Induction of host cell protein synthesis by human herpesvirus 6.

We observed an increase in host cell protein synthesis in human cord blood lymphocytes (CBL) infected with human herpesvirus 6 relative to uninfected cultures. The magnitude of this effect could not be explained by a smaller decrease in cell number in the infected cultures. The induction of host cell protein synthesis by HHV-6 does not appear to be mediated by a stable soluble factor present in the infected cell culture supernatant. When CBL were infected with virus that had been exposed to ultraviolet irradiation (UV) for various intervals, we found that the level of increase in cell number, host protein synthesis, viral DNA and viral antigen was inversely proportional to the length of time of virus exposure to UV. No increase in cell number or host cell protein synthesis was seen in CBL infected in the presence of 50 micrograms/ml phosphonoacetic acid, an inhibitor of HHV-6 DNA replication. These results indicate that components of input virions do not induce the increased protein synthesis and that the induction is dependent on viral DNA replication.

Frequent isolation of human herpesvirus 7 from saliva.

We obtained isolates of human herpesvirus 7 (HHV-7) from 6 of 8 healthy adults by culturing saliva with human umbilical cord blood lymphocytes. These isolates were identified as HHV-7 on the basis of comparisons of restriction endonuclease fragment profiles and hybridization with HHV-7 strain RK DNA. The isolates could be differentiated from HHV-7 strain RK and from each other by their restriction endonuclease fragment profiles. We confirm the finding of frequent isolation of HHV-7 from saliva of healthy adults and report the first dual isolation of human herpesvirus 6 (HHV-6) and HHV-7 from a single saliva specimen. We also describe an in situ hybridization assay that can distinguish between HHV-6 and HHV-7.

Biologic properties of human herpesvirus 7 strain SB.

The growth characteristics of human herpesvirus 7 strain SB (HHV-7 (SB)) were studied in human umbilical cord blood lymphocyte (CBL) cultures. The virus has approximately a 4-day growth cycle, as measured by immunofluorescence analysis, quantitation of the relative viral DNA concentration, and examination of infected cells by electron microscopy on consecutive days post-infection. By systematically varying the culture media components, improved culturing conditions were established. Activated lymphocytes were required for virus growth. HHV-7(SB) grew best in phytohemagglutinin-stimulated CBL cultured in media containing 0.01 mg/ml hydrocortisone. Addition of recombinant human interleukin 2 (IL-2) at concentrations exceeding 1-10 U/ml inhibited virus growth in most CBL cultures. Addition of exogenous IL-2 to the culture media had no effect on viral DNA production. However, the percentage of virus antigen-positive cells was highest when 0.1-1 U/ml was added to the media. Differences in the ability of individual CBL cultures to replicate HHV-7(SB) was not explained by differing CD4+ cell concentrations. However, individual cultures varied in the level of endogenous IL-2 production, which may contribute to the virus growth variability in CBL. HHV-7(SB) grew in the CD4-positive T-cell line SupT1, but not in a variety of other lymphocyte, fibroblast, or epithelial cell lines. Nine compounds were tested for antiviral activity against HHV-7 in vitro. Phosphonoformic acid inhibited virus growth with a 50% effective concentration of 4.8 microM. Ganciclovir (200 microM) and phosphonoacetic acid (100 microM) inhibited more than 90% of virus production. None of the compounds were cytotoxic at concentrations which inhibited the virus. A generalized increase in host cell protein synthesis was also observed in virus-infected cells similar to that seen in CBL infected with human herpesvirus 6.

Growth properties of human herpesvirus-6 strain Z29.

Experiments performed to optimize the growth conditions of HHV-6(Z29) revealed that the virus grows best in phytohemagglutinin-stimulated umbilical cord blood lymphocytes (CBL) cultured in media containing 32 units/ml interleukin-2 and 0.01 mg/ml hydrocortisone. The titer of maternal antibody in the plasma of the cord blood cells does not affect the ability of the cells to support virus growth. DEAE-dextran and polybrene do not increase virus growth in umbilical cord blood lymphocytes. Phorbol myristate acetate abolishes virus growth. The HHV-6(Z29) growth cycle in CBL was approximately 5 days; capsids were not seen before day 3, and mature virions were not seen before day 5.

