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Media Statement For Immediate Release Wednesday, December 18, 2024, Contact: CDC Media Relations (404) 639-3286           CDC Confirms First Severe Case of H5N1 Bird Flu in the United States December 18, 2024-- A patient has been hospitalized with a severe case of avian influenza A(H5N1) virus (“H5N1 bird flu”) infection in Louisiana. This marks the first instance of severe illness linked to the virus in the United States. The case was confirmed by the Centers for Disease Control and Prevention (CDC) on Friday, December 13. Since April 2024, there have been a total of 61 reported human cases of H5 bird flu reported in the United States. Partial viral genome data of the H5N1 avian influenza virus that infected the patient in Louisiana indicates that the virus belongs to the D1.1 genotype related to other D1.1 viruses recently detected in wild birds and poultry in the United States and in recent human cases in British Columbia, Canada, and Washington state. This H5N1 bird flu genotype is different than the B3.13 genotype detected in dairy cows, sporadic human cases in multiple states, and some poultry outbreaks in the United States. Additional genomic sequencing and efforts to isolate virus from clinical specimens from the patient in Louisiana are underway at CDC. While an investigation into the source of the infection in Louisiana is ongoing, it has been determined that the patient had exposure to sick and dead birds in backyard flocks. This is the first case of H5N1 bird flu in the U.S. that has been linked to exposure to a backyard flock. A sporadic case of severe H5N1 bird flu illness in a person is not unexpected; avian influenza A(H5N1) virus infection has previously been associated with severe human illness in other countries during 2024 and prior years, including illness resulting in death. No person-to-person spread of H5 bird flu has been detected. This case does not change CDC’s overall assessment of the immediate risk to the public’s health from H5N1 bird flu, which remains low. This case underscores that, in addition to affected commercial poultry and dairy operations, wild birds and backyard flocks also can be a source of exposure. People with work or recreational exposures to infected animals are at higher risk of infection and should follow CDC’s recommended precautions when around animals that are infected or potentially infected with H5N1 avian influenza virus. This means that backyard flock owners, hunters and other bird enthusiasts should also take precautions. The best way to prevent H5 bird flu is to avoid exposure whenever possible. Infected birds shed avian influenza A viruses in their saliva, mucous, and feces. Other infected animals may shed avian influenza A viruses in respiratory secretions and other bodily fluids (e.g., in unpasteurized cow milk or ‘raw milk’). As a general precaution, whenever possible, people should avoid contact with sick or dead animals, in particular wild birds, and poultry. For individuals with direct/close contact with wild birds or sick or dead poultry or other animals, wearrecommended personal protective equipment (PPE). Wild birds can be infected with avian influenza A viruses even if they don't look sick. Do not touch surfaces or materials (e.g., animal litter or bedding material) contaminated with saliva, mucous, or animal feces from wild or domestic birds or other animals with confirmed or suspected avian influenza A virus infection. For more information on H5 bird flu in the U.S. and CDC’s response, including regularly updated case counts, visit theH5 Bird Flu: Current Situation page.

December 18, 2024-- A patient has been hospitalized with a severe case of avian influenza A(H5N1) virus (“H5N1 bird flu”) infection in Louisiana. This marks the first instance of severe illness linked to the virus in the United States. The case was confirmed by the Centers for Disease Control and Prevention (CDC) on Friday, December 13. Since April 2024, there have been a total of 61 reported human cases of H5 bird flu reported in the United States. Partial viral genome data of the H5N1 avian influenza virus that infected the patient in Louisiana indicates that the virus belongs to the D1.1 genotype related to other D1.1 viruses recently detected in wild birds and poultry in the United States and in recent human cases in British Columbia, Canada, and Washington state. https://www.cdc.gov/media/releases/2024/m1218-h5n1-flu.html

Two cats suspected of contracting H5 bird flu die after drinking recalled raw milk LA County Public health urges people to avoid consuming raw milk or feeding it to pets By Benjamin Gamson • Published 14 seconds ago Getty Images Raw milk from Raw Farm is displayed for sale at a grocery store on November 29, 2024 in Torrance, California. Certain lots of the Fresno-based Raw Farm cream top, whole raw milk have been recalled by California public health and agriculture authorities amid ongoing concerns about possible H5N1 bird flu infections amont the farm’s cattle. (Photo by Patrick T. Fallon / AFP) (Photo by PATRICK T. FALLON/AFP via Getty Images) Two cats have died after being suspected of contracting H5 bird flu after drinking raw milk that had been recalled, the Los Angeles County Department of Public Health announced Thursday. “The risk of H5 bird flu remains low in Los Angeles County, but this suspected case of the virus in a pet cat that consumed raw milk is a reminder that consuming raw dairy products can lead to severe illness in cats," said Dr. Barbara Ferrer, Director of the Los Angeles County Department of Public Health. Both cats died after drinking the raw milk from Raw Farm, LLC that had been recalled on Dec. 4. People who were in contact with the cats are currently being monitored but no cases of bird flu in humans have been reported yet. “To avoid the spread of disease, including H5 bird flu, we strongly encourage residents and their pets to avoid raw dairy and undercooked meat products, limit contact with sick or dead animals, report sick or dead birds and keep pets or poultry away from wild animals and birds,” said Ferrer. https://www.nbclosangeles.com/news/local/two-suspected-of-contracting-h5-bird-flu-die-after-drinking-recalled-raw-milk/3581842/

Cats die after drinking recalled raw milk In a statement, Los Angeles County health officials said the two cats developed symptoms after consuming milk from Raw Farms. Their symptoms included appetite loss, fever, and neurologic signs. Both died after their symptoms worsened. Testing revealed influenza A, which is rare in cats. Officials said the cases are considered suspected H5 avian flu cases and that confirmation tests will be done. Officials noted that, in earlier US dairy farm outbreaks, cats were known to be infected after drinking raw milk from infected cows. People exposed to the sick cats are under monitoring and have been offered antiviral prophylaxis (prevention). When raw milk from Raw Farms was recalled following positive tests from product on retail shelves, officials urged people to avoid drinking raw milk or giving it to their pets. Barbara Ferrer, PhD, MPH, MEd, LA County public health director, said, the cases are a reminder that the virus can cause severe disease in cats. "To avoid the spread of disease, including H5 bird flu, we strongly encourage residents and their pets to avoid raw dairy and undercooked meat products, limit contact with sick or dead animals, report sick or dead birds and keep pets or poultry away from wild animals and birds." https://www.cidrap.umn.edu/avian-influenza-bird-flu/avian-flu-suspected-cats-drank-raw-milk-virus-kills-animals-arizona-zoo

Could Cats Become a Carrier of Bird Flu? A new study highlights the need for public health officials to ramp up bird flu surveillance in our feline companions. Share full article Scientists worry that if a cat were simultaneously infected with H5N1 and a seasonal flu virus, the H5N1 virus could acquire mutations to spread efficiently among people.Credit...Arin Yoon for The New York Times By Emily Anthes and Apoorva Mandavilli Dec. 11, 2024 Domestic cats could provide an unexpected new route for the bird flu virus H5N1 to evolve into a more dangerous form, according to a new study published on Monday. In the year since the virus began circulating in dairy cattle, it has killed many cats, primarily on farms with affected herds. It has also sickened at least 60 people, most of whom had close contact with infected dairy cows or poultry. The new study highlights the need for public health officials to ramp up bird flu surveillance in cats, which tend to have frequent contact with both wild animals and people, said Dr. Suresh Kuchipudi, a veterinary microbiologist at the University of Pittsburgh and an author of the paper. For months, the testing of cows and people for H5N1 has been limited, leaving experts in the dark about the true scale of the dairy outbreak. Last week, the U.S. Department of Agriculture announced that it would begin testing the national milk supply to help identify infected herds. But officials have not addressed the need to expand monitoring of other farm animals, let alone household pets. The U.S.D.A. is