NIH conference. Chronic fatigue syndrome research. Definition and medical outcome assessment.

A workshop was held 18 to 19 March 1991 at the National Institutes of Health to address critical issues in research concerning the chronic fatigue syndrome (CFS). Case definition, confounding diagnoses, and medical outcome assessment by laboratory and other means were considered from the perspectives of key medical specialties involved in CFS research. It was recommended that published Centers for Disease Control (CDC) case-definition criteria be modified to exclude fewer patients from analysis because of a history of psychiatric disorder. Specific recommendations were made concerning the inclusion or exclusion of other major confounding diagnoses, and a standard panel of laboratory tests was specified for initial patient evaluation. The workshop emphasized the importance of recognizing other conditions that could explain the patient's symptoms and that may be treatable. It was viewed as essential for the investigator to screen for psychiatric disorder using a combination of self-report instruments followed by at least one structured interview to identify patients who should be excluded from studies or considered as a separate subgroup in data analysis. Because CFS is not a homogeneous abnormality and because there is no single pathogenic mechanism, research progress may depend upon delineation of these and other patient subgroups for separate data analysis. Despite preliminary data, no physical finding or laboratory test was deemed confirmatory of the diagnosis of CFS. For assessment of clinical status, investigators must rely on the use of standardized instruments for patient self-reporting of fatigue, mood disturbance, functional status, sleep disorder, global well-being, and pain. Further research is needed to develop better instruments for quantifying these domains in patients with CFS.

Enzymatic cleavage of a glycoprotein of respiratory syncytial virus.

The 75K glycoprotein of the A2 strain of respiratory syncytial virus was cleaved by digestion with trypsin or Staphylococcus aureus protease V8. The fragments resulting from trypsin digestion were 40K and 29K; those from the Staphylococcal protease were 49K and 37K.

Genetic approaches to attenuation of influenza A viruses for man.

The explosion of new information concerning the influenza A viral genome provides a basis for deliberate manipulation of its genes with the intent of introducing specific mutations that render influenza virus attenuated and useful for prevention of disease. Currently there is considerable effort to develop a defined set of mutant genes that confer a specific and desired level of attenuation upon any viral recombinant into which they are transferred. In this manner new antigenic variants of influenza A virus may be satisfactorily attenuated after transfer of the mutant genes. The mutant genes must be readily identifiable by simple in-vitro techniques, thus enabling the genetic basis of attenuation to be monitored directly during all phases of vaccine development, manufacture and utilization in man. We describe our experience with two sets of ts mutant genes which affect viral RNA transcription or synthesis and which effect a reproducible level of attenuation in wild-type influenza A virus.

Cold-adapted variants of influenza A virus: evaluation in adult seronegative volunteers of A/Scotland/840/74 and A/Victoria/3/75 cold-adapted recombinants derived from the cold-adapted A/Ann Arbor/6/60 strain.

Influenza A/Scotland/74 (H3N2) and A/Victoria/75 (H3N2) cold-adapted (ca) recombinant viruses, prepared by mating the A/Ann Arbor/6/60 (H2N2) ca donor virus and influenza A wild-type virus, were evaluated in adult seronegative volunteers (serum hemagglutination-inhibiting antibody titer,

Temperature-sensitive mutants of influenza A virus: production and characterization of A/Victoria/3/75-ts-1[E] recombinants.

The Hong Kong/68-ts-1[E] virus, which has a 38 degrees C shutoff temperature for plaque formation, has been proposed as a donor of its two ts lesions to new variants of influenza A virus that pose an epidemic threat. To further examine whether the acquisition of the two ts-1[E] lesions will predictably attenuate new influenza A variants, the HK/68-ts-1[E] virus was mated with the A/Vic/3/75 wild-type virus. The Vic/75-ts-[E] recombinants that had the two ts-1[E] lesions also had a 38 degrees C shutoff temperature. Two Vic/75-ts-1[E] recombinants (clones 81 and 113) that had the two ts-1[E] lesions, a 38 degrees C shutoff temperature, and the Vic/75 hemagglutinin and neuraminidase glycoproteins were similar to each other and to their ts-1[E] parent in the pattern of replication and genetic stability in hamsters. These findings support the hypothesis that the acquisition of the two ts-1[E] lesions will predictably attenuate wild-type influenza A virus. Each Vic/75-ts-1[E] recombinant virus that possessed only the group 1 ts-1[E] lesion had a 39 degrees C shutoff temperature. Two of three of the Vic/75-ts-1[E] recombinants that had only the group 2 ts-1[E] lesion had a 39 degrees C shutoff temperature. This suggests that the HK/68-ts-1[E] donor virus contains two ts genes each of which by itself restricts plaque formation at 39 degrees C and above. The HK/68-ts-1[E] parent virus and its Vic/75 recombinant clones 81 and 113 were evaluated in ferret tracheal organ cultures maintained at permissive and restrictive temperatures. The Vic/75-ts-1[E] clone 81 differed from its parent and sister clone 113 in that it replicated readily and caused ciliostasis at 37 degrees C, a temperature restrictive for the replication of other ts-1[E] recombinants with a 38 degrees C shutoff temperature. The genetic basis underlying this difference was not elucidated.

