First detection and characterization of hepatitis E virus (Rocahepevirus ratti) from urban Norway rats (Rattus norvegicus) in the Republic of Korea.

Hepatitis E virus (HEV), an emerging zoonotic pathogen, poses a significant public health concern worldwide. Recently, rat HEV (Rocahepevirus ratti genotype C1; HEV-C1) has been reported to cause zoonotic infections and hepatitis in humans. Human infections with HEV-C1 are considered to be underestimated worldwide due to limited knowledge of transmission routes, genome epidemiology, and the risk assessment of zoonosis associated with these viruses. A total of 186 wild Norway rats (Rattus norvegicus) were collected from the Republic of Korea (ROK) between 2011 and 2021. The prevalence of HEV-C1 RNA was 8 of 180 (4.4%) by reverse-transcription polymerase chain reaction. We first reported three nearly whole-genome sequences of HEV-C1 newly acquired from urban rats in the ROK. Phylogenetic analysis demonstrated that Korea-indigenous HEV-C1 formed an independent genetic group with those derived from R. norvegicus rats in other countries, indicating geographical and genetic diversity. Our findings provide critical insights into the molecular prevalence, genome epidemiology, and zoonotic potential of Rocahepevirus. This report raises awareness of the presence of Rocahepevirus-related hepatitis E among physicians in the ROK.

Isolation and genetic characteristics of Novel H4N1 Avian Influenza viruses in ChongQing, China.

BACKGROUND: Avian influenza viruses (AIVs) constitute significant zoonotic pathogens encompassing a broad spectrum of subtypes. Notably, the H4 subtype of AIVs has a pronounced ability to shift hosts. The escalating prevalence of the H4 subtype heightens the concern for its zoonotic potential, signaling an urgent need for vigilance. METHODS: During the period from December 2021 to November 2023, we collected AIV-related environmental samples and assessed them using a comprehensive protocol that included nucleic acid testing, gene sequencing, isolation culture, and resequencing. RESULTS: In this study, a total of 934 environmental samples were assessed, revealing a remarkably high detection rate (43.66%, 289/662) of AIV in the live poultry market. Notably, the H4N1 subtype AIV (cs2301) was isolated from the live poultry market and its complete genome sequence was successfully determined. Subsequent analysis revealed that cs2301, resulting from a reassortment event between wild and domesticated waterfowl, exhibits multiple mutations and demonstrates potential for host transfer. CONCLUSIONS: Our research once again demonstrates the significant role of wild and domesticated waterfowl in the reassortment process of avian influenza virus, enriching the research on the H4 subtype of AIV, and emphasizing the importance of proactive monitoring the environment related to avian influenza virus.

Molecular characterization and zoonotic potential of Entamoeba spp., Enterocytozoon bieneusi and Blastocystis from captive wild animals in northwest China.

BACKGROUND: Parasites Entamoeba spp., Enterocytozoon bieneusi and Blastocystis are prevalent pathogens causing gastrointestinal illnesses in animals and humans. Consequently, researches on their occurrence, distribution and hosts are crucial for the well-being of both animals and humans. Due to the confined spaces and frequent interaction between animals and humans, animal sanctuaries have emerged as potential reservoirs for these parasites. In this study, the wildlife sanctuary near the Huang Gorge of the Qinling Mountains in northwest China is chosen as an ideal site for parasite distribution research, considering its expansive stocking area and high biodiversity. RESULTS: We collected 191 fecal specimens from 37 distinct wildlife species and extracted genomic DNA. We identified these three parasites by amplifying specific gene regions and analyzed their characteristics and evolutionary relationships. All the parasites exhibited a high overall infection rate, reaching 90.05%. Among them, seven Entamoeba species were identified, accounting for a prevalence of 54.97%, with the highest infection observed in Entamoeba bovis. In total, 11 Enterocytozoon bieneusi genotypes were discovered, representing a prevalence of 35.08%, including three genotypes of human-pathogenic Group 1 and two novel genotypes (SXWZ and SXLG). Additionally, 13 Blastocystis subtypes were detected, showing a prevalence of 74.87% and encompassing eight zoonotic subtypes. All of the above suggests significant possibilities of parasite transmission between animals and humans. CONCLUSIONS: This study investigated the occurrence and prevalence of three intestinal parasites, enhancing our understanding of their genetic diversity and host ranges in northwest China. Furthermore, the distribution of these parasites implies significant potential of zoonotic transmission, underscoring the imperative for ongoing surveillance and implementation of control measures. These efforts are essential to mitigate the risk of zoonotic disease outbreaks originating from wildlife sanctuary.

