Detection of an avian lineage influenza A(H7N2) virus in air and surface samples at a New York City feline quarantine facility

Background In December 2016, an outbreak of low pathogenicity avian influenza (LPAI) A(H7N2) occurred in cats at a New York City animal shelter and quickly spread to other shelters in New York and Pennsylvania. The A(H7N2) virus also spread to an attending veterinarian. In response, 500 cats were transferred from these shelters to a temporary quarantine facility for continued monitoring and treatment. Objectives The objective of this study was to assess the occupational risk of A(H7N2) exposure among emergency response workers at the feline quarantine facility. Methods Aerosol and surface samples were collected from inside and outside the isolation zones of the quarantine facility. Samples were screened for A(H7N2) by quantitative RT‐PCR and analyzed in embryonated chicken eggs for infectious virus. Results H7N2 virus was detected by RT‐PCR in 28 of 29 aerosol samples collected in the high‐risk isolation (hot) zone with 70.9% on particles with aerodynamic diameters >4 μm, 27.7% in 1‐4 μm, and 1.4% in <1 μm. Seventeen of 22 surface samples from the high‐risk isolation zone were also H7N2 positive with an average M1 copy number of 1.3 × 103. Passage of aerosol and surface samples in eggs confirmed that infectious virus was present throughout the high‐risk zones in the quarantine facility. Conclusions By measuring particle size, distribution, and infectivity, our study suggests that the A(H7N2) virus had the potential to spread by airborne transmission and/or direct contact with viral‐laden fomites. These results warranted continued A(H7N2) surveillance and transmission‐based precautions during the treatment and care of infected cats.


| INTRODUC TI ON
In December 2016, the Centers for Disease Control and Prevention

Detection of an avian lineage influenza A(H7N2) virus in air and surface samples at a New York City feline quarantine facility
care of cats at a quarantine facility operated by the American Society for the Prevention of Cruelty to Animals (ASPCA). 1,2,4 Genome analysis of the initial feline isolate (A/feline/New York/16-040082-1/2016) 5 and subsequent virological characterization of the human isolate (A/ New York/108/2016) 6 showed it to be phylogenetically related to the North American lineage low-pathogenic avian influenza (LPAI) A(H7N2) viruses circulating in live poultry markets during [1996][1997][1998][1999][2000][2001][2002][2003][2004][2005]. Recent analysis of the human A(H7N2) isolate showed it to transmit poorly in ferrets but, unlike previously identified isolates, displayed an increased ability to replicate in human airway cells. 7 While it is unknown how the index cat was exposed to the A(H7N2) virus, the high rate of transmission among cats and concern about a possible zoonotic transmission mandated quarantine precautions.
In an effort to better understand transmission of the 2016 in- Additionally, aliquots of some of these aerosol samples were first concentrated and then passaged in embryonated chicken eggs. Viral quantification was performed by qPCR and hemagglutination assay.
F I G U R E 1 Floor plan of NYC quarantine facility. (A) The majority of cats were housed on the first floor, while (B) some were housed on the second floor. Cats were housed in cages (1-7 cats/cage depending on the cage size). The cages were grouped into pods (6-8 cages/pod). Cats were examined in the treatment rooms (Tx), and the sickest ones were housed individually in 15 cages, some stacked 2 high, in the critical care area. The locations of NIOSH samplers (N01-N20) are shown. NIOSH samplers were in used in pairs with 1 sampler above the other. The locations of SKC BioSamplers (K01-13) also are shown. SKC BioSamplers were used 1 at a time. Donning of personal protective equipment (PPE) was carried out in the cold zone. When exiting the hot zone, workers removed their PPE in the warm zone 2 | E XPERIMENTAL PROCEDURE S

