Invest Clin 66(4): 467- 478, 2025 https://doi.org/10.54817/IC.v66n4a09

Corresponding author: Flor H. Pujol. Laboratorio de Virología Molecular, CMBC, IVIC, Caracas, Venezuela.

Email: fhpujol@gmail.com

The ongoing panzootic of avian Influenza

A (H5N1) and its potential pandemic threat.

Flor Helene Pujol1 and José Esparza2

1Laboratorio de Virología Molecular, CMBC, Instituto Venezolano de Investigaciones

Científicas (IVIC), Caracas, Venezuela.

2Institute of Human Virology, University of Maryland School of Medicine, Baltimore,

MD, USA.

Keywords: Avian Influenza; Pandemics; Panzootic; Influenza A Virus, H5N1 Subtype.

Abstract. The influenza virus is one of the most significant pathogens re-

sponsible for respiratory infections and is the human pathogen most frequently

associated with epidemics and pandemics. The epidemiological record of in-

fluenza suggests that future pandemics caused by this virus are inevitable,

even though their timing, origin, and severity remain uncertain. This review

focuses on the ongoing panzootic of avian influenza A (H5N1), which is cur-

rently spreading across much of the globe. The ongoing panzootic of Influenza

A (H5N1) clade 2.3.4.4b has spread rapidly worldwide and is causing concern.

The virus has already crossed species barriers, infecting multiple mammalian

hosts and causing human cases with varying degrees of severity. While sustained

human-to-human transmission has not yet occurred, an increasing frequency

of spillover events and the emergence of genotypes with mutations associated

with mammalian adaptation are of concern. We assess the potential for this

panzootic to evolve into a pandemic and examine the critical measures needed

for preparedness and prevention, following a One Health approach.

468 Pujol and Esparza

Investigación Clínica 66(4): 2025

La panzootia actual de influenza aviar A (H5N1) y su potencial

amenaza pandémica.

Invest Clin 2025; 66 (4): 467 – 478

Palabras clave: Influenza Aviar; Pandemias; Panzootia; Epizootia; Subtipo H5N1

del Virus de la Influenza A.

Resumen. El virus de la influenza es uno de los patógenos más importan-

tes, causante de infecciones respiratorias, y el agente humano más frecuente-

mente asociado con epidemias y pandemias. El registro epidemiológico de la

influenza sugiere que las futuras pandemias causadas por este virus son inevi-

tables, aunque su momento, origen y gravedad siguen siendo inciertos. Esta

revisión se centra en la panzootia actual de la influenza aviar A (H5N1), que ac-

tualmente se propaga por gran parte del mundo. La panzootia actual del virus

de la influenza A (H5N1), del clado 2.3.4.4b, se ha extendido de manera dramá-

tica a nivel mundial y está generando gran preocupación. El virus ya ha cruzado

las barreras entre especies, provocando infecciones en múltiples hospedadores

mamíferos y causando casos humanos con distintos grados de gravedad. Aun-

que aún no se ha producido una transmisión sostenida de persona a persona,

preocupa la creciente frecuencia de eventos de salto interespecie y la aparición

de genotipos con mutaciones asociadas a la adaptación en mamíferos. En esta

revisión, evaluamos el potencial de esta panzootia para evolucionar hacia una

pandemia y examinamos las medidas críticas necesarias para la preparación y

la prevención, siguiendo un enfoque de Una Sola Salud.

Received: 07-10-2025 Accepted: 17-11-2025

INTRODUCTION

The influenza virus is one of the most

significant pathogens responsible for respi-

ratory infections, distinguished not only by

the severity of the illnesses it causes but also

by its role in numerous historical epidemics

and pandemics 1,2.

While aquatic birds are the natural res-

ervoirs of Influenza A viruses (IAV), these

viruses can also infect a broad range of

mammalian species, including humans. The

epidemiological record of influenza suggests

that future pandemics caused by this virus

are inevitable, even though their timing,

origin, and severity remain uncertain. This

review focuses on the ongoing panzootic of

avian influenza A (H5N1), which is current-

ly spreading across much of the globe. The

virus has already crossed species barriers,

leading to infections in multiple mammalian

hosts and causing human cases with varying

degrees of severity 3. We assess the potential

for this panzootic to evolve into a pandemic

and examine the critical measures needed

for preparedness and prevention.

