Enzyme-linked immunosorbent assay for identification of rotaviruses from different animal species.

Rotaviruses cause gastroenteritis in man and a wide variety of animal species. They cross-react in many immunologic tests and have a similar appearance by electron microscopy, making differentiation among them difficult. Rotaviruses derived from different host species were distinguished by postinfection serum blocking virus activity in an enzyme-linked immunosorbent assay (ELISA). Thirty-three rotavirus isolates from children living in three different parts of the world could not be differentiated by this technique, but they were distinct from four strains recovered from calves, and a series of strains isolated from piglets, foals, monkeys, and infant mice. The four bovine strains were similar, but they could be differentiated from the other animal strains, each of which exhibited a distinct pattern when tested by the ELISA blocking technique.

Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus.

The possibility of immunizing human infants against rotaviruses, which cause severe dehydrating diarrheal disease, may depend on the use of a related rotavirus, derived from another animal species, as a source of antigen. To test the feasibility of this approach, calves were infected in utero with a bovine rotavirus and challenged with bovine or human type 2 rotavirus shortly after birth. Infection in utero with bovine rotavirus induced resistance to diarrheal disease caused by the human virus as well as the homologous bovine virus. These data suggest that the bovine virus is sufficiently related antigenically to the human type 2 virus to warrant further evaluation of the former as a source of vaccine.

Human rotavirus type 2: cultivation in vitro.

A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Reoviruslike agent in stools: association with infantile diarrhea and development of serologic tests.

Reoviruslike particles were visualized by electron microscopy in stool filtrates prepared from stools of infants and young children with severe acute gastroenteritis. Patients who had such particles in their stools and whose paired acute and convalescent serums were tested developed an antibody response to the reoviruslike agent, which was measured by immune electron microscopy and by complement fixation. The reoviruslike agent was antigenically related to the epizootic diarrhea of infant mice virus and the Nebraska calf diarrhea virus.

Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys.

An influenza A reassortant virus that contained the hemagglutinin and neuraminidase genes of a virulent human virus, A/Udorn/72 (H3N2), and the six other influenza A virus genome segments from an avirulent avian virus, A/Mallard/New York/6750/78 (H2N2), was evaluated for its level of replication is squirrel monkeys and hamsters. In monkeys, the reassortant virus was as attenuated and as restricted in its level of replication in the upper and lower respiratory tract as its avian influenza virus parent. Nonetheless, infection with the reassortant induced significant resistant to challenge with virulent human influenza virus. In hamsters, the reassortant virus replicated to a level intermediate between that of its parents. These findings suggest that the nonsurface antigen genes of the avian parental virus are the primary determinants of restriction of replication of the reassortant virus in monkeys. Attenuation of the reassortant virus for primates is achieved by inefficient functioning of the avian influenza genes in primate cells, while antigenic specificity of the human influenza virus is provided by the neuraminidase and hemagglutinin genes derived from the human virus. This approach could lead to the development of a live influenza A virus vaccine that is attenuated for man if the avian influenza genes are similarly restricted in human cells.

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype.

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat.

Human convalescent antiserum to respiratory syncytial virus (RSV) administered intraperitoneally to cotton rats prior to RSV challenge provided near-complete protection from pulmonary infection. Antiserum given subsequent to viral challenge reduced pulmonary viral titers 100-fold or greater within 24 h. Sandoglobulin, a preparation of purified human IgG with high titer of anti-RSV neutralizing activity, produced the same effects as convalescent antiserum. Sandoglobulin was absorbed rapidly and produced a significant therapeutic reduction in virus titer within 3 h. The level of virus reduction in pulmonary and nasal tissues was directly proportional to the neutralizing antibody titer in the cotton rat serum, and was always greater in the lungs than the nose. Animals treated therapeutically with Sandoglobulin had a depressed primary antibody response to infection, but were completely resistant to reinfection with RSV. Histologic examination of pulmonary tissues from Sandoglobulin-treated animals showed no pathologic changes.

An update on approaches to the development of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccines.