The structure of the salivary glands of the human soft palate.

The glandular layer constitutes the greatest bulk of the human soft palate and is composed of individual compound tubulo-acinar salivary glands. Connective tissue partitions of the submucosa divide the glandular layer into lobules of irregular shapes and sizes. The glands are interwoven and bound firmly together by a connective tissue stroma rich in elastic fibers. The secretory units consist of elongated, branched, and sometimes convoluted tubules lined by a single layer of pyramidal mucous cells. Mucous secretion by acini is supplemented to some degree by mucous acinar cells, which were found as epithelial components of all ducts except the main excretory ducts, suggesting a diffuse distribution of progenitor cells. Some mucous acini communicate with highly convoluted intercalated ducts which occupy partially isolated positions within inter- and intralobular connective tissue septa. These ducts follow the connective tissue septa and eventually join the main duct system. The significance of this system of intercalated ducts is not known. A supplemental functional role is hypothesized.

Antiviral screening assays for human herpesviruses 6 and 7.

Human herpesviruses 6 and 7 are ubiquitous herpesviruses that normally infect their hosts early in life. There are two variant groups of human herpesvirus 6 (HHV-6): variants A (HHV-6A) and B (HHV-6B). Variant A has not been unambiguously associated with a specific disease but may contribute to disease in immunocompromised patients; variant B is the major etiologic agent of roseola (roseola infantum or exanthem subitum) and other febrile illnesses of young children, and has been associated with disease in immunocompromised patients. HHV-6B is frequently present in plaque regions in the brains of multiple sclerosis patients, although an etiologic association has not been proven. Human herpesvirus 7 (HHV-7) has been associated with some cases of roseola. The clinical spectrum of these viruses remains to be completely defined. Braun et al. (1) recently described three clinical scenarios that might warrant the use of antivirals to treat HHV-6 infections: (1) transplant recipients with idiopathic pneumonitis (2), multiple sclerosis patients, and (3) patients with HHV-6-associated encephalitis. For HHV-7, cases of neurologic involvement during primary infection might warrant investigation (2).

Reduced motility longevity in thawed human spermatozoa.

Fifty ejaculates from donors serving on an AID panel were divided into two 0.5 aliquots and evaluated for initial motility and kinetics. The first aliquot was then diluted with Tyrode solution and incubated in a Tyrode atmosphere at 37 degrees C. Motility and kinetics were evaluated at 1, 3, 6, and 9 hr. The second aliquot was frozen and thawed 24 hr later. The aliquot was then diluted with Tyrode solution, incubated, and evaluated as before. The results indicate a significant reduction in motility longevity of the thawed aliquots. A significant reduction in kinetics of the thawed aliquots was also discovered. These findings suggest that decreased motility longevity of thawed semen may offer an explanation for reduced pregnancy rates using cryopreserved semen.

Human herpesvirus 7.

Human herpesvirus 7, reported in 1990 is a lymphotropic member of the betaherpesvirus subfamily of herpesviruses. The virus is highly seroprevalent, primary infection usually occurs during childhood, and it has been associated with cases of exanthem subitum, pityriasis rosea, neurological manifestations and transplant complications. The latter two may warrant antiviral intervention, in vitro studies have shown that HHV-7 is susceptible to several nucleoside phosphonate compounds. In vitro, the virus has approximately a 5 day growth cycle in cultured lymphocytes; in vivo, latency is established in peripheral blood T-cells and a persistent infection is established in salivary gland tissue from which infectious virus is constitutively shed in saliva. The HHV-7 genome is approximately 145 kb and encodes at least 84 different proteins. Studies characterising HHV-7 gene products and the required interactions between viral and cellular genes necessary for virus replication, persistence and latency are in their infancy. HHV-7 infection has a variety of effects on host cells including upregulation of interleukin 15 and down-modulation of the cell surface molecule CD4; the latter serves as the cellular membrane receptor for HHV-7. Since HIV also infects T-cells via the CD4 molecule, the interactions of these viruses within T-cells during the course of AIDS are important areas of investigation.

