The Attribution of Human Seasonal Influenza H3N2 Virus Detection to the Collector, Not Avian Sources, During the 2022 Highly Pathogenic Avian Influenza Outbreak in Pennsylvania, USA—Implications for Biosafety and Biosecurity

Simple Summary

In summary, this report emphasizes the importance of accurate virus characterization using whole-genome sequencing and robust biosecurity practices and training for avian influenza surveillance. Risks must be mitigated to provide accurate disease reporting and limit disease spread, considering this case’s avian influenza detection in submitted poultry samples but its attribution to the collector.

Abstract

Highly pathogenic avian influenza (HPAI) surveillance for influenza A virus (IAV) in the United States is conducted using a National Animal Health Laboratory Network (NAHLN) real-time reverse transcriptase–polymerase chain reaction (rRT-PCR). Samples showing the presence of IAV are confirmed and characterized at the national reference laboratory. During the H5N1 HPAI outbreak in 2022, our laboratory reported the detection of IAV in a PA commercial chicken flock using rRT-PCR targeting the matrix gene, which was negative for the H5/H7 subtypes. IAV was not detected by additional sampling of the birds the following day with rRT-PCR. The virus detected was characterized as a human seasonal H3N2 with whole-genome sequencing (WGS). Further investigation revealed that the collector who visited the farm was diagnosed with an IAV infection. This case report emphasizes the importance of farm biosafety and biosecurity, of conducting regular reviews of worker safety protocols, and of advanced molecular techniques like WGS for viral characterization and epidemiology.

1. Introduction

Highly pathogenic avian influenza (HPAI) is a contagious viral disease impacting domestic and wild birds. While they primarily impact avian species, HPAI viruses have been sporadically detected in mammalian species, including humans [1,2]. Infections in humans have mostly been linked to HPAI strains from the goose/Guangdong lineage of H5 viruses, but infections with other subtypes have been reported [1,3,4]. Additionally, low pathogenic avian influenza (LPAI) viruses have been implicated in zoonotic infections [2,5]. Human infections with avian influenza viruses typically occur through direct contact with infected birds or through contact with contaminated surfaces [6]. Influenza A viruses, including HPAI strains, continue to evolve by mutation and reassortment in wild waterfowl hosts and sometimes in domestic bird populations when allowed to circulate. The H5N1 HPAI outbreak, which started with the appearance of this virus, in late 2021 in the United States has led to outbreaks across all four flyways, causing severe clinical signs and high mortality in domestic birds and subsequently in some wild species [7].

During an HPAI outbreak that began in 2022, IAV surveillance testing in poultry was conducted within HPAI Control Areas for animal and product movement, following the HPAI Response Plan Red Book/Foreign Animal Disease Response and Preparedness Plan (Highly Pathogenic Avian Influenza Emergency Response (Available on: aphis.usda.gov/sites/default/files/hpai_response_plan.pdf, accessed on 12 December 2024). Real-time reverse transcriptase–polymerase chain reaction (rRT-PCR) is the preferred method for the early detection of IAV during outbreaks. For gallinaceous domestic poultry like chickens, oropharyngeal swabs constitute the major sample type for IAV screening by rRT-PCR [6,7,8,9]. While sampling protocols are standardized, biosecurity practices for sample collection can vary among farm collectors [6]. When dealing with HPAI viruses such as the current H5N1 outbreak, ensuring collector biosafety is crucial. This includes wearing appropriate personal protective equipment and considering worker immunization for seasonal influenza virus. These measures are essential for protecting workers from infection and preventing the spread of the virus between humans and birds. Maintaining robust biosecurity practices further restricts viral transmission in both agricultural and natural environments [5].

2. Case Presentation

In this report, we present a case in which a sample collector involved in avian influenza virus surveillance inadvertently contaminated samples collected from chickens. The sample collector obtained three pooled samples, each comprising 11-swab oropharyngeal swabs from a flock of approximately 20,000 chickens housed in a poultry house to meet interstate bird movement requirements.