charged with monitoring livestock and the Centers for Disease Control and Prevention takes the lead on disease surveillance in people, but no government agency is responsible for tracking companion animals. “In the process of addressing the immediate problem — which is dairy farms and the milk as a food safety problem, and then human surveillance — we might be missing a much bigger, evolving story,” Dr. Kuchipudi said. “It may already have been happening in plain sight.” He and his colleagues investigated the death of 10 cats at a home in South Dakota this spring. The cats lived outdoors but were considered pets. They displayed respiratory and neurological symptoms before they died. Virus isolated from the cats closely resembled a version seen in cattle on a dairy farm 50 miles away. The dead cats were discovered with bird feathers nearby, suggesting that they became infected by eating wild birds that had carried the virus off the farm. Bird flu viruses naturally latch on to a type of receptor found in birds, and seasonal flu viruses are more suited to a human-type receptor. Scientists typically worry most about pigs — which carry both types of receptors — as the ideal “mixing vessels” in which two flu viruses might swap genes. But many other animals may host bird flu and seasonal flu viruses at the same time, said Richard Webby, an influenza expert at St. Jude Children’s Research Hospital, who was not involved in the work. Those species may vary in how likely they are to encounter both viruses and to pass pathogens on to people. “Cats seem to be a pretty good candidate,” Dr. Webby said. The new study found that cats carry both types of receptors in their brain, lungs and gastrointestinal system, meaning they can host both viruses. As the flu season picks up over the coming weeks, so do the odds of cats becoming simultaneously infected with H5N1 and a seasonal flu virus. So far, H5N1 does not spread easily among people, although studies have suggested that just one or two key mutations could allow the virus to make that leap. There is no evidence that cats have spread H5N1 to people and they may not represent a major avenue for the evolution of bird flu, experts said. Still, if a cat were simultaneously infected with H5N1 and a seasonal flu virus, the H5N1 virus could potentially acquire the mutations it needed to spread efficiently among people. https://www.nytimes.com/2024/12/11/health/bird-flu-h5n1-cats.html

Research Article Marked Neurotropism and Potential Adaptation of H5N1 Clade 2.3.4.4.b Virus in Naturally Infected Domestic Cats Shubhada K. Chothe , Surabhi Srinivas , Sougat Misra , Noel Chandan Nallipogu , Elizabeth Gilbride , Lindsey LaBella , show all Article: 2440498 | Accepted author version posted online: 09 Dec 2024 Cite this article https://doi.org/10.1080/22221751.2024.2440498 Abstract: In April 2024, ten cats died in a rural South Dakota (SD) residence, showing respiratory and neurological symptoms. Necropsy and laboratory testing of two cats confirmed H5N1 clade 2.3.4.4b infection. The viral genome sequences are closely related to recent SD cattle H5N1 sequences. Cat H5N1 genomes had unique mutations, including T143A in haemagglutinin, known to affect infectivity and immune evasion, and two novel mutations in PA protein (F314L, L342Q) that may affect polymerase activity and virulence, suggesting potential virus adaptation. Dead cats showed systemic infection with lesions and viral antigens in multiple organs. Higher viral RNA and antigen in the brain indicated pronounced neurotropism. Lectin-histochemistry revealed widespread co-expression of sialic acid α-2,6 and α-2,3 receptors, suggesting cats could serve as mixing vessels for reassortment of avian and mammalian influenza viruses. No differences in clade 2.2 or 2.3.4.4b H5 pseudoviruses binding to cat lung/brain tissues indicated the neurotropism is unlikely mediated by receptor binding affinity. Keywords: Influenza A viruses A(H5N1) clade 2.3.4.4b neurotropism influenza A virus evolution avian influenza cat Disclaimer As a service to authors and researchers we are providing this version of an accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proofs will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to these versions also. Introduction Since the first discovery of the highly pathogenic avian influenza virus (HPAIV) of H5N1 subtype from a goose in Guangdong, China, in 1996, the virus has diversified into ten clades (Clades 0-9) and subclades [1]. In 2020, the H5N8 strain belonging to the 2.3.4.4b clade caused infections among domestic and wild birds across Europe, Africa, and Asia, leading to new reassortant strains like H5N1 clade 2.3.4.4b [2]. Since the first detection in the Netherlands in October 2020, H5N1 clade 2.3.4.4b virus outbreaks have been reported in multiple European countries, Africa, Asia, and America [3]. The geographic spread and the range of species affected by the clade 2.3.4.4b H5N1 viruses far exceed those of previous H5N1 clades. H5N1 clade 2.3.4.4b infections have been reported in over 90 species of wild and domestic birds and more than 21 mammalian species, including cattle, foxes, skunk, sea lions, mink, dolphins, raccoon dogs, cats, and seals [4-10], including several human infections [11]. Most mammalian infections likely occurred through ingestion or direct contact with infected birds, exacerbated by widespread infection and mortality of avian species [12]. The unprecedented risk of H5N1 clade 2.3.4.4b is highlighted by the massive deaths among sea lions between January and October 2023 across Peru, Chile, Argentina, Uruguay, and Brazil [6]. Animals infected with clade 2.3.4.4b H5N1 viruses commonly exhibited pneumonia and meningoencephalitis, with neurological signs predominating in several animal species. Notably, dolphins [8], skunks [13], minks [14], red foxes [15], and sea lions [6] showed significant neurological signs such as tremors, convulsions, and ataxia, with viral presence mainly in the brain. Though neurotropism and neurological signs were observed during the outbreaks of previous clades of H5N1viruses, the pronounced neurotropism of the current H5N1 clade 2.3.4.4b is highlighted by high viral loads in the brain and minimal or no viral presence in the lungs of several species [8, 16], suggesting a significant shift in virus behavior. There have been 21 documented cases of H5N1 clade 2.3.4.4b infection in cats in the USA since March 2024 [17]. The susceptibility of cats to avian influenza H5N1 viruses became known in 2004. The first documented case of natural H5N1 infection in cats was in 2004 in Thailand, where fourteen cats died in a household [18]. At the same time, severe pneumonia in tigers and leopards fed on infected poultry carcasses in Thailand extended the susceptible host range of H5N1 viruses [19]. Experimental infection of cats with the H5N1 isolate from a fatal human case in Vietnam (clade 2.2 - A/Vietnam/1194/04) revealed that while the virus caused lower respiratory tract disease, it resulted in the death of only one out of three infected cats. Notably, there was no evidence of neurological deficits [20]. A subsequent report from Germany on the natural infection of domestic Shorthair cats with HPAIV H5N1 A/swan/Germany/R65/06 showed a higher viral load in the lungs compared to the brain, with 3.5 logs more virus in the lungs, associated with broncho-interstitial pneumonia [21]. Earlier H5N1 clades in cats led to either subclinical infections [22] or clinical disease characterized by pneumonia and encephalitis [23], and the currently circulating reassortant clade 2.3.4.4b H5N1 virus has also been heavily linked to respiratory and neurological signs in cats in France [24], Poland [25], South Korea [26], and the USA [4]. In this study, we report marked neurotropism of clade 2.3.4.4b H5N1 viruses in cats with an in-depth investigation of the histopathological lesions and viral detection in multiple organs. A key determinant of the host susceptibility and tissue tropism of influenza viruses is the ability of the virus hemagglutinin (HA) to bind to host sialic acid (SA) receptors [27]. While human and other mammalian influenza viruses show preferential binding to SA receptors with α-2,6Gal linkage, avian influenza viruses, including H5N1, show preferential tropism to SA receptors with α-2,3Gal linkage [28]. The distribution of these receptors varies among species; for example, human upper respiratory tract epithelial cells mainly contain SA α-2,6Gal receptors [29], whereas SA α-2,3Gal is the predominant receptor in the trachea of duck and all species of Passeriformes [30, 31]. A shift in the binding affinity of the H5N1 virus's HA from SA α-2,3 to α-2,6-receptors is crucial for it to gain the ability of human-to-human transmission [32]. Several amino acid changes (N182 K, Q222L/G224S, or the combination N182 K/Q222L/G224S) in the H5 protein have been identified that enhance binding to SA α-2,6-receptors [32]. A recent study reported that clade 2.3.4.4b viruses recovered from mesocarnivores in Canada retained amino acid residues in their HA protein that may enable binding to SA α-2,6-receptors. Notably, the substitutions S137A and T160A