I. L. Chrystie

The Effect of Trypsin on the Growth of Rotavirus

It has been found that 1000-fold more bovine rotavirus is obtained when trypsin is incorporated in the maintenance medium and allowed to remain throughout the growth cycle. This holds true for primary calf kidney (CK) cells and also for several continuous and semi-continuous cell lines. In the presence of trypsin it has been possible to pass the virus serially on continuous cell lines seven times. Concentrations of 1 to 10 microgram/ml of trypsin are found to be effective. Preliminary results suggest that the same technique will be effective for the in vitro propagation of human rotavirus.

ROTAVIRUS INFECTIONS IN A MATERNITY UNIT

Between May and August 1975, rotaviruses were detected in the stools of 76 out of 174 (44%) newborn babies in the maternity unit at this hospital. Infection occurred less frequently in breast-fed than in bottle-fed babies (P less than 0.001). However, only 7 out of 76 (8%) babies who excreted rotaviruses had symptoms and these were mild. Complement fixation tests did not show any apparent difference in the antibody titres or serological responses between mothers of rotavirus positive or negative babies. When 68 faecal extracts known to contain rotaviruses by electron microscopy were inoculated by centrifugation on to monolayers of continuous pig kidney cell cultures (IB-RS-2), rotavirus antigen was detected by immunofluorescence in 65 (95.5%) specimens, 58 being positive after centrifugation at 3000 g and a further 7 after centrifugation at 10 000 g. Antigen was first detected 6 hours after inoculation of specimens, maximum levels being detected at 24 hours.

Assisted conception in HIV discordant couples: evaluation of semen processing techniques in reducing HIV viral load

Artificial insemination with motile spermatozoa prepared from HIV-infected men using standard procedures has been employed with many HIV-discordant couples. We have demonstrated that processing semen from HIV positive men can reduce HIV levels, measured as HIV1 RNA copies/ml using nucleic acid based sequence amplification (NASBA), to undetectable levels (less than 400 copies/ml) but not in all samples. We believe that all processed samples should be tested prior to insemination.

Cord blood and breast-milk antibodies in neonatal rotavirus infection.

Studies were carried out during an outbreak of rotavirus type 2 infection in a neonatal nursery to determine the protective role of antibodies in cord blood and breast milk. The range, distribution, and geometric mean titres of rotavirus-specific antibody in the cord blood were similar among rotavirus-positive and rotavirus-negative neonates, and the amount of virus excreted did not correlate with antibody levels. Despite the protective effect of breast feeding, the pattern of rotavirus-specific IgA and IgG antibodies in the expressed breast milk of mothers of babies who were rotavirus excreters and non-excreters was similar. Nevertheless, a higher proportion of expressed breast milk samples contained rotavirus-specific IgA group 2 (92%) and type 2 (97%) specific antibodies than type I (67%) antibodies, and the geometric mean titres of group 2 and type 2 specific antibodies were tenfold higher than type I antibodies. Among breast-fed babies who excreted rotavirus there was no correlation between type 2 rotavirus-specific IgA antibodies in expressed breast milk and the amount of neonatal virus excretion. These studies suggest that factors other than the rotavirus antibodies in expressed breast milk are of importance in preventing rotavirus infection in newborn infants.

K65R and Y181C are less prevalent in HAART-experienced HIV-1 subtype A patients.

The vast majority of HIV-1 infections globally are caused by subtype A or C, although little is known about their drug resistance profiles. We found that HAART-experienced patients infected with subtype A had a lower prevalence of K65R and Y181C than those with subtypes B or C, despite similar exposure to antiretroviral agents that select for these mutations. If confirmed, this information may be important in the planning of antiretroviral regimens in patients infected with HIV-1 subtype A.

Voluntary, named testing for HIV in a community based antenatal clinic: a pilot study

Despite the increasing advantages of identifying HIV infection in pregnant women, only some 12% of HIV positive women attending antenatal clinics in London have been identified by named testing. As virtually all antenatal care will be community based within the next two to three years, we assessed the problems of introducing named HIV testing during pregnancy into the primary care setting. Planning the service took a considerable time and required the production of educational material for both staff and pregnant women and some reorganisation of procedures. Over a one year period an uptake of 44% was noted. Several problems were encountered including an average of 21 minutes needed to give information on AIDS and HIV, an adverse effect on the midwife-mother relationship, and anxiety (affecting both women and midwives). Possible solutions to this difficult problem are discussed.

Problems in the interpretation of HIV-1 viral load assays using commercial reagents.

