Antigenic characterization of flavivirus structural proteins separated by isoelectric focusing.

Isoelectrofocusing of nonionic-detergent-disrupted flaviviruses separated the envelope glycoprotein of 53,000 to 58,000 daltons and the nucleocapsid protein of 14,000 daltons. The envelope protein and nucleocapsid protein were isolated at isoelectric points of pI 7.8 and 10.3, respectively. The antigenic determinants of St. Louis encephalitis, Japanese encephalitis, and dengue virus envelope and nucleocapsid proteins were examined by solid-phase competition radioimmunoassay. By the appropriate selection of antiserum and competing proteins, it was possible to distinguish type-specific, complex-reactive and flavivirus group-reactive antigenic determinants. The envelope glycoproteins of St. Louis encephalitis, Japanese encephalitis, and dengue viruses were found to contain each of these three classes of antigenic determinants. Most of the determinants on the envelope protein were type specific, some were complex reactive, and a small fraction were flavivirus group reactive. The nucleocapsid protein contained only flavivirus group-reactive antigenic determinants.     

Enhancement of St. Louis Arbovirus Plaque Formation by Neutral Red

Experiments with St. Louis encephalitis (SLE) virus have shown that neutral red enhances the plaque size in duck embryo cell cultures. This may represent a new method for genetic studies of SLE virus population. In a mosquito pool specimen NR(+) and NR(-) particles were demonstrated. By intracerebral passage in mice, there is a selection of NR(+) particles. Similar effects were not shown for eastern, western, and California encephalitis viruses. Plaques formed by the latter virus, however, were significantly reduced in number by neutral red.     

St. Louis encephalitis in southern Ontario: laboratory studies for arboviruses.

The first reported outbreak of St. Louis encephalitis in Canada occurred in the summer of 1975 in southern Ontario -- in the Windsor-Sarnia-Chatham area, the Niagara region and the city of Toronto. Hemmagglutination inhibition and complement fixation testing of serum samples collected during the outbreak confirmed that St. Louis encephalitis virus was the etiologic agent. Furthermore, this virus was isolated from brain tissue of a patient who died. This outbreak was probably an extension of the outbreak that occurred in the United States that summer. It was the first outbreak of arbovirus encephalitis in the province of Ontario.     

Serologic evidence of West Nile virus infection in black bears (Ursus americanus) from New Jersey

Serum samples obtained from 51 free-ranging black bears (Ursus americanus) in northwestern New Jersey in February and March 2002 were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis virus. Three (6%) of the black bears tested positive for WNV-neutralizing antibodies. One additional sample was positive for flavivirus-neutralizing antibodies but could not be differentiated for a specific virus type. This is the first report of WNV infection in black bears.     

Unexpected altered specificity is responsible for St. Louis encephalitis virus recombinant protease autoproteolysis

We report herein the study of the cleavage fragments generated by autoproteolysis of the St. Louis encephalitis virus recombinant protease. The cleavage sites leading to truncated forms were identified by microsequencing, which revealed an unexpected altered specificity of the recombinant proteinase towards unusual sequences.     

A focus assay method for Japanese encephalitis virus using complement and anti-virus serum

A sensitive, quantitative, short-time, and reproducible focus assay for Japanese encephalitis (JE) virus is described. After 2 or 3 days of incubation, the infected cells were treated with anti-JE virus serum and complement, and subsequently stained with trypan blue; then clear foci were produced. This method made it easy to titrate the infectivities not only of all seven JE virus strains tested but also of West Nile (WN), Murray Valley encephalitis (MVE), and St. Louis encephalitis (SLE) viruses using hyperimmune anti-JE virus serum for the latter. Moreover, even cell lines which hardly formed plaques by the agar overlay method easily produced foci within 2 or 3 days by this method.     

The 1952 Outbreak of Encephalitis in California (A Symposium): EPIDEMIOLOGIC ASPECTS

For the most part, epidemiologic phenomena observed in the outbreak of encephalitis in 1952 accorded with patterns that had been apparent in previous years. Ninety-seven per cent of the 414 laboratory-confirmed cases of western equine and St. Louis encephalitis in humans occurred in the 20 Central Valley counties. The cases of western equine encephalomyelitis in horses were generally scattered over the state. In the Central Valley most of the cases in horses were in animals less than two years of age; elsewhere the incidence was higher in older horses.There were no laboratory-confirmed cases of western equine or St. Louis encephalitis in humans earlier than June or later than October.In 1952 there were far more cases of western equine than of St. Louis encephalitis—a departure from the pattern in the previous seven years when there were about as many of one as of the other. No known satisfactory index is available for the prediction of the extent or type of outbreaks in humans.Approximately one-third of the cases of western equine encephalitis were in patients less than one year of age, whereas there were no cases of the St. Louis disease in patients that young.The incidence of western equine encephalitis in persons under 5 years of age was about the same for girls as for boys. In higher age brackets, males with western equine encephalitis outnumbered females 2 to 1. The corresponding ratio for St. Louis encephalitis was only 1.2 to 1.     