Effects of temperature, potassium concentration, and sugar on human spermatozoa motility: a cell preservation model from reproductive medicine.

Two experiments were performed in vitro with human sperm from a panel of 11 donors. In both experiments, washed sperm from each donor were divided into four equivolume samples, three of which were resuspended in various formulations of Tyrode's solution and the fourth was resuspended in its own seminal plasma. Each of the four samples was then stored at 37 or 3 degrees C for fixed intervals of 0, 2, 6, 12, and 24 h. Motility was assessed at 37 degrees C for all samples at the end of each interval. The results indicate that sperm survival in vitro at 3 degrees C was significantly enhanced by 20 mM K+ in Tyrode's solution relative to Tyrode's with less K+. A metabolizable sugar such as glucose was essential to maintaining sperm viability in K(+)-free media. The addition of raffinose to media containing glucose improved motility of sperm stored at 3 degrees C for 6 h.

Semen cryopreservation: an update.

Cryopreservation of semen has a place in reproductive medicine. It is done best using liquid nitrogen as the refrigerant and the pre-freeze semen should be of good quality. High quality spermatozoa survive the freezing-thawing process and ordinarily result in good babies. Abnormal sperm generally do not survive the freezing-thawing process, which, consequently, results in a more viable union and outcome from the germ cells. Cryopreservation of semen can be used to preserve semen before medical or surgical sterilization, to augment the sperm count in patients with certain types of oligospermia and to manage the childless couple by donor semen artificial insemination in those instances in which the husband is infertile. Cryopreserved semen produces babies and perhaps thousands of humans have been the result of conception that resulted from cryopreserved spermatozoa.

An immunoblot assay for detection of immunoglobulin M antibody to human herpesvirus 6.

We identified the human herpesvirus 6 (HHV-6)-dominant immunoglobulin M (IgM)-reactive virion protein as being the same 101-kDa protein (101K) previously identified as the major IgG immunoreactive protein and a specific serologic marker of HHV-6 infection. An immunoblot assay (IB) to detect HHV-6-specific IgM antibodies against the 101K protein in human serum samples was developed. The assay was validated by using acute- and convalescent-phase serum collected from children under 2 years of age in which we previously detected IgG seroconversion to the HHV-6 101K protein. Of 32 serum pairs which previously demonstrated IgG seroconversion to the 101K protein, 29 had IgM reactivity to the same protein in the acute-phase sample and the remaining 3 had reactivity in the convalescent-phase sample. We also detected HHV-6 IgM activity in sera collected from individuals > or =4 years of age who were also IgM seropositive to measles or rubella. Results of cross-adsorption studies using measles virus-, rubella virus-, and HHV-6-infected cells as the adsorbing antigen indicated no cross-reactivity between measles or rubella IgM and HHV-6 IgM in human serum samples. The IgM IB detected HHV-6-specific IgM antibody to the 101K protein in 78% (63 of 81) of tested acute-phase serum collected from young children with an undifferentiated rash illness by using a single serum dilution.

Intragenomic linear amplification of human herpesvirus 6B oriLyt suggests acquisition of oriLyt by transposition.

We identified some passage lineages of human herpesvirus 6 variant B (HHV-6B) strain Z29 that contain as many as 12 tandem copies of a genomic segment that corresponds almost precisely to a previously identified minimal efficient origin of lytic replication (oriLyt). Analysis of nucleotide sequences in the vicinity of the amplified segment suggests that the amplification occurred as a two-step process, with the first step being a rare sequence duplication mediated through directly repeated sequences located near the termini of the amplified segment and the second step occurring via homologous recombination through the duplicated sequence. These results demonstrate that oriLyt has been amplified in some virus stocks and indicate that (i) origin amplification confers a growth advantage on the virus in cell culture and (ii) laboratory-passaged HHV-6B genomes can accommodate additional nucleotide sequences and thus may be useful gene transfer vectors. The structures of the amplified segment and its adjacent sequences together suggest that HHV-6B or a progenitor virus acquired oriLyt by transposition from an unknown source.