Zoonotic Cryptosporidium and Giardia in marsupials-an update.

Marsupials, inhabiting diverse ecosystems, including urban and peri-urban regions in Australasia and the Americas, intersect with human activities, leading to zoonotic spill-over and anthroponotic spill-back of pathogens, including Cryptosporidium and Giardia. This review assesses the current knowledge on the diversity of Cryptosporidium and Giardia species in marsupials, focusing on the potential zoonotic risks. Cryptosporidium fayeri and C. macropodum are the dominant species in marsupials, while in possums, the host-specific possum genotype dominates. Of these three species/genotypes, only C. fayeri has been identified in two humans and the zoonotic risk is considered low. Generally, oocyst shedding in marsupials is low, further supporting a low transmission risk. However, there is some evidence of spill-back of C. hominis into kangaroo populations, which requires continued monitoring. Although C. hominis does not appear to be established in small marsupials like possums, comprehensive screening and analysis are essential for a better understanding of the prevalence and potential establishment of zoonotic Cryptosporidium species in small marsupials. Both host-specific and zoonotic Giardia species have been identified in marsupials. The dominance of zoonotic G. duodenalis assemblages A and B in marsupials may result from spill-back from livestock and humans and it is not yet understood if these are transient or established infections. Future studies using multilocus typing tools and whole-genome sequencing are required for a better understanding of the zoonotic risk from Giardia infections in marsupials. Moreover, much more extensive screening of a wider range of marsupial species, particularly in peri-urban areas, is required to provide a clearer understanding of the zoonotic risk of Cryptosporidium and Giardia in marsupials.

Is the European badger a new host for Dirofilaria immitis? The first records in Greece.

Dirofilaria immitis is a ubiquitous nematode parasite with zoonotic potential, transmitted by mosquitoes, that causes heartworm disease in various animal species. Dogs are the parasite's typical final host, and wild carnivores represent the parasite's reservoir in nature. Studies on D. immitis infections in wild animals are essential to assess infection pressure for domestic animals, and until now, there has been only one infection case reported in a European badger (Meles meles). The current report describes the first two European badger cases with cardiovascular dirofilariosis in Greece. Two adult male badgers were rescued in Heraklion and Chania, Crete Island, and admitted to "ANIMA -Wildlife Rehabilitation Centre" in Athens. The detailed clinical examination revealed that the first badger suffered from severe broncho-pneumonitis while the second one displayed clinical signs associated with severe brain trauma. Blood samples were taken for haematology and biochemistry analyses during their short hospitalisation period. In addition, different routine diagnostic tests were carried out, including heartworm antigen testing (ELISA) and the modified Knott's test for microfilariae. Both badgers were positive in both tests. The animals died a few hours after their admission and the detailed necropsies followed, revealed the presence of three parasites in each animal's right heart, morphologically identified as adults of D. immitis. These findings add the European badger in the list of additional potential reservoir hosts for D. immitis and highlight the potential role of wildlife for companion animals and human health.

Picobirnaviruses in animals: a review.