The 1952 outbreak of encephalitis in California; epidemiologic aspects

For the most part, epidemiologic phenomena observed in the outbreak of encephalitis in 1952 accorded with patterns that had been apparent in previous years. Ninety-seven per cent of the 414 laboratory-confirmed cases of western equine and St. Louis encephalitis in humans occurred in the 20 Central Valley counties. The cases of western equine encephalomyelitis in horses were generally scattered over the state. In the Central Valley most of the cases in horses were in animals less than two years of age; elsewhere the incidence was higher in older horses.There were no laboratory-confirmed cases of western equine or St. Louis encephalitis in humans earlier than June or later than October. In 1952 there were far more cases of western equine than of St. Louis encephalitis-a departure from the pattern in the previous seven years when there were about as many of one as of the other. No known satisfactory index is available for the prediction of the extent or type of outbreaks in humans. Approximately one-third of the cases of western equine encephalitis were in patients less than one year of age, whereas there were no cases of the St. Louis disease in patients that young.The incidence of western equine encephalitis in persons under 5 years of age was about the same for girls as for boys. In higher age brackets, males with western equine encephalitis outnumbered females 2 to 1. The corresponding ratio for St. Louis encephalitis was only 1.2 to 1.     

Prevalence of selected zoonotic diseases in vertebrates from Haiti, 1972.

Vertebrate animals collected in Haiti in 1972 were tested for selected zoonotic diseases. No rabies virus or neutralizing (N) antibody was detected in bats (Artibeus jamaicensis). However, N antibody against St. Louis encephalitis, Western equine encephalitis (WEE), and Eastern equine encephalitis were detected in resident species of birds and WEE antibody in bats. No N antibody against Venezuelan equine encephalitis was found. The possible introduction by migratory birds and local transmission of these arboviruses is discussed.     

Exposure of red knots (Calidris canutus rufa) to select avian pathogens; Patagonia, Argentina

As part of the shorebird surveillance, Red Knots (Calidris canutus rufa) were sampled in two Patagonian sites in Argentina, Río Grande and San Antonio Oeste, during 2005-2006. Cloacal swabs and serum samples were collected from 156 birds and tested by virus isolation (Newcastle disease virus), polymerase chain reaction (PCR; avian influenza virus and Plasmodium/Hemoproteus), and for antibodies to St. Louis encephalitis virus. All test results were negative.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

A simple method for the inactivation of St. Louis encephalitis virus preparations for immunofluorescent microscopy.

A study was made of different treatments for the inactivation of St. Louis encephalitis virus in smears prepared for immunofluorescence microscopy. Treatment of infected cells with 0.3% betapropiolactone at 56 degrees C for 40 min resulted in an inactivated virus suitable for immunofluorescence studies.     

Role of peridomestic birds in the transmission of St. Louis encephalitis virus in southern California

In response to the 1984 St. Louis encephalitis (SLE) epidemic in the Los Angeles Basin of southern California (USA), an investigative program was initiated to evaluate the interactive components of the SLE virus transmission cycle. From 1987 through 1996 (10 yr), 52,589 birds were bled and their sera tested for SLE and western equine encephalomyelitis (WEE) virus antibodies by the hemagglutination inhibition (HAI) test. Eighty-three percent of the birds tested were house finches (Carpodacus mexicanus) (48.7%) and house sparrows (Passer domesticus) (34.6%); 1.1% of these birds were positive for SLE antibodies. Prevalence of WEE antibodies was negligible. The analysis of 5,481 sera from rock doves (Columbia livia) yielded 3.6% SLE positives and 0.4% WEE positives. Collection sites were maintained as study sites when identified as positive bird, mosquito, and SLE virus activity localities; others were abandoned. Serial serum samples from 7,749 banded house sparrows and 9,428 banded house finches from these selected sites demonstrated year-round SLE virus transmission. One location exhibited significant numbers of house finches undergoing annual SLE seroconversion and a number of seroconversion-reversion-reconversion sequences suggesting either viral reinfection from mosquitoes or recrudescence by latent virus. A proportion of both bird species also lived for longer than 1 yr, thus, increasing the possibility of virus carry-over from autumn to spring. Assessment of concurrently collected mosquitoes indicated no correlative association between mosquito populations and SLE seroconversion and reconversion. European house sparrows introduced in the 1800's may have provided a supplemental link to the existing SLE virus enzootic cycle involving endemic house finches. Meteorological factors are reviewed as possible important correlates of SLE epidemics. The house finch/house sparrow serosurveillance system is also evaluated for use as an "Early Warning" indicator of SLE virus activity.     

