Approaches to the treatment of pulmonary fibrosis.

There was a general consensus at the workshop that pulmonary fibrosis is a highly lethal lung disorder and that current therapies for this disease have little effect on the natural history of the disease. There was also broad consensus that much has been learned about the pathogenesis of this disease in recent years and this information can be used to direct new therapies for this disease. As a complement to the findings from studies on pulmonary fibrosis itself, there has also been significant development of new therapies for other disorders that might be useful in treating patients with pulmonary fibrosis. The following is a list of some of the issues and recommendations that were generated from this workshop.

The challenge of Pneumocystis carinii culture.

Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.

Bronchoalveolar lavage. The report of an international conference.

The application of the method of bronchoalveolar lavage to an increasing array of pulmonary diseases was evident, and the use of sophisticated technology to study cells and measure minute amounts of protein and other components in bronchoalveolar lavage fluid indicated that more meaningful information may be gained about diseased airways and alveolar spaces than suspected. That new techniques are being developed to make assays more sensitive and specific was evident. The popularity of this research approach was underscored by the interest and participation of colleagues in the United States and especially in Europe (notably France, England, Italy, and West Germany) and in Japan. This meeting was an opportune time to reflect on what bronchoalveolar lavage analysis has contributed to date and to focus attention on new applications that will be forthcoming and will improve our understanding of immunopathogenic mechanisms in an expanding number of pulmonary diseases.

Isolation and characteristics of an equine reovirus type 3 and an antibody prevalence survey to reoviruses in horses located in New York State.

Reoviruses have been isolated from a number of species including human, bovine, feline, canine and equine. In most species they seem to produce mild to inapparent disease. We have isolated a reovirus type 3 from a foal with diarrhea. The virus designated the Ralph strain has been propagated in both the MA-104 and A-72 cell lines. The strain produced cytoplasmic inclusion bodies in these cell cultures. Tissue-cultured virus fixed complement in the presence of reovirus antibodies, but failed to do so in the presence of rotavirus antiserum. By electron microscopy the viral particle measured +/- 65 nm. The virus hemagglutinated pig erythrocytes, but not human O, human A, calf, cow, chicken or guinea pig erythrocytes. In the hemagglutination test there was complete reciprocal crossing between the Ralph strain and the NIH reovirus type 3, but there was no crossing with the NIH reovirus types 1 and 2. A limited serological survey was completed on serum samples from New York State horses collected in 1976-1977 and 1981 using the hemagglutination-inhibition test. The percentage with antibodies to reovirus types 1, 2 and 3 for 1976-1977 was 24.5, 42.2 and 3.9% and in 1981, 8.8, 9.8 and 3.9%, respectively.

The isolation, propagation and characterization of tissue-cultured equine rotaviruses.

From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.

Isolation, propagation, and characterization of a second equine rotavirus serotype.

A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.

Isolation and characterization of an equine rotavirus.

A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell culture-adapted strain hemagglutinated erythrocytes of human group O, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests.

Direct isolation in cell culture of human rotaviruses and their characterization into four serotypes.

Of 73 rotavirus-positive fecal specimens tested, 39 yielded a human rotavirus that could be cultivated serially in MA104 or primary African green monkey kidney cells or both; 18 were serotyped. Four distinct serotypes were identified by plaque reduction or tube neutralization assay or both, and three of these serotypes were the same as those established previously by plaque reduction, using human rotaviruses cultivated by genetic reassortment with a cultivable bovine rotavirus. Ten human rotavirus strains received from Japan were found to be similar, if not identical, to our candidate prototype strains representing these four human rotavirus serotypes.

Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus.