Picobirnaviruses (PBVs) are small non enveloped viruses with bi-segmented ds RNA. They have been observed in a wide variety of vertebrates, including mammals and birds with or without diarrhoea, as well as in sewage samples since its discovery (1988). The source of the viruses is uncertain. True hosts of PBVs and their role as primary pathogens or secondary opportunistic agents or innocuous viruses in the gut remains alien. The mechanisms by which they play a role in pathogenicity are still unclear based on the fact that they can be found in both symptomatic and asymptomatic cases. There is a need to determine their tropism since they have not only been associated with viral gastroenteritis but also been reported in the respiratory tracts of pigs. As zoonotic agents with diverse hosts, the importance of epidemiological and surveillance studies cannot be overstated. The segmented genome of PBV might pose a serious public health issue because of the possibility of continuous genetic reassortment. Aware of the growing attention being given to emerging RNA viruses, we reviewed the current knowledge on PBVs and described the current status of PBVs in animals.

North American and Norwegian Chronic Wasting Disease Prions Exhibit Different Potential for Interspecies Transmission and Zoonotic Risk.

BACKGROUND: Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting various species of wild and captive cervids. The risk that CWD poses to cohabiting animals or more importantly to humans is largely unknown. METHODS: In this study, we investigated differences in the capacity of CWD isolates obtained from 6 different cervid species to induce prion conversion in vitro by protein misfolding cyclic amplification. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at expenses of normal prion proteins from various mammals and human, respectively. RESULTS: Our data suggest that reindeer and red deer from Norway could be the most transmissible CWD prions to other mammals, whereas North American CWD prions were more prone to generate human prions in vitro. CONCLUSIONS: Our results suggest that Norway and North American CWD prions correspond to different strains with distinct spillover and zoonotic potentials.

Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD.

Prions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management.

Genetic diversity of Blastocystis subtypes in the Alpine musk deer (Moschus chrysogaster) in Gansu province, northwestern China.

Blastocystis sp. is a gastrointestinal pathogen that is frequently found in humans and animals worldwide. In this study, 201 fecal samples were collected from captive Alpine musk deer (Moschus chrysogaster) at three farms in Gansu province. Blastocystis was detected and subtyped by amplifying and sequencing the small subunit ribosomal DNA gene. The overall prevalence of Blastocystis was 39.8% (80/201). Five known Blastocystis subtypes (STs), including ST1 (n = 1), ST4 (n = 12), ST10 (n = 50), ST14 (n = 6), and ST24 (n = 11) were identified using subtyping and evolutionary analysis. ST10 was the most common ST observed in each farm. This study showed the infection status and genetic characteristics of Blastocystis in M. chrysogaster. Based on the surveyed data, because various potentially zoonotic STs, such as ST1, ST4, ST10, ST14, and ST24, were detected, it is believed that the zoonotic risk of Blastocystis from the Alpine musk deer in this area cannot be ignored.

The first decade of research advances in influenza D virus.

From its initial isolation in the USA in 2011 to the present, influenza D virus (IDV) has been detected in cattle and swine populations worldwide. IDV has exceptional thermal and acid stability and a broad host range. The virus utilizes cattle as its natural reservoir and amplification host with periodic spillover to other mammalian species, including swine. IDV infection can cause mild to moderate respiratory illnesses in cattle and has been implicated as a contributor to bovine respiratory disease (BRD) complex, which is the most common and costly disease affecting the cattle industry. Bovine and swine IDV outbreaks continue to increase globally, and there is increasing evidence indicating that IDV may have the potential to infect humans. This review discusses recent advances in IDV biology and epidemiology, and summarizes our current understanding of IDV pathogenesis and zoonotic potential.

Enterohepatic Helicobacter species - clinical importance, host range, and zoonotic potential.

The genus Helicobacter defined just over 30 years ago, is a highly diverse and fast-growing group of bacteria that are able to persistently colonize a wide range of animals. The members of this genus are subdivided into two groups with different ecological niches, associated pathologies, and phylogenetic relationships: the gastric Helicobacter (GH) and the enterohepatic Helicobacter (EHH) species. Although GH have been mostly studied, EHH species have become increasingly important as emerging human pathogens and potential zoonotic agents in the last years. This group of bacteria has been associated with the development of several diseases in humans from acute pathologies like gastroenteritis to chronic pathologies that include inflammatory bowel disease, and liver and gallbladder diseases. However, their reservoirs, as well as their routes of transmission, have not been well established yet. Therefore, this review summarizes the current knowledge of taxonomy, epidemiology, and clinical role of the EHH group.