were shown in vitro to moderately enhance influenza A virus (IAV) binding to SA α-2,6 receptors [33]. The neurotropism of clade 2.3.4.4b H5N1 viruses in cats could be due to differences in receptor distribution patterns. We conducted an in-depth evaluation of SA receptor distribution across multiple organs in cats and found widespread co-expression of avian and human influenza receptors across multiple cat tissues, with no notable differences between the lung and the brain. These observations supplement a previously published report that showed tissue distribution of human and avian-type sialic acid influenza virus receptors in domestic cats [34]. Further, we found widespread binding of pseudoviruses expressing H1 and H5 from clade 2.2 and 2.3.4.4b to cat tissues. Further, no notable differences in the binding patterns of clade 2.2 and clade 2.3.4.4b to cat lung and brain tissues suggest that neurotropism of clade 2.3.4.4b viruses is unlikely mediated by receptor binding affinity. Materials and methods Animal Tissues Two dead cats, aged six months and one and a half years, were necropsied at the North Dakota Veterinary Diagnostic Laboratory (NDSU VDL) in the month of April 2024. Clinical signs in these cats include reduced appetite, lethargy, and neurological impairments, which lead to mortality. A comprehensive set of tissues including lung, kidney, heart, stomach, ileum, jejunum, duodenum, colon, pancreas, adrenal gland, cerebrum, cerebellum, hippocampus, brainstem, skeletal muscle, thyroid/parathyroid, lymph node, liver, and spleen were fixed in 10% neutral buffered formalin. The fixed tissues underwent routine histological processing and sectioning for hematoxylin and eosin (H&E) staining and immunohistochemical analysis. Lung and brain tissues were collected for nucleic acid extraction and PCR. Formalin-fixed, paraffin-embedded healthy cat tissue sections were acquired from Zyagen in San Diego, California, USA. These included lung (FP-601), cerebral cortex (FP-210), stomach (FP-302), ileum (FP-309), jejunum (FP-308), and duodenum (FP-307). Quantitative RT-PCR and next-generation sequencing The RNA extraction and IAV matrix/H5 PCR were conducted at the NDSU VDL following the NVSL-approved protocols. The RNA samples were subjected to next-generation sequencing at USDA Animal Plant Health Inspection Service-National Veterinary Services Laboratories. The paired-end whole genome sequencing was performed using the NextSeq 2000 Illumina instrument. The sequence reads were submitted to SRA, NCBI database (SRR29040272, SRR29040273), and the corresponding complete gene segments assembled using Geneious Prime version 2024.0.4 were submitted to GISAID (EPI_ISL_19196362 and EPI_ISL_19196363). Phylogenetic analysis H5N1 HA sequences derived from the two cats in this study were analyzed with a total of 1443 HA sequences belonging to the H5N1 clade 2.3.4.4b that were downloaded from GISAID on June 7, 2024. These sequences originated from humans (global), chicken (North America), duck, goose, and all mammals from North and South America, including the recently reported sequences originating from infected dairy cattle. Duplicate sequences with identical nucleotide composition were removed from the analyses, yielding a total of 745 sequences available for the construction of a phylogenetic tree. Sequences were aligned using the following parameters: gap open cost = 10 and gap extension cost = 1.0 with accurate alignment mode (CLC Genomics Workbench, version 24). The coding sequence of HA from all the sequences was extracted from the alignment and subjected to nucleotide substitution testing employing the following models: Jukes-Cantor [35], Felsenstein81 [36], Kimura80 [37], Hasegawa-Kishino-Yano (HKY) [38], and General Time Reversible (GTR) (also known as the REV model) [39]. The outlined nucleotide substitution models were tested using the Hierarchical likelihood ratio test (hLRT), Bayesian information criterion (BIC), Minimum theoretical information criterion (AIC, Akaike information criterion), and minimum corrected theoretical information criterion (AICc, AIC with a correction for the sample size) to identify the best model for phylogenetic tree construction. The phylogenetic tree for the HA gene was constructed using the neighbor-joining method employing the GTR nucleotide substitution model, which yielded the best model. Rate variation was included in the maximum likelihood phylogeny analysis with the number of substitution rate categories set to 4, and the gamma distribution parameter was set to 1.0. Bootstrap analysis was performed with 100 replicates that afforded computationally feasible phylogenetic tree construction. Structure prediction and visualization The monomeric and multimeric structures of the feline influenza virus proteins were predicted with AlphaFold version 2.3.2, using databases downloaded on March 24, 2024. For both monomers and multimers, AlphaFold was run using the full genetic databases (–db_preset = full_dbs), with relaxation enabled on the best-scoring model (–models_to_relax = best) and templating disabled (–max_template_date = 1900-01-01). AlphaFold-predicted structures were visualized in PyMOL 2.5.4. The ‘super’ command was used to superposition comparable structures from PDB. Lectin histochemistry Lectin histochemistry was carried out on paraffin-embedded tissue sections as previously described [31]. Briefly, sections of 5 µm thickness were first deparaffinized in xylene and rehydrated through a series of ethanol treatments. The rehydrated sections underwent a 5-minute pre-soak in Tris-buffered saline (TBS), followed by antigen retrieval using eBioscience™ IHC Antigen Retrieval Solution - Low pH (10X) (ThermoFisher Scientific, catalog# 00-4955-58), according to the manufacturer's instructions. Sections were then blocked using goat serum (Thermo Fisher Scientific, catalog# PCN5000) diluted 1:40 with TBS and further blocked using a Streptavidin/Biotin Blocking Kit (Vector Laboratories, SP2002), following the manufacturer's guidelines. The sections were incubated with Sambucus Nigra Lectin (SNA), Fluorescein-labeled (Vector Laboratories, FL13012), Maackia Amurensis Lectin II (MAA II), Unconjugated (Vector Laboratories, L-1260-2), and Maackia Amurensis Lectin I (MAA I), Fluorescein (Vector Laboratories, FL13112). SNA binds specifically to sialic acid linked to galactose via an alpha-2,6 linkage (SA α-2,6-Gal), while MAL II and MAL I bind to sialic acid via an alpha-2,3 linkage (SA α-2,3-Gal), while MAL I has a preference for distinct glycan structures compared to MAL II. Each lectin was used at a concentration of 10 μg/ml and incubated overnight at 4°C. After three TBS washes, sections were treated with Streptavidin, Alexa Fluor 594 Conjugate (Thermo Fisher Scientific, catalog# S32356) for 2 hours at 4°C to stain the lectins. Following three washes with TBS, sections were mounted using ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, catalog# P36931). After curing for 24 hours at room temperature, the sections were imaged with an Echo Revolve Fluorescent Microscope. Negative control staining, performed by omitting the lectins, showed minimal background (Suppl. Fig. 5). Influenza pseudovirus production The 3rd generation lentiviral plasmids obtained from BEI resources (catalog # NR-53816) were used to produce H5 and H1 pseudotyped viruses. The influenza A H5N1 HA sequences of clade 2.2 and clade 2.3.4.4b were retrieved from NCBI, and expression constructs were designed using SnapGene software. The constructs were synthesized by GenScript, USA, and cloned into a pHCMV vector under the control of a CMV promoter. H1 expression plasmid. pHCMV_FLUAV_HA was a gift from Feng Zhang (Addgene plasmid # 207273; http://n2t.net/addgene:207273; RRID: Addgene_207273)[40]. Lentiviral helper plasmids, reporter plasmids, and plasmids encoding H5 or H1 were co-transfected into HEK 293 T cells using Fugene6 transfection reagent (Promega, USA, catalog # E2691). Pseudoviruses were collected 48 hours post-transfection, and titers were determined by infecting the HEK-293 T cells and measuring the luciferase expression using BrightGlo reagent (Promega, USA, catalog # E2650). The relative luminescence units (RLU) were measured using BioTek Cytation 7 Cell Imaging Multimode Reader. The pseudoviruses with infectivity titer between 104 and 105 RLU were used for the virus binding assay. Virus binding assay A virus binding assay was conducted using Influenza A pseudoviruses (H5N1 HA, clade 2.2; H5N1 HA, clade 2.3.4.4b; and H1). The deparaffinized and antigen-retrieved tissue sections were incubated with 250μl of Influenza A pseudoviruses at 37°C for 2 hours. The RLU of H5 clade 2.3.4.4b, H5 clade 2.2, and H1 pseudoviruses used in the virus binding assay were 109591, 88124, and 39879, respectively. Further, the sections were blocked with inactivated goat serum (Thermo Fisher Scientific, catalog # PCN5000) at a 1:40 dilution overnight. The sections treated with H5N1 were immunostained using a primary mouse monoclonal anti-Influenza Virus H5 Hemagglutinin (HA) Protein (VN04-8), A/Vietnam/1203/2004 (H5N1) from BEI resources (NR-2733), while those treated with H1 were stained with