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Volume 1, Number 4—October 1995

Dispatch

Are North American Bunyamwera Serogroup Viruses
Etiologic Agents of Human Congential Defects of the
Central Nervous System?

Charles H. Calisher* and John L. Sever†
Author affiliations: *Colorado State University, Fort Collins, Colorado, USA; †George Washington University Medical Center, Children's National Medical Center, Washington, D.C., USA

Main Article

Table 1

Antibody to Cache Valley, Tensaw, La Crosse, Jamestown Canyon, Lokern, or Buttonwillow viruses in mothers of microcephalic or macrocephalic infants and matched controls

Antibody to virus Infant's condition X ²>(p)
Cache Valley Microcephaly 3.840 (0.05)
  Macrocephaly 4.797 (<0.05)
  Either 0.915 (>0.20)
Tensaw Microcephaly 4.891 (<0.05)
  Macrocephaly 4.071 (<0.05)
  Either 0.329 (>0.20)
Cache Valley or Tensaw Microcephaly 5.983 (<0.02)
  Macrocephaly 4.806 (<0.05)
La Crosse Microcephaly 0.211 (>0.20)
  Macrocephaly 1.481 (>0.20)
Jamestown Canyon Microcephaly 0.709 (>0.20)
  Macrocephaly 0.037 (>0.20)
Lokern Microcephaly 1.010 (>0.20)
  Macrocephaly 2.041 (>0.10)
Buttonwillow Microcephaly 2.041 (>0.10)
Macrocephaly <0.001(>0.20)

Bunyaviruses used for all tests were prototypes: (Bunyamwera serogroup) CV (strain 6V-633), TEN (A9-171b), Lokern (FMS-4332), Main Drain (BFS-5015), (California serogroup) La Crosse (Original), Jamestown Canyon (61V-2235), (Simbu serogroup), Buttonwillow (A-7956), and Mermet (AV-782). Samples were tested for antibody by serum dilution-plaque reduction neutralization (20). Briefly, diluted and heat- inactivated °C/30 min) serum was added to an equal volume of virus containing approximately 200 plaque-forming units (PFU), such that the final virus dilution was 100 PFU. Fresh human serum at a final concentration of 8% was added to all virus suspensions. Serum-virus mixtures were incubated at 4ºC for 18 h, and 0.1-ml aliquots were dropped in the center of Vero cultures grown in 6-well plastic plates. After a 45-min period of adsorption, cells were overlaid with medium containing 2% agar and further incubated for 70 to 75 h at 37º C in 5% CO2-95% air. A second overlay, containing medium, 2% agar, and 1:25,000 neutral red was then added, and the plates again were incubated until plaques were readable, usually 12 to 36 h later. A serum specimen was considered positive when it reduced the number of plaques 90% relative to control titrations, which ranged from 80 to 150 plaques. Samples that were positive in a screening (1:10) test were titrated for end-point, and the serum titer was taken as the highest twofold dilution of serum that reduced the number of plaques 90%.
Although a human positive control for detecting IgM antibody by capture enzyme-linked immunosorbent assays was not available, serum specimens were tested by a modification of a published technique for detecting IgM antibody to California serogroup viruses (21). Mouse or sheep serum samples containing IgM antibody to CV virus or mouse serum with IgM antibody to TEN virus served as positive and negative IgM antibody controls. Statistical analyses were done by chi-square or McNemar's chi-square.

Main Article

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