Per NAHLN protocols, the samples were screened by an rRT-PCR assay that targeted a conserved region of the IAV matrix gene [10]. Further avian influenza subtyping was performed using H5 subtyping assays, including the NAHLN H5 rRT-PCR assay, which targeted H5 viruses from North America, Eurasia, and Mexico [9]. Samples with non-negative results for IAV were sent to the National Veterinary Services Laboratories (NVSL, Animal and Plant Health Inspection Service, USDA in Ames, IA, USA) for confirmation and further characterization.

Confirmation testing at NVSL consistently showed positive results with a reproducible cycle threshold (lowest Ct values—26.4) in all three samples. Samples positive for IAV were sequenced directly from samples as previously described [11]. Whole-genome sequencing identified the virus to be a human seasonal H3N2 virus belonging to clade 3C.2a1b.2a.2b (GSAID reference-epi_isl_19186485). This particular clade was predominant in 2019, as indicated by NextClade analysis (Figure 1), although viruses from this clade have been circulating since 2018 and were sporadically reported in 2022. BLASTN analysis showed 100% pairwise identity between H3 and N2 sequences from humans collected in November and December of 2022 in the United States (

Table 1. BLASTN analysis of human H3N2 A/22-041776-001/Pennsylvania/2022 (H3N2) virus recovered from submitted poultry samples.

Segment	Subtype	% Pairwise Identity (BLAST)	Isolate with Highest Nucleotide Identity	Best Top Hit Accession
PB2	NA *	99.6	A/Tennessee/30/2022 (H3N2)	OQ234616.1
PB1	NA	99.7	A/Texas/USAFSAM-13648/2022 (H3N2)	OQ249496.1
PA	NA	99.1	A/Florida/97/2022 (H3N2)	OQ665535.1
HA	H3	100	A/Tennessee/30/2022 (H3N2)	OQ234619.1
NP	NA	99.87	A/Colorado/35/2022 (H3N2)	OQ232802.1
NA	N2	100	A/Texas/USAFSAM-13648/2022 (H3N2)	OQ249492.1
MP	NA	99.9	A/Colorado/35/2022 (H3N2)	OQ232804.1
NS	NA	100	A/Georgia/14/2022 (H3N2)	OQ245245.1

* NA = not available.

).

Further investigation into the collector revealed that they had been diagnosed with IAV infection by a rapid test at a clinic later on the same day that they visited the farm. The specific viral strain from the collector was not sequenced. In efforts to clarify the status of the farm, the same birds were resampled by a different collector and tested the following day, with negative results for IAV by rRT-PCR. The initial detection of an H3 subtype virus, followed by phylogenetic analysis identifying it as a human seasonal H3N2 influenza virus, alongside subsequent negative testing at the farm, strongly suggests that the collector inadvertently contaminated the bird swabs during collection. Although chickens are generally resistant to human seasonal influenza, this incident underscores the importance of sample collectors strictly adhering to good biosafety practices, alongside maintaining robust biosecurity measures [12,13].

This report shows the benefits and critical importance of IAV characterization and of conducting epidemiologic investigations. Misclassification of the farm’s influenza status could have potentially led to unnecessary quarantine measures for the flock. Therefore, accurate virus characterization through methods like phylogenetic analysis is crucial to build an appropriate response. Furthermore, this incident highlights the necessity for farms to review, monitor, harmonize, and enforce stringent biosecurity practices. Effective biosecurity measures are essential to prevent pathogen introduction and must be robust to minimize the risk of sample contamination during collection. Continuous training and awareness among all stakeholders involved in disease prevention are paramount. Recommendations such as immunizing workers against seasonal IAV and ensuring the use of personal protective equipment (PPE) can significantly reduce the risk of infection during fieldwork. Previous studies have generally considered human infections with avian influenza strains to pose a lower risk [4]. However, it is essential to remain vigilant, and both the Centers for Disease Control and Prevention (CDC) and the USDA recommend self-monitoring for illness among responders and sample collectors for ten days following their involvement in highly pathogenic avian influenza (HPAI) events (https://www.cdc.gov/bird-flu/caring/infected-birds-exposure.html?CDC_AAref_Val=https://www.cdc.gov/flu/avianflu/h5/infected-birds-exposure.htm, accessed on: 12 December 2024) despite the current strains being associated with lower risks, although some human infections have been reported recently.