A series of monoclonal antibodies was isolated which reacted with one of two major surface proteins of rhesus rotavirus. Thirty-six monoclonal antibodies immunoprecipitated the 82-kilodalton outer capsid protein, the product of the fourth gene, the viral hemagglutinin. These monoclonal antibodies exhibited hemagglutination inhibition activity and neutralized rhesus rotavirus to moderate or high titer. Three monoclonal antibodies immunoprecipitated the 38-kilodalton outer capsid glycoprotein, the eighth or ninth gene product. These three monoclonal antibodies neutralized rhesus rotavirus to high titer and also inhibited viral hemagglutination.

Serological comparison of canine rotavirus with various simian and human rotaviruses by plaque reduction neutralization and hemagglutination inhibition tests.

By the plaque reduction neutralization test, the CU-1 strain of canine rotavirus was similar, if not identical, to three strains (no. 14, no. 15, and P) of the tentatively designated third human rotavirus serotype. In addition, strain CU-1 demonstrated a one-way antigenic relationship with two other strains (M and B) of the third human rotavirus serotype. The CU-1 strain of canine rotavirus hemagglutinated human group O, rhesus monkey, dog, sheep, and guinea pig erythrocytes. A two-way antigenic relationship between canine (CU-1) and simian (MMU 18006 and SA11) rotaviruses demonstrated previously by the plaque reduction neutralization test was confirmed further with two additional isolates (A79-10 and LSU 79C-36) of canine rotavirus by the plaque reduction neutralization test and the hemagglutination inhibition test. The CU-1 strain of canine rotavirus, which is known to be distinct from two well-characterized human rotavirus serotypes (Wa and DS-1), was also found to be distinct from the St. Thomas no. 4 strain, which is a newly defined fourth human rotavirus serotype. Thus, this canine strain, which is related antigenically to one of four human rotavirus serotypes, is another example of an animal rotavirus which shares serotype specificity with a human rotavirus.

Gene coding assignments for growth restriction, neutralization and subgroup specificities of the W and DS-1 strains of human rotavirus.

Gene coding assignments for growth restriction, neutralization and subgroup specificities were determined for two human rotavirus strains, DS-1 and W, which represent two distinct serotypes. The 4th gene segment of both viruses was associated with restriction of growth in cell culture. The 9th gene segment of W virus and 8th segment of DS-1 were associated with serotype specificity, while the 6th gene segment of W virus was associated with subgroup specificity.

Identification of the rotaviral gene that codes for hemagglutination and protease-enhanced plaque formation.

Temperature-sensitive mutants of bovine rotavirus, UK Compton strain, and rhesus monkey rotavirus, MMU18006 strain, were used to derive 16 reassortants by coinfection of MA104 cells. The parental viruses differed phenotypically in their neutralization specificity, their ability to hemagglutinate, and their requirement for exogenous trypsin for infectivity. When the reassortants were assayed for neutralization specificity and hemagglutination, four phenotypes were observed, indicating that these two rotaviral functions segregated independently. Protease-enhanced infectivity phenotype segregated with the HA phenotype indicating that these two functions were manifestations of the same gene product. In order to determine the gene responsible for these rotaviral functions, the reassortants were genotyped by hybridizing 32P-labeled parental transcripts and denatured reassortant genomic RNAs and analyzing the resulting hybrids by gel electrophoresis. The fourth RNA segment was clearly shown to code for HA and protease enhanced plaque formation in MA104 cells. The neutralization antigen was linked to the eighth and ninth RNA segments that comigrated during gel electrophoresis and thus could not be differentiated.

Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies.

Ten monoclones directed to the 42,000-dalton inner structural protein of rotavirus were analyzed. Eight monoclones reacted broadly with antigenic domains common to virtually all mammalian rotaviruses. Two monoclones had specificities similar or identical to previously characterized subgroup specificities. These subgroup monoclones were more efficient in detecting subgroup antigen than either hyperimmune or postinfection antisera. Using the subgroup monoclones, we determined that some animal as well as human rotavirus strains carry subgroup 2 specificity and that epizootic diarrhea of infant mice virus and turkey rotavirus are antigenically distinct from other mammalian rotavirus strains.