Comparative studies of wild-type and 'cold-mutant' (temperature sensitive) influenza viruses: geneology of the matrix (M) and non-structural (NS) proteins in recombinant cold-adapted H3N2 viruses.

The matrix (M) protein of the H2N2 virus A/Ann Arbor/6/60 may be distinguished from M protein of several H3N2 viruses and A/New Jersey/76 (HSWINI) by SDS acrylamide gel electrophoresis using a discontinuous buffer system. The smallest RNA (RNA 8) of the A/Ann Arbor/6/60 virus may be distinguished from RNA 8 of several H3N2 viruses by acrylamide gel electrophoresis in 3% or 3-6% gels in the absence of urea, if electrophoresis is done at 30 to 36 degrees C or 20 degrees C respectively. Ten clones of conditionally-lethal temperature-sensitive (ts) mutants were studied, which derived their cold-adaption and ts genes from mutant A/Ann Arbor/6/60, and their haemagglutinin from the H3N2 virus A/Scotland/840/74. Each clone was found to derive its M protein from A/Ann Arbor/6/60 mutant, and its RNA 8 from A/Scotland/840/74. The only assignment of genes 7 and 8 consistent with these findings for the recombinants is that in each parent virus (and in the recombinants) gene 7 codes for M protein, and gene 8 for NS protein. Furthermore, it may be concluded from the results that the biologically important ts lesions in the A/Ann Arbor/6/60 mutant parent are not present in the NS gene. In addition to the recombinants of A/Ann Arbor/6/60 and A/Scotland/840/74, five independent ts/cold-adapted recombinants of A/Ann Arbor/6/60 mutant with H3N2 and HSWINI wild-type viruses were examined, and all were found to contain the M protein of the A/Ann Arbor/6/60 mutant parent. This is suggestive that M protein may be at least partially responsible for the cold-adaptation and/or ts properties of the A/Ann Arbor/6/60 mutant and the recombinants.

Cold adapted variants of influenza A. II. Comparison of the genetic and biological properties of ts mutants and recombinants of the cold adapted A/AA/6/60 strain.

The genetic and biological properties of 13 recombinant influenza A clones derived at 25 degrees C from the A/AA/6/60-cold variant (by crosses with 4 different wild type strains) were compared with a set of 5-FU induced ts-mutants. The 5-FU mutants had previously been placed into 7 complementation-recombination groups; the A/AA/6/60-cold parent (PI-7) and the 12 cold recombinant clones which were ts were shown to share a lesion with only one of these groups. The parental strain and 5 recombinant clones were evaluated for replication in the lungs and nasal turbinates of hamsters. Each virus appeared to be attenuated; genetic stability correlated with the level of viral replication in the hamster lung, i.e., viruses which grew best showed a tendency to revert to the ts+ phenotype. Characterization of the ts+ revertants for the presence of the cold adaptation property revealed that these viruses exhibited a spectrum of cold adaptation properties. Two viruses, PI-7 (the parental cold variant) and the CR6 recombinant (A/Queensland/6/72) did not revert in either the lungs or nasal turbinates of hamsters.

Temperature-sensitive mutants of influenza A virus. XI. Transfer of ts lesions in the Hong Kong/68-ts-1 [a] virus to the influenza A/Udorn/72 wild type.

The presence of the temperature-sensitive (ts) lesions of complementation-recombination groups 1 and 5 in the Hong Kong/68-ts-1[A] virus was confirmed by genetic analysis of ts recombinants of the Hong Kong/68-ts-1[A] virus and a Udorn/72 wild-type virus. Three classes of Udorn/72-ts recombinants were found. One class possessed both ts genes of the Hong Kong/68-ts-1[A] parent, a second class possessed the ts lesion characteristic of group 1, and a third class possessed the ts lesion of group 5. The Hong Kong/68-ts-1[A] parent and the Udorn/72-ts recombinants exhibited a 10,000-fold or greater restriction of replication in the lungs of hamsters than did the homologous wild-type virus. All isolates from the lungs and nasal turbinates of recipients of two of the four Udorn/72-ts-1[A] recombinants contained only ts virus. These two properties, restricted replication and genetic stability after replication in vivo, suggest that the Udorn/72-ts-1[A] recombinants should be considered for evaluation as vaccines for use in humans.