Avian Influenza

Influenza viruses belong to the family

Orthomyxoviridae and are enveloped viruses

with a segmented, negative-sense RNA ge-

nome. Four genera of influenza viruses have

been identified: Influenza A, B, C, and D. In-

fluenza A and B viruses possess eight RNA

The ongoing panzootic of avian Influenza A (H5N1) and its potential pandemic threat 469

Vol. 66(4): 467 - 478, 2025

segments, whereas Influenza C and D viruses

typically contain seven segments, although

they frequently package eight 4,5. Influenza

A viruses (IAVs) exhibit a broad host range,

infecting birds, humans, and various other

mammals. In contrast, Influenza B and C

viruses primarily infect humans, while Influ-

enza D virus is predominantly found in bo-

vine hosts 4,6. In humans, influenza disease

is caused mainly by viruses from the A and

B genera, with IAVs being of particular con-

cern due to their pandemic potential. These

genera are distinguished by the antigenic

properties of their internal virion proteins,

while IAVs are further differentiated into

subtypes by the antigenic properties of their

two external proteins 4,6.

The segmented nature of the influenza

virus genome facilitates frequent reassort-

ment events. This process occurs when a

host is co-infected with two different influ-

enza viruses; during replication, genome

segments from both viruses can be packaged

together into progeny virions 4,7. Pigs, which

are susceptible to multiple influenza virus

subtypes, have traditionally been considered

key hosts for reassortment and are often

referred to as “mixing vessels” 7. IAVs dis-

play substantial antigenic diversity in their

two principal surface glycoproteins: hemag-

glutinin (H) and neuraminidase (N). This

variability allows for a binary classification

of IAVs into serotypes, comprising 19 H sub-

types and 11 N subtypes, with more than 130

distinct subtype combinations identified to

date 8,9. The primary reservoir of this genetic

and antigenic diversity is avian IAVs, which

have been the source of multiple influenza

pandemics—often through reassortment

events between avian and human influenza

viruses 7.

Human influenza viruses bind prefer-

entially to α-2,6-linked sialic acid receptors

on the surface of epithelial cells, whereas

avian influenza viruses have a higher affinity

for α-2,3-linked sialic acids. These glycosidic

linkages play a critical role in host specificity

and cross-species transmission of influenza

viruses. Consequently, both the variant and

structural configuration of sialic acid recep-

tors influence viral binding affinity to the H

glycoprotein of influenza viruses 10.

Avian influenza A viruses (AIAVs) can

evolve from low pathogenicity (LPAIV) to

highly pathogenic forms (HPAIV), a classifi-

cation based on their virulence in chickens.

A defining molecular feature of HPAIV is the

presence of a furin cleavage site in the H

protein 3. Proteolytic cleavage of H into HA1

and HA2 subunits is a crucial step in viral

replication, triggering a pH-dependent con-

formational change that exposes the fusion

peptide, enabling fusion with endosomal

membranes and subsequent release of the vi-

ral genome into the host cytoplasm. HPAIVs

contain a polybasic cleavage site that allows

processing by furin-like proteases, which are

expressed in multiple tissues, thereby facili-

tating systemic viral dissemination 11.

Notably, HPAIVs have emerged multiple

times independently from ancestral LPAIVs,

but only among viruses of the H5 and H7

subtypes 12. Since the first emergence of

HPAI H5N1 in 1996, nearly 1,000 human

infections have been reported across 24

countries, with a case fatality rate of around

50%13,14.

The influenza pandemics of the past

Over the past three centuries, ten major

influenza pandemics have been documented,

averaging approximately three per century

(Fig. 1). These events have varied widely in

intensity, morbidity, and mortality 1.

In the 18th century, three pandem-

ics were recorded. The first, in 1729–1730,

had a global reach, with high morbidity

but relatively low mortality—a pattern that

was repeated in the subsequent 1732–1733

outbreak. The 1781–1782 pandemic was

particularly extensive, reportedly infecting

70–80% of the population, though it also ex-

hibited low mortality 1.

