Identification of a Kunjin/West Nile-like flavivirus in brains of patients with New York encephalitis.

Molecular analysis of brains from patients of the recent New York City encephalitis outbreak reveals the presence of a flavivirus not previously described in the Americas.

Human and rodent hantavirus infection in New York State: public health significance of an emerging infectious disease.

BACKGROUND: A case of hantavirus pulmonary syndrome with possible exposure in New York and/or Rhode Island was confirmed in February 1994. OBJECTIVE: To conduct four studies to determine the historical and geographic distribution of human and small-mammal infection with hantaviruses in New York State. METHODS: Enzyme-linked immunosorbent assays were performed on serum samples obtained from 130 humans during a 1978 babesiosis survey, 907 small mammals collected in New York State since 1984, 12 rodents collected in 1994 near the residences of the patients with hantavirus pulmonary syndrome, and 76 New York patients with acute respiratory distress syndrome-like illness (as suspected cases of hantavirus pulmonary syndrome). RESULTS: None of the human serum samples from the 1978 serosurvey showed evidence of hantavirus exposure by enzyme-linked immunosorbent assay. Statewide historical serum samples from white-footed mice showed evidence of Sin Nombre virus infection in 12.0% (97/809) and Seoul-like virus infection in 9.6% (78/809). Site-specific seropositivity rates were as high as 48.5% with Sin Nombre virus during 1 year (1984). Two of 12 mice captured near the residences of a human patient were positive for Sin Nombre virus by enzyme-linked immunosorbent assay, yet were negative for viral RNA by polymerase chain reaction. None of the patients with suspected hantavirus pulmonary syndrome was serologically reactive for Sin Nombre virus. CONCLUSIONS: We provide serologic evidence of small-mammal infection with hantaviruses in New York State as long ago as 1984. Human cases of hantavirus pulmonary syndrome are rare in New York, and data indicate that transmission to humans is probably infrequent. A unique set of host, agent, and environmental factors may be necessary to cause hantavirus pulmonary syndrome in humans.

Presumptive identification of common adenovirus serotypes by the development of differential cytopathic effects in the human lung carcinoma (A549) cell culture.

The neutralization test is commonly used in clinical virology laboratories for the identification by serotype of adenovirus isolates. In an effort to conserve reagents and reduce the amount of time in the performance of this assay, we evaluated the significance of differential cytopathic effects for the presumptive identification of lower-numbered adenovirus serotypes that are commonly encountered in the clinical setting. Utilizing the human lung carcinoma (A549) cell culture as our indicator system, two viral induced monolayer degenerations (i.e., cytopathic effects or CPEs) were recognized. Among our wild and the laboratory adapted (i.e., ATCC) adenovirus isolates tested in this study, serotypes 1, 2, 4, 5, 6, 8, 11, 19, 21, 27, and 31 were expectedly characterized by the typically enlarged, rounded, and refractile cells, which eventually aggregated into irregular 'grape-like' clusters. Adenovirus types 3 and 7, however, were characterized by the development of distinct intranuclear inclusions, a flattening and then a web or net-like monolayer degeneration. Differences in the intensity of intranuclear granulation were related by electron microscopy to differences in the quantity of viral crystalline aggregates within the host cell nucleus. A presumptive identification of the commonly encountered adenovirus serotypes 3 and 7 prior to the performance of the neutralization test would result in a conservation of type-specific antiserum, a decreased use of cell cultures and medium, and lastly, reduced medical technologist workload.

The sequence of the M RNA of an isolate of La Crosse virus.

The middle-size (M) genomic RNA of a New York State, U.S.A. isolate of La Crosse (LAC) virus has been cloned by a random priming procedure and its nucleotide sequence determined by the dideoxy method. The RNA was found to be 4526 nucleotides in length and to have a base composition of 34.2% U, 27.8% A, 20.6% C and 17.4% G. There is a single, long open reading frame in the viral complementary RNA that contains sufficient information to code for a protein of 1441 amino acids. In these respects, as in many others, the LAC virus M RNA and its encoded protein were very similar, if not identical, to those previously reported by other investigators for the closely related snowshoe hare virus. The M RNAs of the two viruses show 79% nucleotide sequence homology and 89% homology in the amino acid sequence of their encoded proteins. Several algorithms for predicting surface residues, as well as the Chou-Fasman rules for predicting secondary structure, were used to compare the LAC virus and snowshoe hare virus M gene proteins. These analyses identified 39 sites on the proteins as those most likely to be linear antigenic determinants that might contribute to the differences between the two viruses.

Two monoclonal antibodies against La Crosse virus show host-dependent neutralizing activity.

Mammalian and arthropod cell cultures were used to assess the neutralizing activity of six monoclonal antibodies specific for the G1 glycoprotein of La Crosse virus. Four antibodies, two neutralizing and two non-neutralizing, showed no host-dependent differences, giving similar results when post-treatment infectivity was determined using either Aedes albopictus cells or BHK-21 cells. For two other antibodies, however, dissimilar activities were observed between the vertebrate and invertebrate cell lines. One of these antibodies was positive when BHK-21 cells were employed as the post-treatment host and negative when mosquito cells were used; the other antibody was the converse. The epitope for this last antibody was present on all California serogroup viruses examined, which suggests that it may have a special significance in the natural life-cycle of the virus.

Replication of non-repetitive DNA during early, middle and late S phase. The general class and the subset transcribed.