Molecular-based identification and phylogeny of genomic and proteomic sequences of mosquito-borne flavivirus

Mosquito-borne flaviviruses (MBFVs) are important cause of emerging and re-emerging human diseases nearly worldwide, transmitted by arthropod vectors (mostly aedes and culex mosquitoes), with particular reference to yellow fever virus, Japanese encephalitis virus, dengue fever virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, etc. In over 100 countries, more than 2.5 billion people are at risk of infection, and approximately 20 million infections are reported annually. Through the analysis of gene sequence data of these virus populations it is possible to infer phylogenetic relationships, which in turn can yield important epidemiological information, including their demographic history. Early attempts to define the evolutionary relationships and origins of viruses in the genus flavivirus are hampered by the lack of genetic information particularly amongst the MBFVs. In this study, complete genome, translated polyprotein, structural and non-structural proteins of MBFVs have been targeted and revealed an extensive series of clades defined by their epidemiology and disease associations. The branching patterns of at the deeper nodes of the resultant trees were different from those reported in the previous study. The significance of these observations is discussed.     

Seroconversion for West Nile and St. Louis encephalitis viruses among sentinel horses in Colombia

We prospectively sampled flavivirus-na?ve horses in northern Colombia to detect West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) seroconversion events, which would indicate the current circulation of these viruses. Overall, 331 (34.1%) of the 971 horses screened were positive for past infection with flaviviruses upon initial sampling in July 2006. During the 12-month study from July 2006-June 2007, 33 WNV seroconversions and 14 SLEV seroconversions were detected, most of which occurred in the department of Bolivar. The seroconversion rates of horses in Bolivar for the period of March-June 2007 reached 12.4% for WNV and 6.7% for SLEV. These results comprise the first serologic evidence of SLEV circulation in Colombia. None of the horses sampled developed symptoms of encephalitis within three years of initial sampling. Using seroconversions in sentinel horses, we demonstrated an active circulation of WNV and SLEV in northern Colombia, particularly in the department of Bolivar. The absence of WNV-attributed equine or human disease in Colombia and elsewhere in the Caribbean Basin remains a topic of debate and speculation.     

High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus.

A detailed restriction map of the genome of Rhodobacter capsulatus SB1003 was constructed recently by using an ordered set of overlapping cosmids. Pulsed-field gel electrophoresis-generated restriction patterns of the chromosomes of 14 other R. capsulatus strains were compared. Two of them, St. Louis and 2.3.1, were chosen for high-resolution alignment of their genomes with that of SB1003. A 1-Mb segment of the R. capsulatus SB1003 cosmid set was used as a source of ordered probes to group cosmids from the other strains. Selected cosmids were linked into one 800-kb contig and two smaller contigs of 100 kb each. EcoRV and BamHI restriction maps of the newly ordered cosmids were constructed by using lambda terminase. Long-range gene order in the new strains was mainly conserved for the regions studied. However, one large genome rearrangement inverted a 470-kb DNA fragment of the St. Louis strain between the rrnA and rrnB operons. A 50-kb deletion covering three SB1003 probes was found in strain 2.3.1 near rrnB. Conservation of about 50% of the positions of restriction sites in all these strains and nearly 80% for the pair 2.3.1- St. Louis made it possible to produce high-resolution alignment of the contiguous 800-kb genome segment. Ten deletions of 2 to 27 kb, one 30-kb inversion, and three translocations were found in this region. Strong clustering of the positions of polymorphic restriction sites was observed. For a 50-kb size interval, two patterns of the distribution of restriction sites were found, one with about 90% and the other with 5 to 30% conservation of sites. This structure may be explained by independent acquisition of these divergent regions from other Rhodobacter strains.     

West nile virus antibodies in bats from New Jersey and New York

Eighty-three serum samples were obtained from big brown (Eptesicus fuscus), little brown (Myotis lucifugus), and northern long-eared (Myotis septentriotalis) bats (Chiroptera: Vespertilionidae), from New Jersey and New York (USA) between July and October 2002. Samples were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis (SLE) virus. One little brown bat and one northern long-eared bat tested positive for WNV neutralizing antibodies. No bats had antibodies to SLE virus. This was the first large-scale investigation of WNV infection in bats in New Jersey. Additional work is needed to determine the effects of WNV on bat populations.     

The Wilhelmine E. Key 1999 Invitational lecture. Predicting the evolution of human influenza A

We studied the evolution of the HA1 domain of the H3 hemagglutinin gene from human influenza virus type A. The phylogeny of these genes showed a single dominant lineage persisting over time. We tested the hypothesis that the progenitors of this single evolutionarily successful lineage were viruses carrying mutations at codons at which prior mutations had helped the virus to avoid human immune surveillance. We found evidence that eighteen hemagglutinin codons appeared to have been under positive selection to change the amino acid they encoded in the past. Retrospective tests show that viral lineages undergoing the greatest number of mutations in the positively selected codons were the progenitors of future H3 lineages in nine of eleven recent influenza seasons. Codons under positive selection were associated with antibody combining sites A or B or the sialic acid receptor binding site. However, not all codons in these sites had predictive value. Monitoring new H3 isolates for additional changes in positively selected codons might help identify the most fit extant viral strains that arise during antigenic drift.