No evidence for zoonotic hepatitis E virus infection through dairy milk in Germany
Authors: Christine Baechlein, Paul Becher
Journal: Hepatology (2016)

Recently, Huang et al. reported that hepatitis E virus (HEV) genotype 4 (HEV-4) is highly prevalent in dairy cows in China and that infectious virus is excreted into milk.1 To follow up this topic, we analyzed bulk milk samples from Germany for the presence of HEV RNA. Milk samples had been collected in 2008 from dairy farms in Germany. Immediately after collecting, the bulk milk was supplemented with sodium azide or boric acid. After centrifugation, the liquid phase between the sediment and the creamy top was harvested and stored at –20°C. Here, 400 bulk milk samples from 400 independent dairy herds originating from eight different federal states were investigated for the presence of HEV RNA. Five independent bulk milk samples were pooled and viral RNA was extracted with the Viral RNA Mini Kit (Qiagen, Hilden, Germany). Real-time TaqMan reverse-transcriptase polymerase chain reaction (RT-PCR) was run with primers and probe described by Jothikumar et al. targeting an open reading frame 3 sequence highly conserved among HEV genotypes.2 By using an RNA copy standard, the detection limit was estimated at 14 genome equivalents. To analyze the assay's sensitivity and exclude a possible inhibitory effect of the milk on RNA detection, the supernatant of disrupted HEV-positive liver material from swine was diluted serially in phosphate-buffered saline (PBS) or in milk samples followed by RNA isolation and quantitative RT-PCR. Liver homogenate dilutions of up to 10–4, corresponding to 48-92 genome equivalents, were constantly detected out of milk or PBS, respectively. In addition, integrity of extracted RNA was proven by amplification of glyceraldehyde 3-phosphate dehydrogenase messenger RNA. In none of the investigated bulk milk samples could HEV-specific RNA be detected. Pigs represent the main reservoir for zoonotic HEV,3 but high HEV seroprevalence in the human population might be suggestive of further reservoir hosts.4 We used broadly binding primers and probe to ensure the detection of both zoonotic genotypes HEV-3 and HEV-4, but our ad-hoc investigation did not point at dairy cows to act as such a virus reservoir. Although infections with HEV-4 have been reported more frequently in the last years,5 HEV-3 is the predominant zoonotic genotype in Europe. At this time point, it can only be speculated whether among zoonotic genotypes exclusively HEV-4 is able to infect cattle. In their study, Huang et al. analyzed samples originating from a rural area, where cattle are often held in close contact to pigs implying cross-species transmission of HEV.1 Such mixed farming systems are not very common in Germany. Whether this separation of pork and milk production may prevent the establishment of another HEV reservoir in Europe will have to be elucidated in further studies. Christine Baechlein, Ph.D.1,2 Paul Becher, D.V.M.1,2 1Institute of Virology Department of Infectious Diseases University of Veterinary Medicine Hannover, Germany Hannover, Germany 2German Center for Infection Research Partner Site Hannover-Braunschweig Hannover, Germany Author names in bold designate shared co-first authorship.