anti-Influenza A H1N1 hemagglutinin antibody [C102 (IV.C102)] (Abcam, catalog # ab128412), both at a 1:100 dilution for 90 minutes. Post-primary antibody incubation, the sections were washed and incubated with a secondary goat anti-mouse IgG H&L (Alexa Fluor® 647) antibody (Abcam, catalog # ab150115) at a 1:800 dilution for 35 minutes. After three TBS washes, the sections were mounted in ProLong Gold antifade mountant with DAPI. Following a 24-hour curing period at room temperature, the sections were imaged using an Echo Revolve Fluorescent Microscope. Negative control staining, performed by omitting the virus and primary antibody, showed minimal background (Suppl. Fig. 5). Control experiments were conducted by substituting the influenza pseudovirus with a SARS-CoV-2 pseudovirus, demonstrating no cross-reactivity in the staining (data not shown). Immunohistochemistry for the detection of Influenza A virus nucleoprotein Tissue sections were processed as mentioned in the lectin histochemistry methodology. Following antigen retrieval, the sections were blocked with goat serum (Thermo Fisher Scientific, catalog # PCN5000) diluted 1:40 in TBS. Next, the sections were incubated with primary anti-Influenza A virus nucleoprotein antibody, Isotype: IgG2a (1:100) (Abcam, catalog # ab20343) for 90 minutes, followed by secondary goat anti-mouse IgG H&L (Alexa Fluor® 647) antibody (Abcam, catalog # ab150115) at a 1:800 dilution for 35 minutes. After three TBS washes, the sections were mounted in ProLong Gold antifade mountant with DAPI. After a 24-hour curing period at room temperature, the sections were imaged using an Echo Revolve Fluorescent Microscope. Negative control staining, performed by omitting the primary antibody, showed minimal background (Suppl. Fig. 5). Isotype control staining was performed using Mouse IgG2a kappa Isotype Control (eBM2a), eBioscience (Thermo Fisher Catalog # 14-4724-82), which showed no cross-reaction and minimal background staining (data not shown). Fluorescence Intensity Quantification The LHC and IHC images were subjected to quantification of the fluorescence using the software ImageJ (v 1.53). For each tissue section, fluorescence intensity was measured at five distinct sites of identical size within the image, and the mean value of these measurements was calculated to represent the mean fluorescence intensity. For intestinal tissues, the region of interest was focused on the mucosal lining, as this area is likely to come into contact with the virus upon ingestion. This approach ensured consistency in quantification across all tissue types analyzed. The mean fluorescence intensity values were statistically analyzed using one-way ANOVA, followed by post hoc Tukey's test to determine significant differences between groups. Results Outbreak of H5N1 clade 2.3.4.4b virus in outdoor cats in rural South Dakota In a rural residential area in South Dakota (SD), ten outdoor-housed cats aged 6 months to 4 years were found deceased. Most of these cats were domesticated and regarded as family pets. Clinical signs included anorexia, lethargy, and potential neurologic deficits. Two deceased cats, aged six and eighteen months, were submitted to the North Dakota State University Veterinary Diagnostic Laboratory (NDSU VDL) for postmortem examinations. Along with positive Streptococcus canis respiratory cultures, Polymerase Chain Reaction (PCR) results revealed the presence of influenza A virus (IAV) in the brain and lungs. The Cycle threshold (Ct) values for the IAV matrix gene in the brain were 20.1 and 18.1, and in the lungs were 35.93 and 31.93 for cats 1 and 2, respectively, indicating much higher viral load in the brains of both cats. Samples were submitted to the National Veterinary Services Laboratory (NVSL), Ames, Iowa, and further confirmed to be H5N1 clade 2.3.4.4b by PCR and whole genome sequencing. Unique mutations in H5N1 clade 2.3.4.4b sequences from fatal cat cases in SD A phylogenetic tree of the HA gene from the two cat H5N1 clade 2.3.4.4b sequences was constructed alongside 1443 H5N1 clade 2.3.4.4b HA sequences from humans worldwide, chicken (North America), duck, goose, and all mammals from North and South America. All statistical tests indicated that the inclusion of rate and topology variation in the GTR model yielded the best-fit nucleotide substitution model (Suppl. Table 2-4). The resulting maximum likelihood phylogeny tree showed that the HA gene from the South Dakota HPAIV H5N1 clade 2.3.4.4b cat sequences were closest to the H5N1 clade 2.3.4.4b sequences from dairy cattle samples originated from South Dakota and Kansas (Fig. 1, annotated highlighted area). Figure 1. Maximum likelihood-based haemagglutinin (HA) phylogenetic tree originating from 1443 sequences of HPAIV H5N1, clade 2.3.4.4b. The highlighted sub-branch of the unrooted tree contains the HPAIV H5N1 clade 2.3.4.4b sequences from two cats from South Dakota. Display full size Further, eight major IAV protein sequences of the cat H5N1 were analyzed alongside 178 dairy cattle sequences and 13 cat sequences obtained from the GISAID database[41]. Sequence alignment was performed using CLC Genomics Workbench, v24. The consensus sequence from the alignment was used as a benchmark to detect the occurrence of mutations in the eight proteins. Two unique mutations were found in the HA (T143A) and neuraminidase (NA) (N71S) proteins. One of the cats H5N1 sequences (GISAID ID # EPI_ISL_19196362) had a mutation in the polymerase acidic (PA) protein (F314L), whereas L342Q mutation was detected in the sequence originating from the other cat (GISAID # EPI_ISL_19196363). There was no evidence of mutations in other proteins. Structural and functional implications of HA, NA, and PA mutations in the cat H5N1 sequences HA Mutation: The top-ranked AlphaFold model of the trimer had very good agreement with PDB structure 4MHI (RMSD = 1.4  Å). Most of the variance manifested as differentially pitched or bent alpha helices in the stalk of HA; the receptor-binding domain was predicted to adopt an essentially identical conformation to the solved structure (Fig. 2A). Amino acid 143 was positioned very close to the SA binding site (Fig. 2B-C). Figure 2. Exploration of mutational consequences on protein structure. (A) Side-view cartoon representation of the AlphaFold-predicted structure for HA trimer (green) superposed with a solved structure (PDB code: 4MHI; blue). (B) Surface representation of the predicted HA trimer structure, with subunits colored in green, cyan, and magenta. The sialic acid binding pocket is circled and shaded grey near the top of the receptor-binding domain, and the nearby T143A mutation is in yellow with an arrow pointing to it. (C) The same but rotated 90 degrees to view from above the receptor-binding domain; all three sialic acid binding pockets and T143A positions are indicated. (D) Top-down view cartoon representation of the AlphaFold-predicted structure for NA tetramer (green) superposed with a solved structure (PDB code: 5HUG; blue). (E) The surface representation of the predicted NA tetramer structure, with subunits colored green, cyan, magenta, and yellow. The sialic acid binding pocket is circled and shaded grey. (F) The same but rotated 90 degrees to view from the side; the location of the N71S mutation is indicated in blue. (G) Side-view surface representation of a viral polymerase complex previously solved by X-ray crystallography (8H69); the PA, PB1, and PB2 subunits are shaded green, cyan, and magenta, respectively. The locations of the mutations we observed in PA are shaded yellow. (H) The same but rotated 90 degrees to highlight the distance of observed mutations from the catalytic core of the polymerase complex. Display full size NA Mutation: The top-ranked AlphaFold model of the tetramer showed near-perfect agreement with PDB structure 5HUG (RMSD = 0.5  Å) (Fig. 2D). The N71S mutation occurred in the stalk between the catalytic head and the transmembrane helices (Fig. 2E-F), far from the sialic acid binding sites and in a region not modeled in the X-ray diffraction data. PA Subunit Mutations: The two recovered feline influenza viruses had a single substitution in the PA subunit of the viral polymerase. One had an F314L substitution, and the other had an L342Q substitution, both located on the outside of the polymerase complex, far from the catalytic core (Fig. 2G-H). Severe neuropathology with extensive viral nucleoprotein in the brains of cats infected with H5N1 clade 2.3.4.4b We performed a detailed histopathological investigation of the tissues, including lung, kidney, heart, stomach, ileum, jejunum, duodenum, colon, pancreas, adrenal gland, cerebrum, cerebellum, hippocampus, brainstem, skeletal muscle, thyroid/parathyroid, lymph node, liver, and spleen. Supplementary Table 1 details the histopathological lesions found in various organs of cats. Briefly, microscopic evaluation of lung sections revealed mixed mononuclear and neutrophilic inflammatory cells in alveoli with fibrin deposition and edema, alveolar epithelial necrosis, alveolar hyaline membranes, bronchiolitis, bronchiolar epithelial hyperplasia, perivasculitis, and perivascular edema. Bronchitis was observed in one