| Quarantine facility
Sampling was performed in a temporary feline quarantine facility in New York City (NYC), NY, USA, on January 5-6, 2017. Aerosol and surface samples were collected throughout the facility's highrisk containment area (hot zone), moderate-risk decontamination area (warm zone), and low-risk outside containment area (cold zone). A floor plan of the facility is shown in Figure 1A,B. At the time of sampling, the facility housed about 500 cats, about 386 of whom had tested positive for influenza A(H7N2). The quarantine facility was housed in a two-story warehouse and had recirculating unit air heaters. The thermostat controlled unit heaters were mounted at ceiling height and cycled on/off throughout the study period. Although the recirculating unit heaters delivered no outdoor or makeup air to the space, they generated significant discharge velocities when activated, thus adding to room air turbulence and its negative influence upon aerosol settling. Outdoor air was supplied through natural ventilation that infiltrated into the facility. high that housed single cats. Cats in pod A were single-housed in about 54 0.9 m × 1.5 m × 0.9 m tall cages in 3 rows stacked 2 high.
One row was close to the wall, and the other 2 rows were backto-back. Cats were examined in the treatment rooms (Tx), and the sickest ones were housed individually in 0.9 m × 1.5 m × 0.9 m tall cages stacked 2 high in the critical care area. The critical care area had 3 rows of cages. Cages in the middle and right row were stacked 2 high for a total of 6 cages in each row with an access passageway between the right and middle row. The left row was only 1 cage high for a total of 3 cages; access was from the walkway. The overall total was 15 cages in the critical care area, although not all had cats in them. Cages with cats were marked with clipboard containing paperwork hanging from the door of each cage. The locations of NIOSH samplers (N01-N20) are shown ( Figure 1A,B). NIOSH samplers were used in pairs with 1 sampler above the other. The locations of SKC BioSamplers (K01-13) also are shown ( Figure 1A). SKC BioSamplers were used 1 at a time.
During sampling, the interior temperature was 18°C and the relative humidity was 25%. All workers entered and exited the hot zone through the warm zone. Before entering the hot zone, workers donned disposable coveralls, N95 respirators, hair covers, shoe covers, eye protection, and 2 pairs of gloves. After leaving the hot zone, workers removed their personal protective equipment in the warm zone.

| Aerosol sample collection
Aerosol samples were collected using NIOSH BC 251 two-stage cyclone samplers (denoted as NXX) and SKC BioSamplers (denoted as KXX). Sampling locations are shown in Figure 1A,B.
Twenty aerosol samples (N01-08 on day 1, N09-20 on day 2) were collected using the NIOSH BC 251 sampler 8 at a 3.5 L/ min flow rate, except for 2 samples (N02 and N03), which were inadvertently collected at 3.8 and 3.9 L/min. The NIOSH sampler separates the particles into 3 size fractions (≥4 μm, 1 to 4 μm, and ≤1 μm) and conforms to the American Conference of Governmental Industrial Hygienists/International Organization for Standardization criteria for respirable particle sampling. 9 The samplers were mounted on tripods in pairs, with the odd-

| Surface sample collection
Thirty-one surface samples were collected on day 1 of sampling using sterile nylon-flocked swabs (Copan Diagnostics, Corona, CA, USA) moistened with VTM. Surface samples are denoted as SXX.
Samples were collected from areas with direct animal contact such as cages/crates, floors, and water bowls, and from common worker high-contact porous and non-porous surfaces such as door knobs, table tops, and elevator buttons. When possible, a 10 cm × 10 cm cardboard template was placed on flat surfaces and surface sampling was carried out by swiping a swab completely across the template in 3 directions (left to right, perpendicular, and diagonal). In some cases (sampling doorknobs, elevator buttons, water bowls), an area ~50-100 cm 2 was sampled. The sampling method had not been robustly evaluated or validated due to time constraints on obtaining samples. After collection, swabs were placed in 0.5 mL VTM and kept on ice until transport to the laboratory after 1-2 days. The final volume of allantoic fluid collected for each inoculated egg sample was ~ 6 mls.