The 19th century witnessed two notable

pandemics in 1830–1831 and 1833, which

differed in severity, with the latter associ-

470 Pujol and Esparza

Investigación Clínica 66(4): 2025

ated with higher mortality. The 1889–1890

pandemic, commonly known as the “Russian

flu,” was particularly significant because it

coincided with the emergence of the germ

theory of disease. This conceptual shift—

from miasmatic to microbial causation—

marked a turning point in public health, pav-

ing the way for modern preventive strategies

such as improved hygiene and social distanc-

ing 1.

The 20th and 21st centuries saw four

influenza pandemics, all caused by IAVs. The

deadliest of these was the 1918-1919 “Span-

ish flu,” caused by an H1N1 virus. It infected

an estimated 500 million people—about 30%

of the global population—and resulted in 50

to 100 million deaths over three devastat-

ing waves. The impact was magnified by the

worldwide disruption and troop movements

associated with World War I. Later pandem-

ics included the 1957 “Asian flu” (H2N2)

and the 1968 “Hong Kong flu” (H3N2),

which were less severe but still responsible

for an estimated 1–3 million and 1–4 mil-

lion deaths, respectively, despite widespread

transmission 1.

The only influenza pandemic of the 21st

century to date occurred in 2009, caused by

a novel H1N1 virus that originated in North

America. Although initially feared to rival the

severity of the 1918 pandemic, it ultimately

proved relatively mild, with an estimated

125,000 to 400,000 deaths worldwide 1.

The panzootic H5N1 clade 2.3.4.4b

In addition to classification by subtype,

IAV genome sequences are further catego-

rized based on phylogenetic relationships,

including common ancestry and descendant

lineages. These classifications include clades

(e.g., 1.1, 2.2, 2.3), subclades (e.g., 2.3.2.1c,

2.3.3.4b), lineages (such as Eurasian and

American), and genotypes (e.g., A1, B1,

B3.13) 3,15,16. Clades and subclades are de-

fined primarily through phylogenetic analy-

sis of the hemagglutinin (H) gene, reflecting

evolutionary divergence. In contrast, geno-

types are determined by the constellation of

gene segments present in each strain, offer-

ing insight into reassortment events and ge-

nomic composition 16.

The subclade 2.3.4.4b emerged in 2016

(Fig. 1) in an H5N8 virus in China and sub-

sequently spread across Asia and into Eu-

rope. This virus caused an unexpected epi-

demic peak in 2020/2021 17. The 2.3.4.4b

H5N8 rearranged with a LPAI H5N1 around

2020, leading to the 2.3.4.4b H5N1. This vi-

Fig. 1. Major events in human and avian Influenza. The timeline shows the known Influenza pandemics in the

past centuries (in blue) and major events in avian influenza over the last two centuries (in orange).

The subtype of IAV responsible for the pandemic of the previous two centuries are shown 1,12,18,23,30.

The ongoing panzootic of avian Influenza A (H5N1) and its potential pandemic threat 471

Vol. 66(4): 467 - 478, 2025

rus caused the wave of 2021/2022, the cur-

rent panzootic, with several human cases,

and was the most devastating avian influ-

enza epidemic 17,18. After this subclade was

introduced into North America, several reas-

sortment events with circulating LPAIVs led

to the emergence of new genotypes. At least

three genotypes have circulated among in-

fected marine birds in South America since

2022, and these genotypes have also shown

infectivity and virulence toward marine

mammals 19,20.

Both avian- and human-type receptors

are present in swine respiratory epithelia.

Swine are generally the intermediate hosts

involved in the reassortment and adapta-

tion of AIAVs to mammals before spillover

to humans. However, new evolutionary path-

ways may be considered for the emergence

of a pandemic, given the frequent infection

of many mammalian species (both marine

and terrestrial), particularly cattle in the

USA20,21.

Cattle are typically infected with the In-

fluenza D virus but not with IAVs. However,

during the current panzootic, the first doc-

umented spillover of Influenza A into cows

involved an H5N1 virus of genotype B3.13—

originally circulating in wild animals (Fig.