RSV and PIV3 are responsible for about 30% of severe viral respiratory tract disease leading to hospitalization of infants and children. For this reason, there is a need to develop vaccines effective against these viruses. Since these viruses cause severe disease in early infancy, vaccines must be effective in the presence of maternal antibody. Currently, several strategies for immunization against these viruses are being explored including peptide vaccines, subunit vaccines, vectored vaccines (e.g., vaccinia-RSV or adenovirus-RSV recombinants), and live attenuated virus vaccines. The current status of these approaches is reviewed. In addition, the immunologic basis for the disease potentiation seen in vaccinees immunized with formalin-inactivated RSV during subsequent RSV infection is reviewed. The efficacy of immunization in the presence of maternal antibody is discussed. Much progress for a RSV and PIV3 vaccine has been made and successful immunization against each of these pathogens should be achieved within this decade.

Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses.

Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.

Site-directed ELISA identifies a highly antigenic region of the simian immunodeficiency virus transmembrane glycoprotein.

The transmembrane glycoprotein (gp32) of the simian immunodeficiency virus (SIV) contains a highly antigenic region that includes amino acid residues 606-628. A synthetic peptide representing this region was highly immunoreactive with sera from SIV-infected primates in a site-directed enzyme-linked immunosorbent assay (ELISA). This reactivity extended across four primate species from three genera and identified infection with at least two distinct isolates of SIV. This site-directed ELISA represents a simple, accessible method with broad specificity for screening large numbers of primates for antibodies against SIV.

Jennerian and modified Jennerian approach to vaccination against rotavirus diarrhea using a quadrivalent rhesus rotavirus (RRV) and human-RRV reassortant vaccine.

Rotaviruses are the single most important cause of severe diarrhea of infants and young children world-wide. Deaths from rotavirus diarrhea occur infrequently in developed countries; however, in developing countries, rotaviruses are estimated to cause over 870000 deaths in the under five-year age group. There is, therefore, a vital need for a vaccine to prevent severe rotavirus diarrhea in infants and young children. The most extensively evaluated strategy for rotavirus vaccination has been the "Jennerian" approach in which an antigenically related rotavirus strain from an animal host (bovine or simian [rhesus monkey]) is used as the immunogen to induce protection against the four epidemiologically important group A human rotavirus serotypes. These orally administered vaccines were safe and immunogenic but had only limited success because serotype-specific immunity was not induced consistently in the under six-month age group. Therefore, a modified "Jennerian" approach was adopted with the goal of attaining broader antigenic coverage. In this approach four serotypes are combined to form a quadrivalent vaccine comprised of (i) rhesus rotavirus (RRV) which provides coverage for VP7 serotype 3, and (ii) three human-RRV reassortants each with ten RRV genes and a single human rotavirus gene that encodes VP7 serotype 1, 2, or 4 specificity. This modified "Jennerian" approach appears to be quite promising in preventing severe diarrhea in field trials. However, if this approach fails to yield an optimal level of protection consistently, additional modified "Jennerian" strategic, are under development that consider not only human rotavirus VP7 but also human rotavirus VP4, the other outer capsid protein. In addition, a non-"Jennerian" approach includes the development of cold-adapted human rotavirus strains or cold-adapted human rotavirus reassortants as vaccine candidates.

Diarrhea associated with rotavirus in rural Guatemala: a longitudinal study of 24 infants and young children.

A population of 24 infants and young children followed prospectively during the first 3 years of life was studied for the occurrence of rotavirus infection by using enzyme-linked immunosorbent assay to detect virus in stools. Infection with rotavirus was associated with 26 (14.2%) of 183 selected diarrheal episodes. Twenty of the 24 infants and young children had diarrhea associated with rotavirus on at least one occasion and six had two such episodes. Rotavirus infection was documented in over 50% of the dehydrating episodes studied, thus further indicating the importance of rotavirus in this population.

Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region.

The recombinant dengue virus type-4 vaccine candidate 2AA30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2Adelta30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer = 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.