Global prevalence, subtypes distribution, zoonotic potential, and associated risk factors of Blastocystis sp. in domestic pigs (Sus domesticus) and wild boars (Sus scrofa): A systematic review and meta-analysis.

The intestinal parasite Blastocystis sp. is a widely distributed protist among humans and various animal hosts, with significant prevalence in developing countries. Due to the zoonotic nature of its subtypes (STs), we aimed at global estimation of the prevalence, STs distribution, zoonotic potential, and associated risk factors of Blastocystis sp. infection in domestic pigs (Sus domesticus) and wild pigs/wild boars (Sus scrofa). The study was designed and conducted in 2021 via searching articles in PubMed, Scopus, Google Scholar, and Web of Science databases, based on the PRISMA checklist, and meta-analysis was done using a random-effects model to calculate the weighted estimates and 95% confidence intervals (95% CIs). Totally, 43 papers (47 datasets) reported data on 7977 examined pigs in 24 countries with a total prevalence of 50.9% (95% CI: 42.8-59%). In details, prevalence was higher among domestic pigs [52.4% (95% CI: 43.9-60.7%)] than wild boars [31.2% (95% CI: 11.2-62%)], but is poorly statistically supported as far as the CIs largely overlap. Out of 28 reported STs, nine (ST1-ST7, ST10, and ST15) were reported from domestic pigs, while six (ST1, ST3-ST5, ST8, and ST15) had been isolated from wild boars. Among nine zoonotic STs (ST1-ST8, and ST12), all were identified in examined swine populations, except for ST12. As well, ST1 and ST5 were probably the most frequently circulating STs among these animals. In addition, male and older pigs showed higher Blastocystis sp. infection. Altogether, Blastocystis epidemiology and the distribution of its related STs in pigs is still open to question and requires more extensive studies, especially in the neglected regions of the world.

Salmonella enterica Serovar Hvittingfoss in Bar-Tailed Godwits (Limosa lapponica) from Roebuck Bay, Northwestern Australia.

Salmonella enterica serovar Hvittingfoss is an important foodborne serotype of Salmonella, being detected in many countries where surveillance is conducted. Outbreaks can occur, and there was a recent multistate foodborne outbreak in Australia. S Hvittingfoss can be found in animal populations, though a definitive animal host has not been established. Six species of birds were sampled at Roebuck Bay, a designated Ramsar site in northwestern Australia, resulting in 326 cloacal swabs for bacterial culture. Among a single flock of 63 bar-tailed godwits (Limosa lapponica menzbieri) caught at Wader Spit, Roebuck Bay, in 2018, 17 (27%) were culture positive for Salmonella All other birds were negative for Salmonella The isolates were identified as Salmonella enterica serovar Hvittingfoss. Phylogenetic analysis revealed a close relationship between isolates collected from godwits and the S Hvittingfoss strain responsible for a 2016 multistate foodborne outbreak originating from tainted cantaloupes (rock melons) in Australia. While it is not possible to determine how this strain of S Hvittingfoss was introduced into the bar-tailed godwits, these findings show that wild Australian birds are capable of carrying Salmonella strains of public health importance.IMPORTANCESalmonella is a zoonotic pathogen that causes gastroenteritis and other disease presentations in both humans and animals. Serovars of S. enterica commonly cause foodborne disease in Australia and globally. In 2016-2017, S Hvittingfoss was responsible for an outbreak that resulted in 110 clinically confirmed human cases throughout Australia. The origin of the contamination that led to the outbreak was never definitively established. Here, we identify a migratory shorebird, the bar-tailed godwit, as an animal reservoir of S Hvittingfoss. These birds were sampled in northwestern Australia during their nonbreeding period. The presence of a genetically similar S Hvittingfoss strain circulating in a wild bird population, 2 years after the 2016-2017 outbreak and ∼1,500 km from the suspected source of the outbreak, demonstrates a potentially unidentified environmental reservoir of S Hvittingfoss. While the birds cannot be implicated in the outbreak that occurred 2 years prior, this study does demonstrate the potential role for wild birds in the transmission of this important foodborne pathogen.

