CONFIRMATION OF MOTHER‐TO‐CHILD TRANSMISSION OF TT VIRUS

In 1997 Nishizawa et al. isolated a DNA clone of a novel human virus using a representation-difference analysis from serum samples from a patient (TT) with posttransfusion hepatitis of unknown etiology and designated the virus as TTV after the name of the index patient. They showed that TTV DNA was detected in association with elevated transaminase values in three patients with non-A to G posttransfusion hepatitis. Utilizing oligonucleotide primers deduced from the original N22 clone, a TTV isolate (TA278) was recovered from plasma of a blood donor, and its sequence was determined within 3739 bases. Recently Mushahwar et al. revealed that the TTV genome was a circular DNA with antisense polarity and its length was 3852 nucleotides in the case of the isolate GH1. Molecular and virologic characterization of TTV is thus being extended. Transmission routes for TTV remain unclear other than the parenteral transmission resulting from transfusion of blood products. 4 The high prevalence of TTV both in blood donors and in healthy adults suggests the presence of other modes of transmission including mother-to-child infection. 5, 6 The aim of this study was to show mother-to-child transmission of TTV by homology analysis and phylogenetic analysis of the noncoding region (NCR) from the viral genome. Methods. Study group. Blood samples from four children and their mothers were subjected to a molecular analysis. The first patient (Case 1) was a 4-year-old boy who was noticed to have liver dysfunction during a medical examination of his family when his grandfather was diagnosed with active tuberculosis. His liver dysfunction was present for 4 months. The second patient (Case 2) was a 5-year-old boy who was found to have liver dysfunction at surgery for cleft lip and palate. His liver dysfunction had fluctuated in the following 12 months, and he was then lost to follow-up. Four years later he was asked to visit our hospital because his stock serum was found to be positive for TTV DNA. The mothers of Cases 3 and 4 had TTV DNA detected in serum; one was a hepatitis B virus and the other a hepatitis C virus (HCV) carrier. Sera from their babies were assayed for TTV DNA. The two infants had normal liver function. None of the four children had a history of blood transfusion. Hepatitis B surface antigen and anti-hepatitis B surface antibodies were determined by radioimmunoassay and antibodies to HCV by a second generation enzyme-linked immunosorbent assay. The sera from two adults and two children were used as a control for homology analysis. Detection and sequence analysis of TTV DNA. Nucleic acids were extracted from serum (50 m l) with the use of SMITEST EX-R&D (Sumitomo Metal Industries, Ibaragi, Japan), dissolved in 20 ml of Tris-HCl buffer (10 mM, pH 8.0) containing 1 mM EDTA, heated at 95°C for 15 min and then chilled quickly on ice. One-tenth portion thereof was subjected to PCR to detect TTV DNA with a pair of primers derived from the conserved sequence of the noncoding region. Amplified products were electrophoresed on 2% NuSieve 1:1 agarose gel, stained with ethidium bromide and observed under ultraviolet light. Nucleotide sequence of the PCR products was determined by direct sequencing method with the Dye Terminator Cycle Sequencing kit and ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA). The 159-base pair sequences (nucleotides 26 to 184 of the prototype strain, TA278) from each patient were compared with those of their mother and those of TA278 (GenBank Accession Number AB008394) and control subjects. On the basis of the nucleotide sequences of these strains, a phylogenetic tree was constructed by the neighbor-joining method with the use of a computer program (GENETYX-MAC, Version 8, Software Development Co., Ltd., Tokyo, Japan). Results. Homology analysis of the noncoding region of TTV. The nucleotide sequences of NCR of the four children had high homology to those of their mothers (nucleotide difference, 2 to 4; homology, 97.4 to 98.7%) and had relatively low homology to that of TA278 (nucleotide difference, 25 to 29; homology, 81.7 to 83.6%). Furthermore nucleotide mutations found in each pair of mother and child are almost identical and also unique to a respective family. A phylogenetic tree constructed on these 9 nucleotide sequences and 4 control subjects showed that the 4 pairs of mother and child were grouped into the same branches. These findings indicate that these respective sequences were very close in molecular evolution. (Full sequences available on request to the authors.) Clinical course of infants born to TTV viremic mothers. In Case 3 the stock sera collected between 2 and 48 months of age were tested for TTV DNA. It was detected in the first serum at 84 days after the birth and remained positive during the following 46 months. During this period she had normal liver function tests and responded to three doses of a recombinant hepatitis B vaccine with no sign of HBV infection. Jaundice and hepatomegaly were not noted at follow-up visits. Her mental and physical development has been normal. In Case 4 only one serum sample from 9 months of age was available; it tested positive for TTV DNA. Serum transaminases, anti-HCV antibodies and PCR for HCV RNA were assayed seven times between 1 and 24 months of age. He had sustained normal transaminase values, cleared passive anti-HCV antibodies at 7 months of age and has not had HCV viremia. Discussion. We showed mother-to-child transmission of TTV in four children by using homology analysis and phylogenetic analysis of the noncoding region of the TTV genome. Because the prevalence of TTV is common, ranging from 1.9 to 83%, 5, 6 it seems mandatory to prove mother-to-child transmission of TTV by means of a molecular analysis. In our subjects this approach with the use of NCR of the viral genome was valuable in showing mother-to-child transmission of TTV. Because NCR of the genome of TTV utilized in the present study showed a variability of ;80% in nucleotide sequence among our control subjects and very small variability between mother and child, homology analysis of this region provided convincing evidence of vertical transmission in these children. TTV was initially identified as a transfusion-transmissible agent, and the parenteral transmission rate was 49% in 37 patients who underwent surgery and received blood transfusions. Furthermore the possibility of nonparenteral transmission has been suggested by recent reports of a high prevalence among blood donors in Japan and among populations in tropical countries. Our findings provide direct evidence that TTV is transmitted between mother and child. In one of the four children the viral genome was present from 84 days of life to 46 months. These findings suggest that mother-to-child transmission of TTV can occur in the perinatal period and TTV infection can persist for as long as 4 years. Unfortunately we are unable to determine when TTV infection took place in this case. However, the infection in this case might have occurred in utero or around the neonatal period because the incubation period of parenterally transmitted TTV appears to be between 1 and 5 weeks. Acknowledgment. We acknowledge the technical advice of Tatsuya Tanaka, M.S.