Wayne P. Campbell

Detection of arboviral RNA directly from mosquito homogenates by reverse-transcription - polymerase chain reaction.

Many arthropod-borne viruses (arboviruses) are important human pathogens medically. The development of an effective technique to detect the viruses by using nucleic acid amplification, such as polymerase chain reaction (PCR), improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, the development of an improved and simplified assay is described for detection of mosquitoes infected with eastern equine encephalitis (EEE) virus, Cache Valley (CV), and California (CAL) serogroup viruses from field-collected mosquito pools. As little as 5 microl of homogenate from mosquito pools was used in the reverse transcription (RT) reaction followed by the use of three sets of specific primers for the PCR. Positive pools were determined by finding PCR bands of the expected size for each arbovirus. The confirmation and identification of Bunyaviruses was done by sequencing the PCR product. In 1999, West Nile virus (WNV) was identified as the etiologic agent of an outbreak of human encephalitis in New York City. It is shown that this protocol is also able to detect West Nile viral RNA in a pool of 100 mosquitoes containing one infected mosquito.

Detection of California serogroup Bunyaviruses in tissue culture and mosquito pools by PCR

A reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for rapid detection of 14 California serogroup viruses using universal primer pairs. These primers are specific for the small RNA (S RNA) and middle RNA (M RNA) segments. The method has been employed to detect Jamestown Canyon (JC) virus in naturally infected mosquitoes. With this technique, it is possible to detect virus in an amount of material equivalent to as little as 0.03 mosquito.

Sequence comparisons of medium RNA segment among 15 California serogroup viruses.

The complete nucleotide sequences have been determined for the M segment of 12 California (CAL) serogroup bunyaviruses. A method is described here of long reverse transcription-polymerase chain reaction (RT-PCR) that yields the full-length medium (M) RNA genomic segment. A phylogenetic tree was constructed by comparison of the open reading frames (ORFs) in the M RNA segment of 15 CAL serogroup viruses. Three distinct branches were identified and they are represented by the California encephalitis (CE), Melao (MEL), and Trivittatus (TVT) complexes. These groups correspond to those previously established by small (S) RNA genomic sequences. In addition, except for Inkoo virus, the predicted relationship among these viruses agreed with those found by serology.

The distribution of 5-bromodeoxyuridine in the DNA of polyoma-transformed mouse cells and some apparent effects on transcription☆

Abstract The DNA of polyoma-transformed mouse cells grown in the presence of 5-bromodeoxyuridine (BUdR) was examined in neutral and alkaline CsCl gradients. All of the cells had incorporated BUdR into their DNA and no distinct classes of different degrees of substitution were observed. The reassociation kinetics of DNA containing tritiated BUdR demonstrated that the analog did not preferentially replace thymidine in either the repetitive or non-repetitive sequences. RNA-DNA saturation hybridization experiments using RNA extracted from cells grown in medium containing BUdR revealed an apparent reduction in the extent of non-repetitive DNA transcription compared with untreated control cells. The degree to which regulatory genes, as opposed to structural genes, is involved in this decrease cannot be determined from these data.

Evidence that fatal human infections with La Crosse virus may be associated with a narrow range of genotypes

La Crosse (LAC) virus belongs to the California (CAL) serogroup of the genus Bunyavirus, family Bunyaviridae. It is considered one of the most important mosquito-borne pathogens in North America, especially in the upper Mid-West, where it is associated with encephalitis during the time of year when mosquitoes are active. Infections occur most frequently in children and young adults and, while most cases are resolved after a period of intense illness, a small fraction (< 1%) are fatal. At present there have only been three isolates of LAC virus from humans all made from brain tissue postmortem. The cases yielding viruses are separated chronologically by 33 years and geographically from Minnesota/Wisconsin (1960, 1978) to Missouri (1993). The M RNA sequence of the first two isolates was previously reported. The present study extends the observations to the isolate from the 1993 case and includes several mosquito isolates as well. A comparison of the M RNAs of these viruses shows that for the human isolates both nucleotide sequence and the deduced amino-acid sequence of the encoded proteins are highly conserved, showing a maximum variation of only 0.91% and 0.69%, respectively. This high degree of conservation over time and space leads to the hypothesis that human infections with this particular genotype of LAC virus are those most likely to have a fatal outcome. It is also shown that a virus with this genotype could be found circulating in mosquitoes in an area more or less intermediate between the locations of the first and second fatal cases.