Development of thyroidal responsiveness to estrogen in the maturing rat (38471).

The incidence of thyroid disorders around the time of puberty is frequent in human females. The influence of estrogen on thyroid function during this period has been controversial. Female rats hypophysectomized at 21, 30 or 50 days of age were treated with either 17beta-estradiol (E-2) or TSH or a mixture of E-2 and TSH. Determinations were then made of thyrodial 131I-uptake, organic 131I, and 131I excretory patterns. In 21-day old animals, E-2 alone had no effect on thyroidal 131 I uptake, whereas in 30-day or older animals E-2 significantly increased 131I uptake. TSH or TSH-E-2 combinations markedly increased 131I uptake in both groups. Thyroidal 131I was shown to be almost exclusively protein-bound in each instance. No difference was observed between 21 and 30-day old rats with respect to 131I excretory patterns,except for decreased urinary output in the TSH-E-2 groups; this decrease, however, appeared to be partially attributable to an increased thyroidal uptake of iodine in these animals. The results suggest that the thyroid gland becomes responsive to estrogen only at or around the time of puberty.

Identification of a nucleocapsid protein as a specific serological marker of human herpesvirus 6 infection.

Enveloped whole virions and nucleocapsids of human herpesvirus 6 (HHV-6) strain Z29 were purified from supernatant fluids of infected human cord blood lymphocytes by filtration through polyvinylpyrrolidone-treated filters, banding on a Nycondenz step gradient, and centrifugation through two successive continuous sucrose gradients. More than 20 proteins ranging in molecular weight from less than 30,000 to more than 200,000 were identified in preparations of purified whole virions labeled with [35S]methionine and [35S]cysteine. Immunogenic virion proteins of HHV-6 were identified in immunoblot assays with human immune sera, immune sera generated from mice immunized with purified whole virions or purified nucleocapsids, and a monoclonal antibody generated from a mouse immunized with purified nucleocapsids. The sera and the monoclonal antibody reacted strongly with a 101-kilodalton protein in the immunoblots, suggesting that the protein is a component of the nucleocapsid. Human sera lacking HHV-6-specific antibodies and seropositive for one or more of the other human herpesviruses failed to react with this protein, indicating that it is a specific serologic marker for HHV-6 infection.

Physical and genetic maps of the human herpesvirus 7 strain SB genome.

Human herpesvirus 7 (HHV-7) is a close relative of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) based on limited biologic and genetic data. In this work we describe physical and genetic maps for HHV-7 strain SB [HHV-7(SB)], which was obtained from the saliva of a healthy adult. The HHV-7(SB) genome length is approximately 144 kb by clamped homogeneous electric field gel electrophoresis and approximately 135 kb by summation of restriction endonuclease fragments. We constructed plasmid clones and PCR amplimers that span the HHV-7 genome, except for the genomic termini, and determined the maps of the restriction endonuclease cleavage sites for BamHI, PstI, and SacI. The HHV-7(SB) genome is composed of a single unique region of approximately 122 kb bounded at each end by a 6 kb direct repeat. Homologs to thirty-five herpesvirus genes were identified. The highest similarity was with the HHV-6 genes, with an average amino acid identity of 50%, followed by the human cytomegalovirus counterpart. The genomic and genetic maps indicated that the HHV-7 and HHV-6 genomes are colinear. There was no sequence variation in a segment of the gene encoding the DNA polymerase-associated factor homolog among six HHV-7 isolates, while the corresponding segment of the HHV-6A and HHV-6B counterparts differed by 4.6%. These data support previous observations that the closest genetic relatives of HHV-7 are betaherpesviruses.