DISCUSSION

Cache Valley virus is a mosquito-borne arbovirus that is a member of the family Bunyaviridae, genus Bunyavirus, serogroup Bunyamwera. The virus is widely distributed throughout North America.⁷ Neutralizing antibodies to the virus have been detected in deer, sheep, horses, cattle, and in humans.^8–11 Cache Valley virus has been reported to cause disease in humans on at least two occasions. A case of encephalitis and multiorgan failure leading to death of a human was reported in 1997 to be attributed to CVV infection.¹² More recently, a case of acute meningitis in a patient was attributed to CVV.¹³ The CVV isolated from the encephalitis patient in 1997 was found by thin-section electron microscopic analysis of infected Vero cells to contain enveloped viral particles of 55 to 84 nm (average 70 nm) size in intracellular vacuoles of smooth cytoplasmic membranes.¹² In the case of the meningitis patient in 2006, evaluation of negative-stained concentrated cell culture supernatants revealed viral particle sizes ranging from 60–80 nm.^15,13 Although the 80–100 nm particle size observed for the year-2000 isolate in the present study was in the nominal range (80–120 nm) expected for a bunyavirus,⁷ the isolates from 2003 and 2004 displayed particle sizes in the mid-70 to mid-80 nm range, substantially smaller than expected but similar to those noted in the human cases.^12,13 This initially confounded the identification of the viral contaminant for these episodes, because it led us incorrectly in the direction of alphaviruses and togaviruses and appeared to rule out the bunyaviruses. The smaller viral particle sizes observed for CVV in many of these cases do not appear to be an artifact of the method used to size the particles (i.e., thin–section versus negative staining of viral pellets).

The contamination of biologics manufacturing processes with CVV has been attributed to the large volumes of non-gamma-irradiated fetal bovine serum (FBS) used as a cell growth medium component. Although the FBS is tested for viral contaminants before use, the lots of serum involved are large, and potential viral contaminants are likely to be nonhomogenously distributed among the individual bottles comprising each lot. Modeling of CVV growth in CHO cells under the conditions used in biologics production has suggested that very low levels of the CVV in the FBS used during cell expansion can cause the manufacturing failures observed.¹⁶ The rapid lytic infections resulting from introduction of CVV into CHO-cell processes typically manifest themselves through aberrations in process parameters that are monitored during the production runs. Therefore, the presence of the virus typically leads to premature termination of the manufacturing processes, and the investigatory testing that is required leads rapidly to identification of CVV as the root cause of the failed production runs.

Fortunately, the risk of introducing CVV to patients through contaminated biologics should be very low because of: 1) the rarity of the contamination events; 2) the facility with which the infections are detected during production through in process monitoring and in vitro virus screening; 3) the fact that known contaminated lots of product are discarded; and 4) the relatively high susceptibility of any undetected CVV to physical and chemical inactivation strategies used during product purification.

At the time when this article was written, Raymond W. Nims, PhD, was a scientific director, Sandra K. Dusing, PhD, was a scientific director, and Wang-Ting Hsieh, PhD, was a senior scientist, all at BioReliance, Rockville, MD. Archie Lovatt, PhD, was a scientific director, G. Gordon Reid, PhD, was a scientist, David Onions, PhD, FRSE, was the chief scientific officer, and Euan W. Milne was a senior scientist, all at BioReliance UK, Glasgow, Scotland, UK. Corresponding author Nims currently is at Amgen, Longmont, CO, 303.401.2354, rnims@amgen.com