During routine monitoring of human immunodeficiency virus (HIV) viral load, two problems arose. First, a number of patients, the majority being African, were found to have low viral loads by the Chiron branched‐chain DNA assay in conjunction with low CD4+ cell numbers. In order to determine whether this was due to failure of the branched‐chain DNA assay to detect non‐B subtypes of HIV, selected samples were subtyped and HIV RNA quantified by branched‐chain DNA, NASBA, and the Roche Monitor RT‐PCR assay. Twenty‐eight (97%) of 29 Africans were infected with a non‐B subtype of HIV and 15 (93.7%) of 16 non‐Africans with subtype B. Twenty‐three samples had a low viral load by branched‐chain DNA, which was confirmed by the NASBA and RT‐PCR assays. All three assays detected B and non‐B subtypes with similar efficiency; NASBA failed to detect HIV RNA in a small number of non‐B samples. Discrepancies between viral load and CD4+ cell numbers did not appear therefore to be related to subtype. Second, while quantification of HIV RNA was being conducted using version 2 of the branched‐chain DNA assay (lower detection limit 500 HIV RNA copies/ml) the manufacturers had developed a more sensitive assay and a comparative evaluation was therefore conducted. In approximately 30% of samples the viral load was up to 10 times higher with the more sensitive assay. These experiences emphasise the importance of close collaboration between the clinic and the laboratory. J. Med. Virol. 61:187–194, 2000.

Genotyping of B and Non-B Subtypes of Human Immunodeficiency Virus Type 1

ABSTRACT Current HIV-1 genotyping assays were developed using subtype B viruses prevalent in Western countries. It is not clear whether these assays are appropriate for use among African patients, who are likely to be infected with non-B subtypes. We evaluated the Bayer TRUGENE HIV-1 genotyping (TG) assay using prospectively collected samples from HIV-1-infected individuals who acquired infection in either sub-Saharan Africa or the West (Europe, North America, and Australia). Plasma samples from 208 individuals with an HIV-1 viral load of >1,000 copies/ml were tested using version 1 primers supplied with the TG assay. If these failed, an alternative primer set version 1.5 was used. Of the 208 individuals, the likely origin of infection was Africa (n = 104), Western (n = 87) and “Others” (i.e., all other geographic locations or origin not certain; n = 17). Among the three groups, the version 1 primers were successful in 85 (82%), 77 (89%), and 13 (76%) individuals, respectively (P = 0.1). Of the remaining 32 samples, 30 were successfully amplified by using the version 1.5 primers. HIV-1 subtypes deduced from the reverse transcriptase sequences correlated with the likely origin of infection: Africa (28A, 3B, 33C, 13D, 6G, 4J, 2K, 5CRF01_AE, and 10CRF02_AG), Western (86B and 1K), and Others (1A and 16B). The success of the version 1 primers correlated with viral load (P < 0.014) and not with HIV-1 subtypes. A protocol based on version 1 primers, followed by 1.5 primers, was successful in sequencing 99% of the samples in this cohort.

Further studies of Hiv morphology by negative staining

Thin-section studies of HIV-1- and HIV-2-infected cells were used to establish peak virus productivity and distribution of virus on and around infected cells. Maximum virus yields occurred 7 days after passage; cells at that stage were used as a source of virus for negative staining. Various methods of separating virus and cells were assessed: results showed that gentle homogenization in a Tenbroek-type homogenizer yielded considerably more virus than other techniques. Virus obtained in this way mainly appeared in the form of large clumps. Because of the large numbers of virus particles obtained it was possible to visualize what is probably the immature form of the virus. The inner component of this particle is spherical and, as is discussed, is a transient form proceeding to the now well established, mature, cone-shaped virus core.

Antenatal HIV testing: current problems, future solutions. survey of uptake in one London hospital

Pregnant women attending Guys and St Thomass Hospitals Trust have one of the highest prevalence rates for HIV-1 in inner London (0.53% in 1996).1 In 1992 we showed that this was associated with African ethnic origin.2 However, despite the Department of Healths recommendations that named HIV testing be made available to all pregnant women in areas of relatively high prevalence, uptake in our trust is disappointingly low—about 30%—as elsewhere in inner London. In 1995, throughout London, only 26 of 205 (13%) HIV positive pregnant women had been identified antenatally.3 Most were therefore almost certainly unable to benefit from recent advances in treatment and in the prevention of mother to child transmission of HIV. This paper describes uptake of HIV testing among pregnant women between 1991 and 1996 and includes a detailed survey of 789 women, of whom 428 attended antenatal clinics at Guys Hospital, 310 attended six …