that 30 Sri Lankan VZV isolates contained the PstI site in ORF 38 and one isolate and the Oka vaccine strain were negative for the PstI site.

RFLP analysis of ORF 54
PCR amplification of DNA with primers for ORF 54 produced amplicons of 497 bp (Figure 2).The digestion of PCR product with BglI restriction enzyme resulted in two fragments of 256 bp and 241 bp.All of the viruses analyzed, which included 31 Sri Lanka isolates and Oka vaccine strain, were positive for the BglI site.

RFLP analysis of ORF 62
PCR amplification of DNA fragments in ORF 62 produced amplicons with a size of 268 bp.Subsequent SmaI digestion of 30 of 31 analyzed VZV DNA yielded 153, 79 and 36 bp fragments (Figure 3).The PCR product of the Oka vaccine strain and one Sri Lankan isolate were cleaved in a set of 112, 79 and 41/36 bp fragments.These results indicate that one Sri Lankan isolate and the Oka vaccine strain contain SmaI site in ORF 62 whereas others did not have the site.

Discussion
In this study we found that the majority of the VZV strains had the genotype PstI + BglI + SmaI -, which is characteristic of the Asian/African clades [3,11].We found that the wild type strain can be successfully differentiated from Oka strain with PCR-RFLP technique using genetic markers ORF 38, 54 and 62.The majority of wild type VZV strains in tropical or subtropical climate are PstI + BglI + SmaI -while the dominant pattern in countries with temperate climate is PstI + BglI -SmaI - [6,10,11].Predominantly circulating strains in tropical countries such as Zambia, Guinea Bissau, Bangladesh, India and Singapore were BglI + marker in ORF 54 [5,11].The circulating wild type of VZV in India was also similar to the genotype in Sri Lanka which is PstI + BglI + SmaI - [11].
Since the introduction of the live attenuated varicella zoster vaccine to Sri Lanka, the need has arisen for molecular characterisation of the wild type virus isolates in Sri Lanka, to differentiate it from the vaccine strain.Although 30/31 strains were PstI + BglI + SmaI -, one strain was PstI -BglI + SmaI, which is characteristic of the VZV vaccine strain.The live attenuated VZV vaccine is associated with development of a rash in 5% of vaccines [12].In addition, the vaccine virus is still able to establish latency in the vaccinated host and can be transmitted to nonimmune individuals from skin lesions of individuals with vaccine varicella resulting in primary varicella [13][14][15][16].Therefore, the presence of the Oka virus strain in one of our patients (who was a 13 year old otherwise healthy individual) with chickenpox could have been due to transmission of the vaccine virus.
In summary we have identified that the wild type virus strain in Sri Lanka is of the PstI + BglI + SmaI - genptype, which is characteristic of the Asian/African clades.By using RFLP of PCR products of ORF 38, 54 and 62, we are able to differentiate the wild type virus strain from the Oka vaccine strain, which could be used to determine vaccine virus associated varicella infection.

Introduction
Health related quality of life (HRQL) is important in people with chronic diseases.Cirrhosis of the liver has a considerable effect on patients' physical and psychosocial wellbeing.The progression of symptoms, functional Ceylon Medical Journal 2013; 58: 156-62