| NIOSH two-stage cyclone samples
Viral RNA was extracted from NIOSH two-stage cyclone samplers using the MagMAX-96 Viral RNA Isolation Kit (ThermoFisher Scientific) as previously described. 8 Upon thawing and resuspension by vortexing 1 minute, samples were supplemented with a 1:1 volume of 2-propanol (Sigma) followed by the addition of 1 μL carrier RNA and 20 μL of prepared bead mix. Viral RNA was washed and processed according to the manufacturer's protocol.
Eluted viral RNA in total (26.4 μL) was immediately transcribed to cDNA. was immediately transcribed to cDNA.

| Allantoic fluids
Viral RNA was extracted from 1 mL of allantoic fluid derived from surface swab, SKC, and concentrated SKC samples that were passaged in embryonated chicken eggs. The MagMAX-96 Viral RNA isolation Kit protocol was modified comparably to the methods used in isolating viral RNA from SKC aerosol samples. The isolated viral RNA was immediately transcribed to cDNA.

| cDNA transcription
Isolated viral RNA was transcribed to complimentary DNA using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) as described by the manufacturer's protocol. Final cDNA volume on all aerosol, surface swab, and allantoic samples was 40 μL.

| pDNA standards
To quantify Matrix (M1) gene copies during the qPCR analysis of samples, a plasmid DNA standard was used as described previously. 13 For H7 HA-specific quantification, the complete DNA sequence

| Quantitative qPCR
Quantitative PCR of the Matrix M1 gene was performed as previously described. 13

| DNA sequence analysis
For the verification of A/feline/NY/2016, selected qPCR-positive aerosol and surface swab samples were purified according to protocol using the QIAquick PCR Purification kit (Qiagen) and submitted to Genewiz for Sanger DNA sequencing services. Sequence analysis was performed on both the 5′ and 3′ end of submitted DNA using the above mentioned M1 and A(H7N2) HA oligonucleotides.

| Hemagglutination assay and stock viruses
As a measure of viral replication, hemagglutination assays were performed on the collected allantoic fluid as described by Current Protocols in Microbiology 15G.1.8. Chicken red blood cells (RBCs) were obtained from Lampire Biological Laboratories (Everett, PA, USA) and prepared to a final concentration of 0.5%. Briefly, 50 μL of sample allantoic fluid was serially diluted in twofold increments using 1X PBS (ThermoFisher Scientific) followed by the addition of 50 μL 0.5% chicken RBCs. Plates were then incubated for at least 30 minutes at room temperature before agglutination was assessed.

| Aerosol sampling in the quarantine facility
The cat quarantine facility was a two-floor warehouse with the majority of cats housed in the hot zone on the first floor ( Figure 1A) and the remainder in the smaller second floor hot zone ( Figure 1B (Table 2).

| Surface sampling in the quarantine facility
PCR analysis of surface samples from quarantine hot zones showed an average M1 copy number of 885/~100 cm 2 , comparable to M1 copies found in the >4μm and 1-to 4μm particle  Table S4). Surface samples taken from cold zone areas showed no detectable M1 copies. To determine whether infectious virus was also present in surface swab samples from the hot zones, aliquots of swab samples S64, S73, S75, S79, and S89 were passaged in 10-day-old embryonated chicken eggs and A(H7N2) titers were measured after 48 hours.
Infectious virus was found in all 5 swab samples, and overall titers increased 26-651-fold (Table 3). Aliquots of the allantoic fluid from the first passage of virus-derived swab samples S75 and S89 were passaged again in eggs and titers increased 42-46-fold (Table 3).

| D ISCUSS I ON AND CON CLUS I ON
The potential for the A(H7N2) virus crossing the species barrier aptly raises concern. Though the LPAI A(H7N2) subtype is primarily found TA B L E 1 M1 gene copies in egg-propagated SKC samples-1st and 2nd passages    Prompt risk assessment of any pathogenic microorganism is key to preventing occupational exposure, controlling disease transmission, and maintaining public health.

ACK N OWLED G EM ENTS
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention (CDC). The authors would like to thank the ASPCA for allowing us access to their facility and for their cheerful cooperation and assistance during our study.