1). This genotype is a reassortant between

B3.6 and an LPAIV endemic in the United

States. Phylogenetic evidence suggests that

a single spillover event was responsible for

the emergence of this panzootic clade in

cattle 22. In cattle, the primary site of AIAV

replication appears to be the udder, as mam-

mary tissue expresses avian-type α-2,3-linked

sialic acid receptors. This raises the possibil-

ity of virus transmission through unpasteur-

ized milk. The initial spillover event is be-

lieved to have occurred in Texas, USA, with

subsequent spread to cattle in other states

likely facilitated by contaminated milking

equipment 18. Since February 2024, at least

two additional spillover events involving a

different genotype, D1.1, have been reported

in U.S. cattle 23,24. Alarmingly, the first fatal

human case associated with this panzootic

in the United States was linked to infection

with this genotype 23.

Most human cases of H5N1 infection

during the current panzootic have been mild

so far. However, this apparent low pathoge-

nicity may be partly attributed to host-relat-

ed factors, including the young age of many

patients and the presence of pre-existing im-

munity to N1 and other conserved cellular

immunity epitopes 25,26. In contrast, experi-

mental infection of ferrets with the H5N1

genotype B3.13—currently circulating in

U.S. cattle—resulted in high lethality 27. An-

other study reported reduced mortality in

mice infected with a clade 2.3.4.4b H5N1 vi-

rus, despite the strain exhibiting high patho-

genicity in chickens 28. Notably, differences

in pathogenicity among genotypes within

the panzootic clade have also been observed

in both teals and poultry 29.

These findings underscore the poten-

tial for this lineage’s pathogenicity to evolve

further through reassortment and mutation.

Comprehensive studies are urgently needed

to assess the virulence and transmissibility

of these viruses in humans. Regardless of the

current severity, it is imperative to imple-

ment preparedness and response plans now

to mitigate the risk of a future influenza

pandemic.

Pandemic risk?

The current panzootic caused by Influ-

enza A-H5N1 has been marked by frequent

spillover events into a wide range of terres-

trial and marine mammalian species, with

around 50 species affected to date 3,30,31. The

United States currently reports the high-

est number of human cases associated with

this outbreak, with 70 confirmed infections

between 2024 and the end of April 2025 13.

However, this number remains below the

highest annual record of human H5N1 cas-

es, which occurred in 2015, when 145 cases

were reported globally, 136 of them in Egypt

alone 13.

A prerequisite for the development of

a pandemic is that the panzootic influenza

472 Pujol and Esparza

Investigación Clínica 66(4): 2025

virus acquires the ability not only to infect

humans, but also to sustain efficient human-

to-human transmission—something that has

not occurred to date. Efficient human-to-hu-

man transmission of avian influenza viruses

likely requires the cumulative acquisition of

several key mutations (Fig. 2) 30-33.

H mutations enhancing affinity for hu-

man receptors: Adaptation to the human

α-2,6-linked sialic acid receptor is criti-

cal. Notably, only four mutations were suf-

ficient to render an Influenza A-H5N1 virus

transmissible, via respiratory droplets, in

ferrets34. More recently, a single mutation,

Q226L, in the H of bovine H5N1 (genotype

B3.13) was shown to switch viral specificity

toward mammalian receptors 35. Three addi-

tional mutations have been identified as key

contributors to increased human receptor

affinity 32.

PB2 mutations enabling polymerase

adaptation to mammalian ANP32 proteins:

ANP32A and ANP32B, which serve as host

cofactors for the viral polymerase, differ be-

tween avian and mammalian species. Several

mutations have been characterized that al-

low avian influenza polymerases to utilize

mammalian ANP32 proteins 36 efficiently.

Some of these mutations have already been

detected at low frequency in viruses infect-

ing cattle 22.

Increased viral stability at low pH: Avian

influenza viruses generally exhibit reduced

stability in acidic environments such as the

human upper respiratory tract, posing a bar-

rier to transmission 33.

Host immune history: Pre-existing im-

munity to seasonal human influenza viruses,

whether from prior infection or vaccination,

may influence susceptibility and clinical out-

comes following exposure to H5N1 viruses 37.