of the cats. Within the brain was mononuclear perivascular cuffing in the cerebellum, brain stem, and cerebrum with random foci of necrosis and suppurative inflammation, gliosis, neuronal necrosis, edema, and perivasculitis. Mild mononuclear perivascular infiltrates in the submucosa and tunica muscularis of the small and large intestines were observed in both cats. Aggregates of mixed mononuclear cells and neutrophils and necrosis in the ventricular sub-endomyocardium, as well as random foci of hepatitis, were found in one of the cats. The pancreas (exocrine and ductular regions) and thyroid gland were multifocally expanded by mild to moderate mononuclear infiltrates. The spleen showed marginal hyperemia and follicular lymphoid depletion. Increased numbers of tingible-body macrophages were seen in the cortex and medullary sinus of the lymph node. Figure 3 (A-H) shows the H&E-stained images of the histologic lesions observed in the cats. Figure 3. Histopathological and immunohistochemical analysis of HPAIV H5N1 infected cat tissue sections. H&E-stained tissue sections of HPAIV H5N1 infected cat showed various histologic lesions. (A) Lung exhibited 1) interstitial pneumonia, 2) bronchiolitis, 3) bronchitis. (B) Cerebrum had (4) meningitis and 5) encephalitis whereas (D) hippocampus showed no lesions. The immunohistochemical (IHC) analysis (I-L) revealed the presence of IAV nucleoprotein in each of these organs. Brain tissues showed a higher nucleoprotein staining level than the lung tissue, with the cerebellum and hippocampus exhibiting extensive presence of nucleoprotein. Tissues were primarily stained using an anti-influenza A virus nucleoprotein antibody, followed by a secondary goat anti-mouse IgG H&L (Alexa Fluor® 647) antibody (red) and DAPI nuclear stain (blue). The H&E-stained images were acquired by Epredia Pannoramic MIDI II using Z stacking with extended focus, and the IHC images were acquired by ECHO revolve microscope. Display full size We further conducted immunohistochemistry on the tissues, targeting the IAV nucleoprotein, which revealed intense staining in a higher proportion of cells in various parts of the brain and intestine. Noticeably, the cerebellum and the hippocampus showed an abundance of nucleoprotein compared to the lungs (Fig. 3, I-L), indicating a higher viral load in brain tissues, as confirmed by the PCR assay. Virus nucleoprotein was also found in other organs of the cat, particularly in the jejunum and colon (Suppl. Fig. 1). Abundant co-expression of SA α-2,3-Gal and SA α-2,6-Gal receptors in cat brain and lung Significant co-expression of avian (SA α-2,3-Gal) and human (SA α-2,6-Gal) type influenza -receptors was observed in both healthy (Fig. 4) and infected cat tissues (Suppl. Fig. 2, 3, and 4). SNA staining indicated a widespread presence of α-2,6-linked sialic acids, while MAL I and MAL II lectins provided detailed insights into sialylation patterns: MAL I detected simpler sialylated structures with α-2,3-linked sialic acids [SA α-2,3-Gal-β (1–4) N-acetylglucosamine] and MAL II highlighted complex sialylated structures with the same α-2,3 linkage [SA α-2,3-Gal-β (1–3) N-acetylgalactosamine (GalNAc)]. The abundant co-expression of SA α-2,3-Gal and SA α-2,6-Gal was particularly notable in healthy cats’ cerebral cortex (Fig. 4, E-H, and 1b). Lung tissue from healthy cats also exhibited a high abundance of both avian and human-type influenza receptors, especially in the alveolar epithelium, alveolar duct, and visceral pleura of the lung (Fig. 4, A-D) with no significant quantitative difference within the receptors (Fig. 4, 1a). In the brain tissues of infected cats, including the cerebellum, cerebrum, brainstem, and hippocampus (Suppl. Fig. 2), co-expression of SA α-2,3-Gal and SA α-2,6-Gal receptors was also observed. Figure 4. Co-expression of SA α-2,3-Gal and SA α-2,6-Gal receptors in cat tissues. The composite fluorescent images reveal co-expression of SA α-2,3-Gal (red) and SA α-2,6-Gal (green) influenza receptors in the lung, cerebral cortex, stomach, ileum, jejunum, and duodenum of the cat. The lung and cerebral cortex exhibit co-expression of these receptors, whereas the gastrointestinal tissues display a higher expression of SA α-2,6-Gal (green) receptors, particularly on the luminal mucosal lining of the ileum, jejunum, and duodenum. Tissue sections were stained with FITC-labeled SNA (SA α-2,6-Gal) lectin, FITC-labeled MAA I (SA α-2,3-Gal), biotinylated MAA II (SA α-2,3-Gal), and DAPI nuclear stain (blue). Panels 1a–1f present the quantified mean fluorescence intensity for each corresponding tissue. Bars represent the mean ± standard deviation (SD) for each group (***p < 0.001, ****p < 0.0001). Mock-treated images are available in the supplementary data. Scale bar = 170 µm. Display full size Predominant expression of SA α-2,6-Gal receptors in cat intestines The stomach, ileum, jejunum, and duodenum of healthy cat tissue showed co-expression of SA α-2,3-Gal and SA α-2,6-Gal receptors (Fig. 4, I-X and 1c-1f). Notably, the ileum, jejunum, and duodenum exhibited a predominance of SA α-2,6-Gal receptors on the mucosal lining of the villi (Fig. 4, 1d-1f). These receptors were also present in the goblet cells, lamina propria, muscularis, and intestine serosa. In contrast, the avian-type receptors (SA α-2,3-Gal) were concentrated along the basal region of the villi, the underlying lamina propria, crypts, submucosal regions, and muscularis mucosae. Comparative binding of pseudoviruses with clades 2.2 H5 and clade 2.3.4.4b H5, and H1 to cat brain and lung tissues Virus–receptor binding assays were performed on healthy cat lungs, cerebral cortex, and gastrointestinal tissues (stomach, ileum, jejunum, and duodenum) using pseudoviruses expressing H1 or H5 from H5N1 clade 2.2 or H5N1 clade 2.3.4.4b generated in our laboratory. The cat SA α-2,3-Gal and SA α-2,6-Gal receptors were found to be compatible with binding to the pseudoviruses, and the virus binding pattern correlated with the relative abundance of the receptors, with greater receptor expression, leading to greater virus binding. The H1 pseudovirus exhibited abundant binding to various tissues, reflecting the high abundance of SA α-2,6-Gal receptors in cats. Both the H5 pseudoviruses (clade 2.2 and clade 2.3.4.4b) exhibited binding to multiple tissues; binding was less extensive than the H1 pseudovirus (Fig. 5). No significant difference in binding was observed between pseudoviruses carrying clade 2.2 H5 and clade 2.3.4.4b H5 (Fig. 5, 1a-1f). The H1 and H5 pseudovirus bound extensively to the mucosal lining of the intestines, indicating the potential for enteric pathogenesis upon oral infection. Figure 5. Widespread binding of pseudoviruses carrying H5 of clade 2.2 or clade 2.3.4.4b and human influenza H1 to cat tissues. The fluorescent images illustrate the extensive binding of H5N1 clade 2.2, 2.3.4.4b and human H1 pseudoviruses to cat tissues, including the (A,B,C) lung; (D,E,F) cerebral cortex; (G,H,I) stomach; (J,K,L) ileum; (M,N,O) jejunum; and (P,Q,R) duodenum. This binding pattern correlates with the relative abundance of SA α-2,3-Gal and SA α-2,6-Gal receptors in these tissues. The mucosal endothelial cells lining the alveoli and the luminal mucosal cells of the intestine exhibit a strong preference for virus binding. The tissue sections were primarily stained with mouse monoclonal anti-influenza virus H5 hemagglutinin (HA) protein (VN04-8), A/Vietnam/1203/2004 (H5N1) or anti-influenza A H1N1 hemagglutinin antibody followed by secondary goat anti-mouse IgG H&L (Alexa Fluor® 647) antibody (red) and DAPI nuclear stain (blue). Panel 1A-1F shows the quantified mean fluorescence intensity for each virus binding. Bars represent the mean ± standard deviation (SD) for each group (*p < 0.05, **p < 0.01, ****p < 0.0001). Mock-treated images are provided in the supplementary data. Scale bar = 170 µm. Display full size Discussion Increasing evidence suggests recent shifts in the patterns of mammalian infections with the HPAI H5N1 viruses worldwide, indicating ongoing adaptation to infect mammalian hosts [42] In addition, the host range of the HPAIV H5N1 virus has been expanding, with clade 2.3.4.4b spillovers now detected in various mammalian species. These include both domestic and wild carnivores, such as domestic cats [43], red foxes [44], multiple bear species [45], and seals [10] among others. This growing list of susceptible mammalian hosts highlights the virus's ability to cross species barriers, raising concerns about its potential impact on wildlife and domestic animal populations. In this study, we report a natural H5N1 clade 2.3.4.4b virus infection resulting in the deaths of ten cats in rural South Dakota. The exact source of infection remains unclear; however, phylogenetic analysis of H5N1 sequences from two of the cats reveals a close genetic relationship to clade 2.3.4.4b strains previously detected in local cattle, suggesting a possible link. Additionally, the presence of bird feathers near the deceased cats indicates the likelihood that infection may have occurred through the consumption of virus-infected birds. However, because the disease typically requires several days to