Paolo Pagnotti

Detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy

Detection of a broad number of respiratory viruses is not undertaken currently for the diagnosis of acute respiratory infection due to the large and always increasing list of pathogens involved. A 1‐year study was undertaken on children hospitalized consecutively for acute respiratory infection in a Pediatric Department in Rome to characterize the viruses involved. Two hundred twenty‐seven children were enrolled in the study with a diagnosis of asthma, bronchiolitis, bronchopneumonia, or laringo‐tracheo bronchitis. A molecular approach was adopted using specific reverse transcription (RT)‐PCR assays detecting 13 respiratory viruses including metapneumovirus (hMPV) and the novel coronaviruses NL63 and HKU1; most amplified fragments were sequenced to confirm positive results and differentiate the strain. Viral pathogens were detected in 97 samples (42.7%), with 4.8% of dual infections identified; respiratory syncytial virus (RSV) was detected in 17.2% of children, followed by rhinovirus (9.7%), parainfluenza virus type 3 (PIV3) (7.5%), and influenza type A (4.4%). Interestingly, more than half the patients (9/17) that have rhinovirus as the sole respiratory pathogen had pneumonia. HMPV infected children below 3 years in two peaks in March and June causing bronchiolitis and pneumonia. One case of NL63 infection is described, documenting NL63 circulation in central Italy. In conclusion, the use of a comprehensive number of PCR‐based tests is recommended to define the burden of viral pathogens in patients with respiratory tract infection. J. Med. Virol. 79:463–468, 2007.

Measurement of the sensitivity of different commercial assays in the diagnosis of CMV infection in pregnancy.

To evaluate the performance of different commercial assays for the detection of recent cytomegalovirus (CMV) in pregnancy, the sensitivity and specificity of assays for CMV-specific IgM antibodies were compared. Routine specimens from pregnant women were screened for CMV IgM using the Abbott AxSYM assay. Sera that were reactive according to AxSYM were further tested for IgM by other commercial assays. In selected IgM positive samples a CMV IgG avidity assay (Radim) and virus isolation from urine (shell vial) were also performed. The positivity rate for IgM anti-CMV by AxSYM was relatively high (140 out of 492, combining reactive and grayzone results). Only 26 of the 140 samples were positive for IgM according to Radim. The IgG avidity was low in 16 of the 43 samples tested, and the Radim and DiaSorin IgM assays were negative in 5 of them; 2 of the latter cases were also positive for viral isolation according to a shell vial method. There are differences in the sensitivity of the commercially available tests for CMV antibodies. CMV screening in pregnancy is performed as a first step by immunoassays and the choice of highly sensitive IgM test associated with further serological and virological methods could help to identify early primary infections.

THE DISTANCE BETWEEN THE 3'-PYRIMIDINE-RICH TRACT AND THE AUG CODON MODULATES INTERNAL INITIATION OF TRANSLATION OF HEPATITIS A VIRUS RNA

Protein synthesis directed by hepatitis A virus (HAV) RNA is mediated by a mechanism involving the recognition of internal sequences. Two in‐frame AUG codons initiate the long open reading frame (positions 734–736 and 740–742). The extra‐cistronic region extending between the uncapped 5′‐end and the ORF contains two pyrimidine‐rich tracts (PRTs): one 12 nucleotides in length in the close vicinity of the initiator AUG, and a longer one between bases 94 and 140. In order to study the relative contribution of these elements to the process of internal initiation of translation, cDNA representations of the 5′‐terminal extra‐cistronic region of HAV RNA were inserted in the intergenic region of the bi‐cistronic plasmid pSV‐GH/CAT, between the genes encoding the human growth hormone (GH) and the bacterial enzyme chloramphenicol acetyltransferase (CAT), and following transfection of COS‐1 cells, the transient expression of both genes was quantified. The importance of the 3′‐PRT appeared to be strongly influenced by the length of the ‘spacer’ sequence extending between this structure and the translation initiation site: placed 45 nucleotides upstream from the initiator codon of a reporter gene, its integrity was stringently required for initiation to occur. Bringing the length of the ‘spacer’ back to its actual size in HAV RNA (i.e. 11 or 17 nt) reduced considerably the overall rate of internal initiation of translation, and the relative contribution to this process of the 3′‐PRT became marginal. Concomitantly, the importance of the functional domains previously identified in the 5′‐PRT fluctuated: while integrity of domain 100–106 was always stringently required for initiation to occur, the activity of domain 113–118 paralleled that of the 3′‐PRT, and the opposite applied to domain 121–126, whose contribution became relevant only after switching off the 3′‐PRT. Systematic mutations introduced in the ‘spacer’ sequences suggest that the length of this region may be responsible for the down regulation of translation of HAV RNA and, possibly, for its lengthy replication cycle.