Detection of California serogroup viruses using universal primers and reverse transcription-polymerase chain reaction.

Universal primers have been identified and a protocol developed that are suitable for rapid detection of California encephalitis (CE) complex viruses in a reverse transcription-polymerase chain reaction (RT-PCR) assay. These primers correspond to sequences in the coding regions of the G2 glycoprotein of the middle-size RNA segment. The identities of the amplified products were confirmed by sequencing on the clones or PCR products. The technique is capable of detecting 40 plaque-forming units (PFU) directly on an ethidium bromide-stained agarose gel and the sensitivity increases to 0.4-1 PFU when a radiolabeled probe is used as the detector.

Hantavirus S RNA sequence from a fatal case of HPS in New York.

In April, 1995, the second fatal case of hantavirus pulmonary syndrome (HPS) occurred in the northeast in a New York State resident. Using the patients lung tissue obtained at autopsy, the S genomic RNA segment of a hantavirus, designated H‐NY1, was amplified by reverse transcriptase‐polymerase chain reaction (RT‐PCR), cloned, and sequenced. The S RNA was found to contain 2084 nucleotides, 6 nucleotides longer than reported by Hjelle et al. (1995) for the virus associated with the first northeastern case (RI‐1). There were 101 nucleotide differences in the S RNA between the H‐NY1 and RI‐1, which result in the prediction of a single amino‐acid change in the nucleocapsid (N) protein. Rodents were trapped for serologic and virologic studies at the patients residence and work site. The white‐footed mouse (Peromyscus leucopus) was the most frequently captured species and more than 50% of those trapped near the patients residence showed serologic evidence of hantavirus infection. Using RT‐PCR it was possible to amplify hantavirus S RNA sequence from the lung tissues of 8 out of 11 seropositive animals. No difference in nucleotide sequence was found between the HPS patient sequence and the P. leucopus sequence (nucleotides 189 to 599). These data are consistent with those of Hjelle et al. (1995) in suggesting that P. leucopus is the primary rodent vector for the etiologic agent of HPS in the northeastern United States.

Diagnosis of Jamestown Canyon Encephalitis by Polymerase Chain Reaction

In recent years, polymerase chain reaction (PCR) has been under study as a potential technique to improve the accuracy of diagnosis of suspected central nervous system viral infections. We describe a case of severe encephalitis in a previously healthy 20-year-old woman from New York who presented with headache, fever, and photophobia. Her illness was characterized by progressive worsening of her neurological status, leading to confusion, delirium, and status epilepticus. The diagnosis of Jamestown Canyon encephalitis was established by positive reverse transcriptase (RT)-PCR and nucleic acid sequencing of the band from both cerebrospinal fluid and brain tissue. The nucleotide sequence and the deduced amino acid sequence of the Jamestown Canyon virus from this patient were very similar to Jamestown Canyon virus isolates from mosquito pools in New York. This report suggests that RT-PCR assays could be important tools in the diagnostic workup of cases of encephalitis.

Comparison of the M RNA genome segments of two human isolates of La Crosse virus

The M genomic RNA segments of La Crosse (LAC) virus isolates from the brains of two children autopsied 18 years apart in Wisconsin were molecularly cloned using a reverse transcriptase-PCR assay and the nucleotide sequences of the cDNAs determined. The M RNA of each virus contains 4526 nucleotides, similar to that reported previously for a New York mosquito isolate of LAC. There were 20 nucleotide differences between the two human isolates, which results in the prediction of 7 amino acid changes in the proteins encoded in the single, long open reading frame of the M segment. One of these predicted differences occurs in the G2 glycoprotein and six in the G1 glycoprotein. The two viruses were identical in terms of predicted amino acid sequence in the region believed to represent a nonstructural protein. These data have been further compared to those available for two other California serogroup isolates.

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.