Evidence for Zoonotic Potential of Enterocytozoon bieneusi in Its First Molecular Characterization in Captive Mammals at Bangladesh National Zoo.

To determine the occurrence and genotypes of Enterocytozoon bieneusi in captive mammals at Bangladesh National Zoo and to assess their zoonotic significance, 200 fecal samples from 32 mammalian species were examined using a nested PCR and sequencing of internal transcribed spacer (ITS) gene. Enterocytozoon bieneusi was detected in 16.5% (33/200) of the samples. Seven different ITS genotypes were identified, including two known genotypes (D and J) and five new ones (BAN4 to BAN8). Genotype D was the most common genotype being observed in 19 isolates. In phylogenetic analysis, four genotypes (D, BAN4, BAN5, and BAN6), detected in 30 isolates (90.9%), belonged to Group 1 having zoonotic potential. The sequence of genotype J found in a Malayan pangolin was clustered in so-called ruminant-specific Group 2. The other two genotypes BAN7 and BAN8 were clustered in primate-specific Group 5. To our knowledge, this is the first report of molecular characterization of E. bieneusi in Bangladesh, particularly in captive-bred wildlife in this country. The potentially zoonotic genotypes of E. bieneusi are maintained in zoo mammals that may transmit among these animals and to the humans through environmental contamination or contact.

Prevalence and subtype diversity of Blastocystis in human and nonhuman primates in North China.

Blastocystis is of public health concern due to its global distribution in diverse animals including humans. Here, fecal specimens sampled from human and nonhuman primates were examined for Blastocystis by PCR and sequence analysis of the small subunit ribosomal RNA gene. Among age cohorts, the parasite was positive only for three of 126 (2.4%) adults admitted to a hospital in Harbin, Heilongjiang province, with a less common human subtype (ST), ST5, determined. Blastocystis was identified in 7.0% of nonhuman primates (NHPs), giving an infection rate of 6.8% (4/59) to zoo NHPs in Harbin and 7.1% (9/126) to lab NHPs in Beijing. No significant prevalence differences by macaque species, age, gender, and sample source were observed. Among the subtypes found in NHPs, seven belonged to ST1, three to ST2, one to ST3, and the remaining two to mixed ST1/ST3 and ST2/ST3. Although the data here showed no direct evidence linking human infections to Blastocystis of NHP origin, individuals might acquire colonization of ST5 from livestock sources judged by occurrence of this subtype also in cattle in Harbin and pigs and sheep in unspecified cities of Heilongjiang as noted in previous reports. In addition, given the nonrigid (but sometimes, perhaps cryptic) host specificity of ST1, ST2, and ST3 and their dominant role in human affections globally as discussed in a previous NHP report by Alfellani et al. (Parasitology 140:966-971, 2013a), precautions should be taken to minimize the possible transmission of those subtypes from NHPs to humans in North China.

Schistosome Interactions within the Schistosoma haematobium Group, Malawi.

Molecular analysis of atypical schistosome eggs retrieved from children in Malawi revealed genetic interactions occurring between human (Schistosoma haematobium) and livestock (S. mattheei and S. bovis) schistosome species. Detection of hybrid schistosomes adds a notable new perspective to the epidemiology and control of urogenital schistosomiasis in central Africa.

Genetic Characterization and Zoonotic Potential of Highly Pathogenic Avian Influenza Virus A(H5N6/H5N5), Germany, 2017-2018.

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade 2.3.4.4b isolates found in Germany in 2017-2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.

Comparison of the phylogenetic analysis of PFGE profiles and the characteristic of virulence genes in clinical and reptile associated Salmonella strains.