The eventual emergence of a pandem-

ic influenza virus can result not only from

the gradual accumulation of key mutations

but also, frequently, from reassortment

events20,21. Traditional “mixing vessels” for

such reassortment have been pigs; however,

emerging evidence highlights other hosts

with high reassortment potential, including

humans, minks, ferrets, seals, dogs, cats,

and various bird species—particularly tur-

keys, chickens, quails, and ducks 20,21. Inter-

estingly, current data suggest that pigs may

be less susceptible to the current panzootic

IAV than some other mammals 38,39.

Although several key mutations associ-

ated with mammalian adaptation have been

detected in viruses isolated from infected

mammals during this panzootic 21, none of

the sequenced viral isolates to date possess

the whole constellation of mutations re-

quired for efficient human-to-human trans-

Fig. 2. Major mutations in four of the AIAV associated with efficient mammalian transmission and replication.

Mutations in four viral proteins, potentially related to human adaptation, are shown 22,30,32-36.

The ongoing panzootic of avian Influenza A (H5N1) and its potential pandemic threat 473

Vol. 66(4): 467 - 478, 2025

mission. Nevertheless, the extensive geo-

graphic spread of the panzootic clade and its

ability to infect a broad range of mammalian

hosts raise significant concerns about the

potential emergence of a pandemic strain 40.

Preparedness

According to the World Health Organi-

zation 41, influenza virus activity is classified

into six distinct phases, progressing from

circulation limited to animals to widespread

human transmission at the global level (Ta-

ble 1). This framework is divided into two

overarching stages: Phases 1–3, which focus

on preparedness, and Phases 4–6, which em-

phasize response and mitigation.

In Phase 1, no influenza viruses circu-

lating among animals have been reported

to cause infections in humans. Phase 2 is

characterized by an animal influenza virus

- circulating among domesticated or wild

animals - that has caused human illness

and is therefore considered a potential pan-

demic threat. Phase 3 occurs when an ani-

mal or human-animal reassortant influenza

virus causes sporadic cases or small clus-

ters of disease in people but does not lead

to sustained human-to-human transmission

or community-level outbreaks. In Phase 4,

human-to-human transmission results in lo-

calized community outbreaks, indicating a

significant increase in pandemic risk. Phase

5 is marked by community-level outbreaks in

at least two countries within one WHO re-

gion. In contrast, Phase 6 -defined by com-

munity outbreaks in multiple WHO regions

- signifies the onset of a global pandemic 41.

Although the global spread of the A

(H5N1) panzootic remains a serious con-

cern, no sustained human-to-human trans-

mission has been reported as of May 2025.

Consequently, the human influenza epidem-

ic remains classified as Phase 3, highlight-

ing the urgent need to bolster preparedness

efforts and prevent escalation to a pandem-

ic. To mitigate this risk, countries must de-

velop and implement coordinated response

strategies at both national and international

levels, emphasizing proactive preventive

measures. Since 2021, the World Health Or-

ganization (WHO) has been working with

Member States to draft a global treaty on

pandemic prevention, preparedness, and re-

sponse. This treaty was formally adopted by

consensus at the 78th World Health Assem-

bly in May 2025. It establishes guidelines for

the timely sharing of epidemiological data,

genomic analysis of emerging pathogens,

and critical information related to potential

vaccines and treatments 42.

Table 1. World Health Organization pandemic phases descriptions.

Phase Description Estimated probability

of pandemia

1 No animal influenza virus reported as causing human cases Uncertain

2 An animal influenza virus has caused infection in humans Uncertain

3 An animal or human-animal reassortant virus has caused a small

number of human cases but has not resulted in significant

human-to-human transmission

Uncertain

4 Human-to-human transmission able to sustain

community-level outbreaks

Medium to high

5 Community-level outbreaks in at least two countries

in one WHO region

High to certain

6 Community-level outbreaks in an additional WHO region Pandemic in progress

Adapted from World Health Organization 41.

474 Pujol and Esparza

Investigación Clínica 66(4): 2025

National preparedness plans should

incorporate robust communication strate-

gies to promote public adherence to non-

pharmaceutical interventions (NPIs). For

influenza, these include individual-level pro-

tective behaviors such as staying home when

symptomatic, covering coughs and sneezes,

and practicing frequent hand hygiene. At the

community level, recommended measures

may include temporary school closures, sus-

pending childcare services, and canceling

mass gatherings during periods of height-

ened transmission. When a novel pandemic

influenza virus emerges, the combined im-

plementation of NPIs and antiviral therapies

can significantly reduce transmission rates,

particularly in the early stages before a vac-

cine becomes widely available 41-44.