Synchronized cultures of Chinese hamster ovary cells were pulse-labelled with 5-bromodeoxyuridine (BrdU) during early (0-2.0 h), middle (2.5-4.0 h) and late (4.5-6.0 h) S phase in two successive cell cycles. In each case, the DNA containing BrdU in both strands was duplicated at the same time in both cycles and was isolated for further characterization by centrifugation in CsCl gradients. These DNAs were then radiolabelled by nick-translation and used in either DNA-DNA or RNA-DNA hybridization experiments. In the DNA-DNA experiments, advantage was taken of the substantial rate increases attainable in high concentrations of dextran sulfate to obtain complete reassociation curves with relatively small amounts of material. Assuming that no unresolved low repetition frequency components exist, renaturation kinetics suggest that early replicating DNA contains a greater proportion of non-repetitive sequences than DNA synthesized at later times, the order being early greater than middle greater than late. However, in terms of complexity the non-repeated DNA duplicated early had only 74% of the diverse sequences present in log-phase cells, whereas that replicated in middle and late S phase had 82 and 79.5%, respectively. It therefore appears that while DNA synthesized at different times in S phase may contain varying proportions of non-repetitive sequences, when their diversity is taken into account very few of these sequences (25% or less) exhibit temporal control of replication. Finally, measurements with total cell RNA indicated that the transcribed fraction of non-repeated DNA showed a slight preference for replication in early S phase.

Monoclonal antibodies against La Crosse virus.

Monoclonal antibodies have been used to show that an epitope is present on the G1 glycoprotein of prototype La Crosse virus that is absent or significantly altered on several isolates of La Crosse virus made in New York State, U.S.A. The portion of the G1 protein where this epitope is located plays a role in both virus neutralization and haemagglutination. Additional experiments revealed that under the appropriate assay conditions the monoclonal antibodies permitted discrimination between representatives of the North American members of the California serogroup.

Evidence for three separate antigenic sites on the G1 protein of La Crosse Virus.

Competitive binding assays with monoclonal antibodies have been used to show that there are a minimum of three nonoverlapping antigenic sites on the G1 glycoprotein of La Crosse virus. One of these sites contains the epitopes for three of the five monoclonal antibodies employed and is involved with both hemagglutination and neutralization. A second site contains the epitope for an antibody that inhibits hemagglutination but has no neutralizing activity. The third site encompasses the epitope for an antibody that at present has no identified biological role.

Sequence diversity of nuclear and polysomal polyadenylated and non-polyadenylated RNA in normal and regenerating rat liver.

A DNA probe purified from RNA . DNA hybrids of total sham-operated liver RNA and non-repetitive DNA was used to show that nuclear poly(A)-rich RNA from sham-operated liver and from 2.5-h and 48-h regenerating liver contains about 50% of the complexity of total liver RNA. It was further shown that the differences between normal and regenerating liver, reported earlier from this laboratory, occur in the poly(A)-free fraction of nuclear RNA. At the polysome level it was found that polysomal RNA has one-third of the sequence diversity of total RNA and of this, approximately 65% can be accounted for by poly(A)-rich and 55% by poly(A)-free molecules. When DNA probes were prepared from hybrids formed using polysomal RNA from sham-operated liver and regenerating liver at 2.5 h and 48 h post-hepatectomy and then employed in reactions with homologous and heterologous RNAs, no differences were detectable between either normal and regenerating liver or regenerating liver at times during hypertrophy and hyperplasia.

Ageing studies in rat liver. I. Complexity of RNA from two to ten months of age.

A highly sensitive nucleic acid hybridization assay was used to compare the extent of nonrepetitive DNA transcription in rat liver between the ages of two and ten months. The basic approach consisted on initially purifying the DNA expressed in liver at these ages and then using it in reactions with homologous and heterologous RNAs. Such experiments failed to reveal any differences in nonrepetitive DNA transcription as a function of age. The possibility was also explored that there might be an age-associated variation in the proportion of the total RNA complexity attributable to poly(A+) RNA. These experiments, too, were negative in that the poly(A+) RNA was found in all cases to account for approximately 50% of the total sequence diversity. Overall, these data strongly suggest that ageing is not accompanied by a steady, progressive change in the regions of the genome transcribed, either quantitatively or qualitatively.

Nonrepetitive DNA transcription in normal and regenerating rat liver.

Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNA-DNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcrips present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA.

Complexity of poly(A+) and poly(A-) polysomal RNA in mouse liver and cultured mouse fibroblasts.

RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.

Transcriptional complexity of vaccinia virus in vivo and in vitro.

The transcriptional complexity of vaccinia virus both in vivo and in vitro has been measured by using DNA:RNA hybridization with RNA in excess. In vivo, "early" or prereplicative RNA was found to saturate at 25% or one-half of the viral genome. "Late" or postreplicative RNA from infected HeLa cells saturated at 52% or essentially the entire genome. This well-regulated transcriptional pattern of the virus in vivo was not maintained in vitro. In a number of experiments a range of saturation values from 40 to 50% was obtained for in vitro synthesized RNA. The complexity of polyadenylated and non-polyadenylated RNA, as well as total purified 8 to 12S RNA released from the virus, was indistinguishable from purified high-molecular-weight virion-associated RNA with a sedimentation value of greater than 20S and equivalent to total in vitro synthesized RNA. No additional hybrid formation was observed in experiments in which total in vitro RNA and late in vivo RNA from infected HeLa cells were combined, suggesting that the virus does not transcribe in vitro DNA sequences that are not also transcribed during productive infection. Approximately 15% complementary RNA was detected when radiolabeled total in vitro RNA was allowed to reanneal with late in vivo RNA, while as much as 8% of the in vitro synthesized RNA was found to be complementary.