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Volume 27, Number 11—November 2021
Research Letter

Highly Pathogenic Avian Influenza A(H5N1) Virus in Wild Red Foxes, the Netherlands, 2021

Jolianne M. RijksComments to Author , Hanna Hesselink, Pim Lollinga, Renee Wesselman, Pier Prins, Eefke Weesendorp, Marc Engelsma, Rene Heutink, Frank Harders, Marja Kik, Harry Rozendaal, Hans van den Kerkhof, and Nancy Beerens
Author affiliations: Dutch Wildlife Health Centre, Utrecht University, Utrecht, the Netherlands (J.M. Rijks, H. Hesselink, M. Kik); Stichting Faunavisie Wildlife Care, Westernieland, the Netherlands (P. Lollinga); Stichting Faunavisie Wildlife Care, Blijham, the Netherlands (R. Wesselman); Dierenkliniek Winsum, Winsum, the Netherlands (P. Prins); Wageningen Bioveterinary Research, Lelystad, the Netherlands (E. Weesendorp, M. Engelsma, R. Heutink, F. Harders, N. Beerens); Dutch Food and Consumer Products Safety Authority, Utrecht (H. Rozendaal); Coordination Centre for Communicable Disease Control, National Institute for Public Health and the Environment, Bilthoven, the Netherlands (H. van den Kerkhof)

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We detected infection with highly pathogenic avian influenza A(H5N1) virus clade in 2 red fox (Vulpes vulpes) cubs found in the wild with neurologic signs in the Netherlands. The virus is related to avian influenza viruses found in wild birds in the same area.


Salivating red fox (Vulpes vulpes) cub 1 during a fit, the Netherlands, 2021. Seizure started with retracting lips at 0 sec (A), followed by facial wrinkling with opening of mouth at 0.07 sec (B), closing of the jaws at 0.17 sec (C), then back to “normal” at 0.40 sec (D), before this sequence starts all over at 0.50 sec.

Figure. Salivating red fox (Vulpes vulpes) cub 1 during a fit, the Netherlands, 2021. Seizure started with retracting lips at 0 sec (A), followed by facial wrinkling with opening...


Salivating red fox (Vulpes vulpes) cub 1 during a fit, the Netherlands, May 10, 2021.

Video. Salivating red fox (Vulpes vulpes) cub 1 during a fit, the Netherlands, May 10, 2021.

On May 10, a red fox Vulpes vulpes cub (cub 1) displaying abnormal behavior was found in Bellingwolde, the Netherlands, and taken into care of a wildlife rescue center. Upon entry, the 6- to 8-week-old cub was slightly dehydrated and showed at intervals of <30 minutes lip retraction, rapid opening and closing of mouth, excessive salivation, skin twitching, head shaking, and body tremors (Figure; Video). The cub first seemed to improve, but on May 12 it reacted aggressively when touched. Subsequently, we observed difficult swallowing and labored breathing. The cub seemed blind and stopped eating. As the situation further deteriorated, we humanely euthanized the cub on May 16. On May 13, the center received another 6- to 8-week-old red fox cub (cub 2) found ≈900 m from cub 1. Cub 2 was hypothermic and dehydrated. It had seizures and died overnight.

Retrospectively, we concluded that the mother of the cubs was likely a vixen found walking circles on May 10, ≈975 m direct distance from cub 1 and ≈90 m from cub 2. The vixen reacted very aggressively to capture, responding to sound but blind. The vixen had a fresh elbow fracture, probably caused by a road traffic accident. We humanely euthanized her the same day and sent her carcass for destruction.

Although rabies lyssavirus is unlikely in the Netherlands, European bat 1 lyssavirus is endemic in serotine bats (Eptesicus serotinus) (1). To exclude lyssavirus infection in the fox cubs, we performed a direct fluorescent antibody test on smears of brain tissue in accordance with World Organisation for Animal Health (OIE) protocol ( Test results were negative.

Subsequently, we tested brain samples for avian influenza virus by using a PCR detecting the influenza A virus matrix gene, followed by the subtype-specific H5-PCR on the hemagglutinin gene, as described previously (2). The samples from both cubs tested positive (Table), and we subtyped the virus as highly pathogenic avian influenza (HPAI) influenza virus A subtype H5N1. We isolated the HPAI H5N1 virus from the brain of cub 1 by inoculation of the samples into 10-day-old embryonated special pathogen–free chicken eggs.

During April–May 2021, large numbers of dead barnacle geese (Branta leucopsis) were reported in the northern part of the Netherlands, and later other species of waterfowl and birds of prey were also found dead. A selected number of dead wild birds were submitted for AI diagnostics and tested positive for HPAI H5N1 virus. We performed whole-genome sequencing of the HPAI H5N1 viruses found in wild birds and the 2 foxes as previously described (3) and conducted genetic and phylogenetic analyses to study the relationship between these viruses. Phylogenetic analysis of the gene segments (Appendix 1 Figure 1–8) showed the viruses detected in wild birds and the 2 foxes were in the same cluster and highly related. We classified the viruses as H5 clade viruses, which were related to other HPAI H5N1 viruses detected in wild birds and poultry in Europe during 2020–2021. The HPAI H5N1 viruses detected in the foxes were not related to zoonotic H5N1 strains infecting humans in Asia and did not contain any known zoonotic mutations (data not shown). The sequences of the viruses detected in cub 1 (GISAID [] accession no. EPI_2194218) and cub 2 (GISAID accession no. EPI_2194219) were identical; the closest related virus was identified in a white-tailed eagle (Haliaeetus albicilla) near the village of Noordlaren. We observed only 6 aa differences: mutations A152T and T521I in polymerase basic protein 2 (PB2); M644V in polymerase basic protein 1; A336T in nucleoprotein; L22S in neuraminidase protein; and D209N in nonstructural protein (Appendix 1 Figure 1–8). Whether these changes are associated with adaptation of the avian virus to mammal species remains unknown.