Pandemic preparedness for influenza

depends heavily on early detection of novel

strains, particularly those that originate in

animal reservoirs through spillover events.

Over the past two decades, repeated zoonot-

ic influenza outbreaks have underscored the

vital importance of sustained surveillance

in both human and animal populations, es-

pecially among birds and swine. As noted

earlier in this paper, the most pressing cur-

rent threat is the ongoing panzootic of avian

influenza A (H5N1). However, other avian

influenza subtypes continue to circulate in

more localized contexts. Genetic reassort-

ment during coinfections plays a critical

role in the emergence of pandemic-capable

strains. While the surface glycoproteins H

and N are traditionally viewed as central

to influenza infectivity and host specificity,

increasing evidence indicates that internal

viral genes -particularly those encoding the

polymerase complex- also significantly con-

tribute to virulence and disease severity 44.

Since 1952, the World Health Organization

has led a global initiative to monitor circu-

lating influenza viruses, enabling a coordi-

nated international response to emerging

influenza threats 45.

Given that a future pandemic influenza

virus is likely to emerge from an avian influ-

enza strain affecting poultry, control strat-

egies must integrate biosecurity measures,

epidemiological surveillance, targeted cull-

ing, and vaccination with strain-specific im-

munogens 46-48. Culling should be prioritized

in outbreaks involving HPAI strains, mainly

when rapid transmission occurs within poul-

try populations. Protective measures for

farm workers are also essential to minimize

the risk of zoonotic transmission. A similar

level of urgency applies to controlling H5N1

infections in other livestock species, such as

cattle 49.

During the preparedness phase, early

interventions should focus on securing vac-

cine and antiviral stockpiles 48-51 and on ex-

panding the healthcare system capacity to

ensure effective clinical management during

a pandemic. Notably, at least three experi-

mental vaccines targeting early H5N1 influ-

enza virus strains have demonstrated the

ability to elicit cross-reactive binding and

cross-neutralizing antibodies against the

HPAI clade 2.3.4.4b in humans, suggesting

they could serve as interim protective tools

while updated vaccines are developed52. Fur-

thermore, a novel H5-based mRNA vaccine

has recently been shown to generate a ro-

bust adaptive immune response in cattle53.

Nevert heless, the final formulation of a pan-

demic influenza vaccine must be tailored to

the specific viral strain in circulation at the

time of emergence to ensure optimal effi-

cacy.

CONCLUSIONS

The unprecedented epidemiology of

the panzootic AIAV (H5N1) clade 2.3.4.4b

has already resulted in devastating impacts

on poultry and wild mammal populations. It

now poses an emerging threat to livestock,

further complicating the pre-pandemic land-

scape. Although several human infections

have been reported, the mortality rate to

date has been lower than in previous out-

breaks. While it remains impossible to pre-

dict with certainty when or how a new influ-

The ongoing panzootic of avian Influenza A (H5N1) and its potential pandemic threat 475

Vol. 66(4): 467 - 478, 2025

enza pandemic might emerge, the current

spread of AIAV (H5N1) is deeply concerning

and demands urgent preparedness efforts. A

coordinated One Health approach—integrat-

ing human, animal, environmental health,

and, evidently, vaccines—is essential to ad-

dress this evolving threat effectively. This re-

quires strong collaboration among animal,

environmental, and public health sectors

to assess risks, strengthen early prevention,

coordinate outbreak responses, and develop

effective countermeasures. To reduce future

pandemic risks, urgent action is needed, not

only to protect people at the highest risk of

zoonotic infection (such as farm workers)

but also to prevent transmission among wild

and domestic animals and humans. Efforts

should target the underlying factors that en-

able such outbreaks and ensure rapid risk

assessment and response to every zoonotic

event 54.

ORCID number of authors

• Flor H. Pujol (FHP):

0000-0001-6086-6883

• José Esparza (JE):

0000-0002-2305-6264

Conflict of interest

The authors declare no conflict of in-

terest.

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