manifest post-ingestion, the exact timing of exposure is unclear. This evidence points toward a plausible cattle-to-bird-to-cat transmission pathway, supported by recent studies that identified H5N1 sequences across multiple species on affected farms, including dairy cows, wild birds, domestic cats, and raccoons [46]. Our study provides a significant new insight into the neurotropism of the H5N1 clade 2.3.4.4b virus in naturally infected domestic cats. There is a notable shift in the neurotropism of HPAI H5N1 viruses, particularly with the emergence of clade 2.3.4.4b in cats and wild carnivores like foxes. For example, in cats, experimental infection with the H5N1 Vietnam isolate (clade 2.2 - A/Vietnam/1194/04) showed primarily respiratory disease, with only one of three infected cats succumbing and no neurological symptoms[20]. Similarly, a natural H5N1 infection in Germany (A/swan/Germany/R65/06) in domestic cats displayed higher viral loads in the lungs than in the brain, with infection linked mainly to broncho-interstitial pneumonia [21]. Furthermore, studies on red foxes fed bird carcasses infected with clade 2.2 H5N1 also demonstrated limited clinical impact, with the foxes excreting the virus without developing severe disease. In contrast, recent H5N1 clade 2.3.4.4b infection in two cats from Texas [47] resulted in neurological signs and 50% mortality, likely due to ingestion of unpasteurized milk from infected cattle. Further, recent reports from Europe and the United States involving infection of red foxes with H5N1 clade 2.3.4.4b have shown a marked shift toward neurotropism [44]. These cases have documented viral adaptations that facilitate central nervous system involvement, with some infections exhibiting viral mutations indicative of enhanced neurotropism [46]. We identified several key mutations in the H5N1 sequence from infected cats that may suggest adaptation to cats. We observed a threonine-to-alanine mutation at residue 143 in HA (T143A). In A/Netherlands/219/2003 HA, this threonine (T143) forms a glycosylation motif (N-X-T/S) involving asparagine 141 (N141) around the receptor binding site (RBS), which is known to increase virus infectivity and resistance to neutralizing antibodies [48]. While the potential implications of the T143A mutation in clade 2.3.4.4.b are unclear, it could represent an adaptation mutation in cats that warrants further investigation. Notably, residue 143 in HA has been identified as a major mutation site in the RBS of H5 that contributes to viral escape from neutralizing antibodies among the different subclades, including 2.3.4.4b [49]. The N71S mutation in NA has not been previously reported in H5N1. While this mutation may not likely alter substrate specificity directly, it could potentially reduce efficiency on some substrates because phosphorylation or glycosylation of the serine residue might make the stalk of NA more rigid [50, 51]. The PA mutations (F314L and L342Q) are novel, and their functional implications are unknown. However, it is important to note that mutations in residues adjacent to these positions (343 and 347) in avian H5N1 influenza viruses have been shown to affect polymerase activity and virulence in mice [52]. Therefore, further investigation of these two PA mutations is critical to better understand their impact on the virus's polymerase activity and mammalian pathogenicity. Consistent with our findings, a 2023 study reported H5N1 clade 2.3.4.4b infection in cats in Poland with multi-organ lesions and higher viral load in the brain compared to the respiratory tract [53]. However, all the viruses from Poland had mutation PB2-E627 K, which is an important molecular marker of virus adaptation to mammals [53-55]. However, this mutation was absent in the H5N1 sequences from the cats in our study. The lack of PB2-E627 K in our cases suggests that alternative mechanisms may be driving the virus’s neurotropic behavior, indicating a potentially unique adaptation pathway in these cats that warrants further investigation. The co-expression of avian and mammalian SA receptors in cats identified in this study, combined with their potential exposure to various influenza viruses, poses a significant risk for genetic reassortment of different influenza strains, leading to the emergence of novel viral variants. A previous report examined the tissue distribution of human and avian-type sialic acid influenza virus receptors in cats, showing their expression in various organs [56]. Contrary to our findings, the earlier report indicated absence of alpha-2,3 sialic acid-linked receptors in the gastrointestinal tract and the absence of both alpha-2, 3 and alpha-2, 6 sialic acid-linked receptors in organs of the central nervous system, such as cerebrum and cerebellum. Further, unlike our study, the report did not investigate the binding patterns of MAA I (highly specific to SA α2,3 binding) binding receptors [31]. In the current study, we observed co-expression of SA α-2,3-Gal and SA α-2,6-Gal receptors in the lung, cerebral cortex, and gastrointestinal tissues. The luminal mucosal lining of the ileum, jejunum, and duodenum expressed high SA α-2,6-Gal (green) receptors, which is significant as this surface may come into contact with ingested viral particles. MAA I-specific SA receptors were also detected in the lungs, cerebral cortex, and lamina propria of gastrointestinal tissues. The limited use of lectins, variations in staining methodologies - such as immunochemical (previous study) vs. fluorescent staining (current study) - and differences in the number of animals investigated may have contributed to the discrepancies observed. Furthermore, other factors like sample processing conditions, individual variability among subjects, and differences in tissue collection and handling protocols could also play a role in the variations between our findings and those of previous studies. In many rural households, as was the case with the infected cats in rural South Dakota reported in this study, cats are often housed outdoors, used for pest control, and considered family pets. This unique role exposes them to diverse environments and interactions, including terrestrial, aquatic, wild birds, and other livestock animals and humans. This exposure puts cats at a higher risk of encountering a broad spectrum of avian and mammalian influenza viruses. Notably, a recent study found that stray cats in the Netherlands were frequently exposed to HPAI H5, with a seropositivity rate of 11.8% among clinically healthy individuals [57]. The presence of asymptomatic infections in cats with H5N1 is a significant threat as these cats could serve as silent carriers, transmitting the virus to humans without showing any clinical signs of illness. The continued exposure, viral circulation, and adaptation of the H5N1 virus in cats raise significant concerns for transmission and public health. Cats, common companion animals that frequently interact with humans and other species, could serve as a bridge for cross-species transmission of H5N1 viruses. Infected cats develop systemic infections and shed the virus through both respiratory and digestive tracts [58], potentially creating multiple routes of exposure to humans. Furthermore, the ability of the virus to persist and adapt in mammalian hosts heightens the risk of evolving into strains with increased transmissibility, posing an emerging zoonotic threat with profound public health implications. As H5N1 viruses continue to infect a wide range of avian and mammalian hosts, including an increasing number of human cases, there is an urgent need for coordinated One Health surveillance to monitor the spread of H5N1 among domestic and wild birds, animals, and humans. Supplemental material Marked Neurotropism and Potential Adaptation of H5N1 Clade 2.3.4.4.b Virus in Naturally Infected Domestic Cats