BACKGROUND: Salmonella is generally considered as a human pathogen causing typhoid fever and gastrointestinal infections called salmonellosis, with S. Enteritidis and S. Typhimurium strains as the main causative agents. Salmonella enterica strains have a wide host array including humans, birds, pigs, horses, dogs, cats, reptiles, amphibians and insects. Up to 90% of reptiles are the carriers of one or more serovars of Salmonella. Extraintestinal bacterial infections associated with reptiles pose serious health threat to humans. The import of exotic species of reptiles as pet animals to Europe correlates with the emergence of Salmonella serotypes, which not found previously in European countries. The presented study is a new report about Salmonella serotypes associated with exotic reptiles in Poland. The goal of this research was to examine the zoonotic potential of Salmonella strains isolated from reptiles by comparative analysis with S. Enteritidis strains occurring in human population and causing salmonellosis. RESULTS: The main findings of our work show that exotic reptiles are asymptomatic carriers of Salmonella serovars other than correlated with salmonellosis in humans (S. Enteritidis, S. Typhimurium). Among the isolated Salmonella strains we identified serovars that have not been reported earlier in Poland, for example belonging to subspecies diarizonae and salamae. Restriction analysis with Pulsed-field Gel Electrophoresis (PFGE), showed a great diversity among Salmonella strains isolated from reptiles. Almost all tested strains had distinct restriction patterns. While S. Enteritidis strains were quite homogeneous in term of phylogenetic relations. Most of the tested VGs were common for the two tested groups of Salmonella strains. CONCLUSIONS: The obtained results show that Salmonella strains isolated from reptiles share most of virulence genes with the S. Enteritidis strains and exhibit a greater phylogenetic diversity than the tested S. Enteritidis population.

Prevalence and molecular characterization of Cryptosporidium and Giardia in pre-weaned native calves in the Republic of Korea.

Cryptosporidium spp. and Giardia duodenalis are protozoan parasites that cause diarrhea in humans and animals. Molecular data on Cryptosporidium spp. and G. duodenalis in calves in the Republic of Korea (ROK) is limited; thus, we investigated the prevalence of Cryptosporidium and Giardia in pre-weaned calves, analyzed the association between these parasites and diarrhea, and identified their zoonotic potential for human infection. Fecal samples were collected from 315 pre-weaned calves aged 1-60 days from 10 different regions in the ROK and screened for Cryptosporidium spp. and G. duodenalis using PCR. Overall prevalence of Cryptosporidium spp. and G. duodenalis was 4.4% (n = 14) and 12.7% (n = 40), respectively. Co-infection was not detected. All Cryptosporidium-positive samples were identified as C. parvum after sequence analysis of a small subunit rRNA fragment and further subtyped into zoonotic IIaA15G2R1 (n = 13) and IIaA18G3R1 (n = 1) by DNA sequencing of the 60-kDa glycoprotein gene. To our knowledge, this is the first report of C. parvum IIaA15G2R1 subtype in calves in the ROK. Based on ß-giardin (bg) gene, G. duodenalis-positive samples belonged to assemblages E (n = 36) and A (n = 4), with the latter belonging to subtype A1, the zoonotic genotype. Six subtypes of assemblage E were identified at the bg locus: E1 (n = 6), E2 (n = 3), E3 (n = 13), E5 (n = 1), E8 (n = 1), and E11 (n = 1). The occurrence of C. parvum and G. duodenalis was not associated with diarrhea in pre-weaned Korean native calves. The present results suggest that the prevalence of C. parvum is not related to calf age; in contrast, the prevalence of G. duodenalis was significantly higher in 41-50-day-old calves (odds ratio = 9.90, 95% confidence interval 2.37-41.34; P = 0.001) than in 1-10-day-old calves. Therefore, calves are a potential source of zoonotic transmission, which may have significant public health implications.

Association of Increased Receptor-Binding Avidity of Influenza A(H9N2) Viruses with Escape from Antibody-Based Immunity and Enhanced Zoonotic Potential.

We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014-2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.