Zoonotic infection with swine A/H1avN1 influenza virus in a child, Germany, June 2020

A zoonotic A/sw/H1avN1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.

A zoonotic A/sw/H1 av N1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.
During routine surveillance at the National Influenza Centre in Germany in June 2020, a nasal swab was conspicuous because qPCR for the influenza A virus matrix protein (MP) and N1 neuraminidase (NA) genes were positive, whereas the haemagglutinin (HA) qPCR gave no results. The sample underwent whole genome sequencing and results pointed to a zoonotic influenza virus originating from swine. Here we describe the clinical features of the infection as well as the results of antigenic and genetic characterisation of this zoonotic influenza virus.

Description of the case and setting
The diagnostic sample originated from a 2.5-year-old child who lived on a farm, had regular contact with pigs, most recently 3 days before symptom onset, and was not vaccinated against influenza. The child had influenza-like illness over 3 days, displaying fever up to 40 °C, a sore throat, rhinorrhoea, headaches, myalgias, some fussiness and one episode of emesis, and slept a lot. Afterwards, they recovered quickly and fully. The child was not treated with antiviral drugs. No other family member, including the child's 5-month-old sibling, showed any symptoms, although some of them had been in close contact with the pigs. Four weeks later, 15 pigs of all age groups held at the farm and six family members were swabbed. All nasal swabs were negative, indicating absence of further virus circulation at this location. Four family members tested positive for rhinoviruses, but not the child who had had influenza. Because these swabs were qPCR-negative, virus isolation was not attempted from the pigs' swabs.
The pig herd of the farm has 600 fattening pigs. Every 4 weeks, 120 new pigs (ca 30 kg, 8-9-weeks-old) are introduced from another farm in Germany. The pig farm is situated outside of the village and no one except the farmer, his family and the veterinarian have access to it. The pig feed is generated by the farm from its own harvest. The pigs are not vaccinated against influenza. Two weeks before the child was infected, a new batch of pigs arrived at the farm. At that time, some pigs were displaying a cough, for which they were treated with antibiotics. Thus, the infection was most probably introduced to the herd via the new batch of pigs.

Antigenic characterisation
Virus isolation from the child's nasal swab was successful in MDCK-SIAT cells and embryonated hens' eggs. The virus was termed influenza A/Hessen/47/2020 (HES/2020). Antigenic characterisation showed that cross-reactivity was highest with swine hyperimmune serum directed against influenza A/sw/H1 av N1 virus (Table 1) [1]. Further investigations using ferret antisera demonstrated cross-reactivity with the wildtype and vaccine influenza A(H1N1)pdm09 viruses, but not with the previous seasonal influenza A(H1N1) viruses (i.e. those circulating before 2009).
Sequence analysis showed that the majority of HA antigenic sites were conserved between influenza A/ sw/H1 av N1 and A(H1N1)pdm09 viruses ( Table 2) [2]. In accordance with International Health Regulations, the case was reported to World Health Organization (WHO) via the Early Warning and Response System (EWRS) [3] and the virus was provided to the WHO Collaborating Centre London for further characterisation [4].

Resistance characterisation
While HES/2020 does not exhibit NA or PA mutations conferring resistance against neuraminidase inhibitors or baloxavir marboxil, its M2 sequence contains the AA substitutions L26I, V27A and S31N, all of which are associated with adamantane resistance (amantadine and rimantadine). Phenotypic susceptibility testing against oseltamivir, peramivir and zanamivir confirmed that HES/2020 was sensitive to all neuraminidase inhibitors authorised in Europe.

Discussion
This is the sixth zoonotic swine influenza virus infection in humans investigated at the German National Influenza Centre (in 2007: A/sw/H1 av N1 and A/sw/ H3N2 in Lower Saxony, in 2010: A/sw/H1 av N1 in Lower Saxony, in 2011: A/sw/H1 hu N2 and A/sw/H1 av N1 in Lower Saxony) [14]. Of the five previously reported cases, two occurred in children and one in an immunocompromised adult; influenza A/sw/H1 av N1 infections    were the most common [14]. All previous German cases were detected in Lower Saxony, the federal state with the second largest pig population in Germany. The case described here is the first from a region with a low density of pig holdings, i.e. Hesse.
Swine influenza viruses acquired adamantane resistance in the late 1980s [19]. The influenza A(H1N1) pdm09 virus contains the MP gene from A/sw/H1 av N1 viruses which confers adamantane resistance via the M2-S31N mutation in MP gene 2 [20]. This mutation was common in all seasonal influenza A viruses circulating globally during the last years [21]. In addition to S31N, HES/2020 contains the M2 AA substitutions L26I, V27A which are also associated with adamantane resistance. The M2-L26I and M2-V27A mutations can be found sporadically in influenza A viruses [21].
Swine influenza viruses have acquired some resistance genes against human myxovirus resistance protein MxA during their evolution in pigs, facilitating their transmission to humans [12]. Pig-to-human influenza virus transmissions are not rare, especially in close contact settings such as agricultural fairs [22], and sporadic zoonotic transmission of swine influenza A(H1N1) virus has been reported [23,24]. The farm child was the only member of his family who was infected, although some of the other family members had also been exposed. The infection of a child is not surprising. Because of their limited exposure history, young children display a narrower (if any) immune response to influenza virus than adults [25]. Our serology investigations indicate some level of cross-reactivity between influenza A(H1N1)pdm09 virus and A/sw/H1 av N1 viruses in ferrets. This is in line with previous findings that influenza A(H1N1)pdm09 infection induces broadly neutralising (not strain-specific) antibodies [26]. Antibodies against influenza A/ sw/H1 av N1 viruses in the human population are rare [27,28]. On the other hand, sera of human volunteers collected 3-7 weeks after vaccination with the annual 2017/18 vaccine all reflected antibodies against influenza A/sw/H1 av N1 virus at varying microneutralisation titres and none was negative [15]. Although the family members of the zoonotic case had not been vaccinated, they may have been exposed to human and swine influenza A viruses before, potentially resulting in pre-existing immunity which might impair transmission of influenza A/sw/H1 av N1 influenza virus.
However, the rising genetic diversity among swine influenza viruses, involving antigenic drift and shift, may increase divergence from influenza A/sw/H1 av N1 viruses in the future. In particular, swine reassortant viruses may quickly acquire antigenic changes, and this is where substantial zoonotic potential may arise.