Human infection with avian influenza A(H7N2) virus—Virginia, 2002

Background In March 2002, an outbreak of low‐pathogenic avian influenza (LPAI) A(H7N2) was detected among commercial poultry operations in Virginia. Methods We performed a serosurvey of 80 government workers involved in efforts to control the outbreak. Results One study participant who assisted with disposal of infected birds tested positive for neutralizing antibodies to influenza A(H7N2) by microneutralization assay and H7‐specific IgM antibodies by enzyme‐linked immunosorbent assay (ELISA). The acute infection was temporally associated with an influenza‐like illness that resolved without hospitalization. Conclusion This study documents the earliest evidence of human infection with an H7 influenza virus of the North American lineage.


| INTRODUC TI ON
breaks. [2][3][4] No human infections with LPAI H7N2 viruses had been documented prior to the 2002 Virginia outbreak. We describe a serosurvey conducted among persons involved in control efforts that resulted in identification of the first human LPAI H7N2 infection.

| ME THODS
We performed a serosurvey among eligible workers (≥18 years of age who had participated in control efforts of the LPAI H7N2 poultry outbreak in Virginia for at least 14 days by May 17, 2002) and who gave informed consent to enroll in the study. Outbreak-associated and previous exposure to wild birds and poultry, the use of personal protective equipment during control efforts, and recent ocular and respiratory symptoms were assessed. A blood sample was collected from each participant for serologic testing. A second blood sample was collected from participants whose first sample tested positive for antibody to influenza A(H7N2

Human infection with avian influenza A(H7N2) virus-Virginia, 2002
microneutralization (MN) assays for the presence of neutralizing antibodies to Tky/VA virus using a starting dilution of 1:20 as described previously. 5 Work with live Tky/VA virus was conducted in a biosafety level 3 laboratory with enhancements required by the US Department of Agriculture. Serum samples with duplicate titers of ≥80 were tested further by immunoblot and an enzyme-linked immunosorbent assay (ELISA) detecting IgM also described previously, 5 but using hemagglutinin protein derived from whole purified Tky/VA virus by treatment with bromelain. 6 Additional testing was performed using adsorbed serum samples to rule out the possibility of antibody cross-reactivity between H7 and the A(H3N2) subtype.
A contemporary human A(H3N2) virus was chosen for the adsorption protocol because the individual exhibited substantial titers to A(H3N2) virus (data not shown). In addition, H3 and H7 HA are both phylogenetic group 2 HA subtypes, raising the concern that crossreactive antibody to the H3 HA could possibly interfere with the detection of authentic H7 subtype-specific antibodies. 7 Briefly, a 50 μL volume of serum was incubated with 100 μg of whole purified Tky/VA or purified A/Sydney/5/97 (H3N2) (Syd/97) viruses for 2.5 hours at 4°C followed by ultracentrifugation at 100 000 g for 30 minutes to remove virus-antibody complexes. Viral protein was measured by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). To remove residual virus, sera were then adsorbed for 30 minutes with a one-tenth volume of packed turkey red blood cells, followed by centrifugation at 21 000 g for 2 minutes at 4°C.

| RE SULTS
Of the 80 persons completing testing, 63 (79%) were exposed to poultry or their contaminated environment; 17 (21%) had office duties only. Exposure to potentially infected birds or virus-contaminated environments occurred through outbreak control activities including collection of swabs from dead birds and poultry facilities, disposal of dead birds, incineration of birds, disinfection of equipment, and laboratory testing of poultry specimens. Many study participants (41%) reported >1 influenza-like illness (ILI) symptoms and 18% reported ocular symptoms during their deployment with VAITF. The use of personal protective equipment varied by activity (Table 1).
Serum from 1 study participant tested positive for antibody to Tky/VA virus, achieving titers of 1:80 by MN in several independent assays ( Table 2); sera from the remaining 79 participants tested seronegative. The specificity of the neutralizing antibody from the seropositive individual was determined by adsorption of sera with purified whole H7N2 and H3N2 viruses. Adsorption with H7N2, but not H3N2, removed the neutralizing antibody from the sera ( Table 2). This result was confirmed by immunoblot analysis (data not shown), fulfilling WHO criteria for the serological identification of humans infected with avian influenza subtypes. 8 This person (age mid-20s) reported that he worked disposing of infected birds during the response and used gloves always and dust mask most of the time.
Prior to outbreak, he had hunted waterfowl, but had limited exposure to domestic poultry. He reported an illness with onset 10 days prior to study enrollment with feverishness, cough, sore throat, and headache. He sought medical care at a private facility 1 day after illness onset. Chest X-ray showed a right middle lobe infiltrate, and a diagnosis of lower respiratory tract infection was given. No diagnostic specimens were collected for microbiologic testing. Outpatient treatment consisted of unspecified intramuscular and oral antibiotics. He recovered fully.

| D ISCUSS I ON
While this investigation was carried out in 2002, this report provides evidence of risk of human infection with a North American lineage LP H7N2 virus among persons exposed during control efforts.
Exposure to and infection with an H7 influenza virus before participation in outbreak control efforts cannot be definitively excluded as no blood sample was available from the worker before engaging in  Titers represent the reciprocal of the highest dilution of serum exhibiting 50% neutralization of 100 tissue culture infectious doses of virus. Untreated serum Sample 1 had a neutralizing antibody titer of 1:80 in 4 independent MN assays and was positive for antibody to purified Tk/VA/02 H7 HA by Western blot. c Serum samples were diluted serially fourfold from a starting dilution of 1:100 in the first well. Titers represent the reciprocal of the highest dilution of the test serum achieving a value greater than the mean plus 3 standard deviations of 3-6 negative controls at equivalent dilution.
TA B L E 2 Serological response to H7N2 virus in antibody-positive study participant involved in outbreak control operations a