For Immediate Release: December 12, 2024 Suspected H5 Bird Flu Detected in Los Angeles County Cats That Consumed Recalled Raw Milk The Los Angeles County Department of Public Health is investigating two possible cases of H5 bird flu in cats that consumed recalled raw milk from Raw Farm, LLC. The infected indoor cats consumed raw milk linked to a recall of raw milk and cream products prior to the onset of symptoms, which included lack of appetite, fever and neurologic signs. The infected cats died after severe worsening of their illness, and subsequently tested positive for Influenza A, a rare result in cats. Public Health is considering these suspected H5 bird flu cases and is obtaining confirmatory testing. The nationwide H5 bird flu outbreak has seen other cats infected with the virus after consuming infected raw milk. People who had direct contact with the cats are monitoring for symptoms and have been offered antiviral prophylaxis. There have been no human cases of bird flu associated with exposure to these cats yet identified. The investigation is ongoing. Although human cases of bird flu are rare and the risk to residents remains low, this detection of H5 bird flu in cats who consumed raw milk underscores the importance of being proactive about preventing ongoing transmission of the virus. “The risk of H5 bird flu remains low in Los Angeles County, but this suspected case of the virus in a pet cat that consumed raw milk is a reminder that consuming raw dairy products can lead to severe illness in cats," said Dr. Barbara Ferrer, Ph.D., M.P.H., M.Ed., Director of the Los Angeles County Department of Public Health. “To avoid the spread of disease, including H5 bird flu, we strongly encourage residents and their pets to avoid raw dairy and undercooked meat products, limit contact with sick or dead animals, report sick or dead birds and keep pets or poultry away from wild animals and birds.” Cats may be exposed to H5 bird flu by consuming infected birds or other animals, being in environments contaminated with the virus, and consuming unpasteurized milk from infected cows. Cats infected with H5 bird flu may develop severe illness that can include fever and neurologic signs, and that can rapidly progress to death. Transmission of the H5 bird flu virus from mammal to mammal can occur. Cats have transmitted another influenza strain to humans, but there have been no known cases to date of H5 bird flu transmitted from cats to humans as part of this nationwide H5 bird flu outbreak. Raw milk, which is milk that has not been pasteurized, can carry harmful germs including influenza. These germs can present serious health risks to you, your family, and your pets. Anyone can become sick from drinking raw milk or consuming raw milk products. The people at the highest risk for severe illness include people who are pregnant, adults 65 years and older, children younger than 5 years, and people with weakened immune systems. Public Health continues to strongly encourage residents to avoid consuming raw milk and to not feed it to their pets; this includes frozen raw milk products since freezing does not eliminate harmful germs that can cause illness. Pasteurized milk remains safe to drink. Symptoms of H5 bird flu infection in humans include eye redness or discharge, cough, sore throat, runny or stuffy nose, diarrhea, vomiting, muscle or body aches, headaches, fatigue, trouble breathing and fever. Anyone who has consumed these specific recalled raw milk products and is experiencing symptoms should immediately contact their health care provider or local health department. Samples from birds, cats, and wild mammals in LA County continue to be tested for H5 bird flu at our Public Health Laboratory. In addition, the Public Health Laboratory routinely tests clinical specimens from humans for H5 bird flu as part of ongoing surveillance. Best Practices to Reduce Risk for You and Your Pets While the current risk of transmission of H5 bird flu to LA County residents and pets remains low, Public Health encourages these best practices: · Avoid Raw Dairy and Undercooked Meat Products: Do not drink raw milk or eat raw cheeses and undercooked meat products. Do not feed these to your pets. Raw milk, even from healthy cows, may be contaminated with harmful germs that can make you and your pets very sick. Freezing raw milk does not eliminate the harmful germs that may be in the product. · Limit Contact with Animals: Avoid unprotected contact with sick or dead animals or birds or any materials contaminated with bird feces. Avoid handling wild birds and observe them only from a distance. If you have to handle wild birds, even if they appear healthy, wear a well-fitting mask and gloves, and practice good hand hygiene, as some birds may carry the virus without showing symptoms. · Report sick or dead birds: Contact your local animal control agency if you see sick or dead birds. Symptoms can vary; infected birds or animals may be unable to fly, have seizures, have difficulty walking or be found dead. · Protect pets or poultry: Keep pets or poultry away from wild animals and birds. Ensure that wild birds cannot defecate into areas holding or housing pet birds or poultry. · Remove Bird Feeders and Baths: Take down bird feeders and communal bird baths to reduce the risk of the virus spreading from bird-to-bird. · Get a Seasonal Flu Vaccine: People should receive a seasonal flu vaccine. While this vaccine does not prevent avian influenza infection, it can reduce the risk of getting sick with human and bird flu viruses at the same time. For questions or to find a nearby clinic or doctor, residents can call the Public Health InfoLine at 833-540-0473. Open every day from 8 a.m. to 8 p.m. For more information, visit our websites: Avian flu in animals: publichealth.lacounty.gov/vet/HPAI.htm Avian flu in humans: ph.lacounty.gov/acd/diseases/h5n1.htm

The Los Angeles County Department of Public Health is investigating two possible cases of H5 bird flu in cats that consumed recalled raw milk from Raw Farm, LLC. The infected indoor cats consumed raw milk linked to a recall of raw milk and cream products prior to the onset of symptoms, which included lack of appetite, fever and neurologic signs. The infected cats died after severe worsening of their illness, and subsequently tested positive for Influenza A, a rare result in cats. http://publichealth.lacounty.gov/phcommon/public/media/mediapubhpdetail.cfm?prid=4901

California child ‘may have caught H5N1 from raw milk’ If H5N1 is confirmed, it will mark the first time a person would be known to have contracted the virus directly from drinking raw milk Maeve CullinanGlobal Health Security Reporter Related Topics Bird Flu, California, Global Health Security, Robert F. Kennedy Jr, Viruses 11 December 2024 6:12pm GMT Raw milk sales have grown by 20 per cent over the last year despite H5N1 spreading amongst US dairy cattle Credit: Lisa Baertlein/REUTERS A child who is believed to be infected with H5N1 may have caught the virus from drinking raw milk, health officials said on Tuesday. The child from Marion County, California, began vomiting and developed a high temperature after drinking a glass of unpasteurised milk, local health officer Dr Lisa Santora told reporters. After being taken to A&E, the child tested positive for influenza. Though the exact strain is yet to be determined, officials suspect it is H5N1 – the bird flu that has infected almost one thousand herds of dairy cattle in the US this year, the bulk of which are in California. Other household members who drank the same milk didn’t develop symptoms, but had consumed the milk in much smaller quantities by adding it to their coffees, Dr Santora said. Almost one thousand dairy herds have been infected with H5N1 this year Credit: Natalie Behring/Getty Images North America If H5N1 is confirmed, it will be the first time a person has contracted the virus directly from drinking raw milk. Most of the 60-odd cases detected in the US this year have been from direct contact with sick cattle or poultry. However, in May, 24 farm cats contracted the virus from drinking raw milk from infected cattle. Half of the cats died, and all had severe symptoms including “stiff body movement, ataxia, blindness, circling, and copious oculonasal discharge,” the US Centres for Disease Control said. The news comes after traces of live H5N1 virus were detected in batches of unpasteurised milk sold at retail stores across California last week. The milk was produced by Fresno-based brand Raw Farm, the largest producer of raw milk in the state. While pasteurised milk undergoes a rigorous heating process that kills bacteria and viruses such as H5N1, the raw variety can lead to a number of serious health risks, including infections like salmonella, E. coli, brucella, campylobacter and listeria. For this reason, US health agencies have long since warned of the dangers of consuming raw milk. But several public figures – including incoming US Health Secretary Robert F. Kennedy Jr and celebrity Gwenthyn Paltrow – continue to promote its consumption. Raw milk sales have grown by 20 per cent over the last year despite the risk of H5N1 growing over the same period. https://www.telegraph.co.uk/global-health/science-and-disease/bird-flu-milk-h5n1-california-raw-unpasteurised-cattle/

By Helen Branswell Dec. 11, 2024 Senior Writer, Infectious Diseases An initial test, using a swab swirled around the child’s nostrils, was negative for influenza. But because of the known exposure to raw milk, a second flu test was conducted the next day, where the child’s mouth and throat were swabbed. That test came back positive for flu A, a category of flu viruses that includes the seasonal flu viruses H1N1 and H3N2, but also H5N1 bird flu. But there was little virus in the swab and when the local and state public health laboratories tried to confirm the diagnosis and subtype the virus — that is, to determine which flu A virus was present — they could not do so. “So we only have a positive flu A test, and a symptomatic child,” Santora said. https://www.statnews.com/2024/12/11/california-bird-flu-child-case-details/