These 2 cases of infection with H5N1 clade virus in wild red fox cubs underscore the need to raise awareness that HPAI viruses are not only zoonotic but also infect other mammal species. HPAI infection should be on the list of differential diagnoses for animals that have signs of respiratory or neurologic disease. The detection of virus in the brain suggests systemic infection of the cubs. The clinical signs were largely consistent with those reported in other natural infections of carnivores with HPAI H5 subtypes (47). Whether the fox cubs were infected through the parents or by eating infected bird carcasses is unclear (cubs start eating solid food at 4 weeks of age). Carnivores are known to be at risk for avian influenza virus infection upon ingesting infected birds (4,5,8). We did not test for virus shedding in these cubs, but virus shedding has been observed in experimental infection of 6- to 10-month-old red foxes with HPAI H5N1 clade 2.2 virus (8).

The United Kingdom reported infection of a red fox and seals in an animal shelter with a related HPAI H5N8 clade virus (9), and Russia reported infection in poultry workers (10). These findings suggest that HPAI H5 clade viruses may sporadically transmit from birds to mammals, including humans. Virus evolution and adaptive mutations must be closely monitored to rapidly identify viruses with increased zoonotic potential.

Dr. Rijks is a postdoctoral researcher at the Dutch Wildlife Health Centre in Utrecht, the Netherlands. Her primary research interests are wildlife diseases and epidemiology.



We acknowledge the authors and submitting laboratories of the sequences from the GISAID EpiFlu Database (Appendix 2).

This work was funded by the Dutch Ministry of Agriculture, Nature, and Food Quality (project no. WOT-01-003-012).



  1. Van der Poel  WH, Van der Heide  R, Verstraten  ER, Takumi  K, Lina  PH, Kramps  JA. European bat lyssaviruses, The Netherlands. Emerg Infect Dis. 2005;11:18549. DOIPubMedGoogle Scholar
  2. Beerens  N, Heutink  R, Bergervoet  SA, Harders  F, Bossers  A, Koch  G. Multiple reassorted viruses as cause of highly pathogenic avian influenza A(H5N8) virus epidemic, the Netherlands, 2016. Emerg Infect Dis. 2017;23:197481. DOIPubMedGoogle Scholar
  3. Beerens  N, Heutink  R, Pritz-Verschuren  S, Germeraad  EA, Bergervoet  SA, Harders  F, et al. Genetic relationship between poultry and wild bird viruses during the highly pathogenic avian influenza H5N6 epidemic in the Netherlands, 2017-2018. Transbound Emerg Dis. 2019;66:13708. DOIPubMedGoogle Scholar
  4. Marschall  J, Hartmann  K. Avian influenza A H5N1 infections in cats. J Feline Med Surg. 2008;10:35965. DOIPubMedGoogle Scholar
  5. Songserm  T, Amonsin  A, Jam-on  R, Sae-Heng  N, Pariyothorn  N, Payungporn  S, et al. Fatal avian influenza A H5N1 in a dog. Emerg Infect Dis. 2006;12:17447. DOIPubMedGoogle Scholar
  6. Lee  K, Lee  EK, Lee  H, Heo  GB, Lee  YN, Jung  JY, et al. Highly pathogenic avian influenza A(H5N6) in domestic cats, South Korea. Emerg Infect Dis. 2018;24:23437. DOIPubMedGoogle Scholar
  7. Hu  T, Zhao  H, Zhang  Y, Zhang  W, Kong  Q, Zhang  Z, et al. Fatal influenza A (H5N1) virus Infection in zoo-housed Tigers in Yunnan Province, China. Sci Rep. 2016;6:25845. DOIPubMedGoogle Scholar
  8. Reperant  LA, van Amerongen  G, van de Bildt  MW, Rimmelzwaan  GF, Dobson  AP, Osterhaus  AD, et al. Highly pathogenic avian influenza virus (H5N1) infection in red foxes fed infected bird carcasses. Emerg Infect Dis. 2008;14:183541. DOIPubMedGoogle Scholar
  9. ProMED. Avian influenza (45): Europe (UK, Croatia) seal, fox, wild bird, HPAI H5N8. ProMED 2021 Mar 17., archive no. 20210317.8252821.
  10. ProMED. Avian influenza, human (02): Russia, H5N8, first case. ProMED 2021 Feb 21., archive no. 20210221.8204014.




Cite This Article

DOI: 10.3201/eid2711.211281

Original Publication Date: October 07, 2021

Table of Contents – Volume 27, Number 11—November 2021

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Jolianne Rijks, Dutch Wildlife Health Centre, Utrecht University, Androclus kamer O.177, Yalelaan 1, 3584CL Utrecht, the Netherlands

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