Susanne Rust @susrust · 17h 2) This is all according to Lisa Santora, Marin Co public health officer. Nasopharyngeal swab was negative for Influenza A. Santora said that it has not been uncommon for #H5N1 positive dairy workers to have negative nasopharyngeal tests. 1 10 78 1.5K Susanne Rust @susrust · 17h 3) B/c of "large" raw milk consumption by child, co health officials tested orally a day later. That was positive for Influenza A. No circulating human flu in child's home community or Marin Co area where family was visiting. No one else in family tested positive for Influenza A. 1 14 87 2.7K Susanne Rust @susrust · 17h 4) No one else was symptomatic. Only the child had consumed the milk. Child's mom bought cream top milk, thinking it was organic (kind she usually buys) -- maybe hadn't realized it was raw. It was a Raw Farm product. 2 13 84 1.5K Susanne Rust @susrust · 17h 5) All of these elements: Severe illness, negative naso, positive oral, no flu circulating in location or family, consumption of bird flu recall milk led health officials to call it a "presumptive" case. 1 12 89 4.8K Susanne Rust @susrust · 17h 6) CDC is now testing sample. A lab at Quest did not find H5, but had too little virus to make a determination. County, state and federal officials still investigating. 4 12 92 1.6K

Susanne Rust @susrust 1) Story update coming// Here's why Marin Co and CDPH called the case of the Marin Co child a "presumptive" #H5N1 : Child is a toddler. Child was visiting Marin Co. -- not a resident. Child brought to ER by mom. The child had a "high" fever and had been vomiting for 3 days 12:39 PM · Dec 11, 2024 · 13.8K Views

California investigating possible case of bird flu in child who drank raw milk By Jamie Gumbrecht, CNN 2 minute read Published 9:39 AM EST, Wed December 11, 2024 Video Ad Feedback Government to test milk for bird flu 03:57 - Source: CNN CNN — California health officials are investigating a possible case of bird flu in a child who became ill after drinking raw milk, Marin County Public Health said on Tuesday. The child went to a local emergency department in November with fever and vomiting after drinking raw milk and tested positive for influenza A, the county said. More testing is underway to determine whether the child had H5N1 bird flu. The child recovered and no family members became ill. Related articleUS government to begin expanded testing of milk supply to better track the spread of bird flu California health officials have been warning about the risks of raw milk and other raw dairy products since the virus was identified in products last month. While pasteurized milk goes through a heating process that kills harmful pathogens, raw milk is not pasteurized and can carry listeria, campylobacter, salmonella, E. coli and bird flu virus. Distribution from Fresno-based Raw Farm was halted in November after bird flu was identified in milk products from store shelves, dairy storage and bottling sites. Raw Farm has said it has paused production while its herd is under quarantine. Bird flu has continued to spread in wild birds, poultry and dairy cattle around the United States since spring. There’s no evidence of person-to-person spread but scientists worry the virus can mutate to spread more easily among people. The US Department of Agriculture announced last week a plan to expand testing of milk bound for pasteurization in order to better track the spread of bird flu. Get CNN Health's weekly newsletter Sign up here to get The Results Are In with Dr. Sanjay Gupta every Friday from the CNN Health team. Fifty-eight bird flu cases have been confirmed in humans in the United States so far this year, including 32 in California. Most are linked to farm workers who have been in contact with sick animals. California also reported last month the first US case identified in a child; the US Centers for Disease Control and Prevention said Tuesday that the virus from that case resembled those previously detected in humans, cattle and poultry in California but it’s not clear how the child was exposed. In an alert to health-care providers last week, the California Department of Health said doctors should consider bird flu in people with acute respiratory symptoms or conjunctivitis who’ve had recent exposure to animals with bird flu or who have recently consumed raw dairy products. Bird flu symptoms in humans include typical flu-like symptoms such as eye redness, sore throat, runny nose, cough, diarrhea, vomiting, body aches, fatigue, trouble swallowing or fever. https://www.cnn.com/2024/12/11/health/california-bird-flu-child-raw-milk-marin/index.html

Marin County Public Health is closely monitoring a multi-state outbreak of H5N1 bird flu in dairy cows with transmission to humans primarily in dairy and poultry workers. H5N1 bird flu was first detected among cows in California in August 2024. H5N1 bird flu was first detected among humans in California in October 2024. H5N1 has been detected in both wild birds and poultry in Marin County. H5N1 has also been detected in wastewater. H5N1 has not been detected among livestock or farm workers in Marin. In November, Marin County Public Health (MCPH) was notified of a suspected case of bird flu. The child presented to a local emergency department with fever and vomiting after drinking raw milk. The child tested positive for Influenza A. MCPH is working with California Department of Public Health (CDPH) and the Center for Disease Control (CDC) on additional testing to confirm if this infection was bird flu or seasonal flu. The child has recovered and no other family members became sick, indicating no person-to-person transmission. People rarely get bird flu, but those who interact with infected dairy cows, poultry, or wildlife have a greater risk of infection. The current risk to the public remains low. Health Care Providers: On December 6th, CDPH released an alert(link is external) advising healthcare providers to consider avian flu in symptomatic persons who consumed raw milk products. Contact Marin County Public Health to coordinate testing for suspected avian influenza A (H5N1) in persons with signs and symptoms consistent with acute respiratory tract or gastrointestinal infection and/or conjunctivitis with history of consuming raw milk in the past 10 days. During business hours, call 415-473-4163 to coordinate testing. Dairies & Cattle Farms: Call 1-866-922-2473 to report an unusual number of sick livestock or if you suspect Bird Flu in your livestock. For more information, visit: CDC News Release 11.22.2024(link is external) CDFA - AHFSS - AHB - H5N1 Bird Flu Virus in Livestock(link is external) CDPH Current Bird Flu Situation(link is external) CDC’s H5N1 Bird Flu: Current Situation Summary (link is external) Last reviewed and updated: December 10, 2024 What is H5N1 Bird Flu? H5N1 bird flu is a specific strain or type of influenza virus. H5N1 bird flu is also called highly pathogenic avian influenza (HPAI). H5N1 bird flu can infect wild birds, poultry, and mammals such as cows. Human infections with H5N1 bird flu are rare, but spread of the virus may lead to changes that make it more likely to infect humans. Public Health Recommendations CDC has indicated that the current risk to the general public from H5N1 bird flu is low. People should: Wash your hands regularly, especially before eating and after interacting with animals. Avoid unprotected exposures to sick or dead animals including wild birds, poultry, and other domesticated birds. Handling sick or dead animals safely requires personal protective equipment and training. Do not drink or eat unpasteurized (raw) milk or raw cheese. Raw milk and cheese have not gone through a process called pasteurization that kills disease-causing germs. The milk of cows infected with H5N1 carries live virus. For more information about how raw milk can make you sick, visit the CDC’s Raw Milk web page(link is external). Recommendations for Agricultural Workers People who have job-related or recreational exposure to infected animals, including wild birds, poultry, and dairy cows, are at greater risk of being exposed to H5N1 bird flu. People at higher risk should: Follow all CDC recommendations(link is external) for worker protection to reduce the risk of infection Call your health care provider if you feel sick. Symptoms of H5N1 bird flu can include red or watery eyes, cough, sore throat, and fever. The California Department of Public Health has also created an educational flyer for agricultural workers about H5N1 bird flu. Please access the PDF here: English(link is external) / Spanish(link is external) Additional Resources Avian Influenza (Bird Flu) Fact Sheet(link is external) (California Department of Food and Agriculture) Avian Influenzas Updates(link is external) (California Department of Food and Agriculture) Poultry Products Transportation(link is external) (California Department of Food and Agriculture) Updates on Highly Pathogenic Avian Influenza (HPAI)(link is external) (Food and Drug Administration)

In November, Marin County Public Health (MCPH) was notified of a suspected case of bird flu. The child presented to a local emergency department with fever and vomiting after drinking raw milk. The child tested positive for Influenza A. MCPH is working with California Department of Public Health (CDPH) and the Center for Disease Control (CDC) on additional testing to confirm if this infection was bird flu or seasonal flu. The child has recovered and no other family members became sick, indicating no person-to-person transmission. https://www.marinhhs.org/h5n1-bird-flu