China reported the world’s first human infection of the H10N3 bird flu strain on Tuesday but said the risk of it spreading widely among people was low.
A 41-year-old man was admitted to hospital with fever symptoms in the eastern city of Zhenjiang on April 28 and was diagnosed with H10N3 a month later, China’s National Health Commission (NHC) said in an online statement.
“The risk of large-scale spread is extremely low,” the NHC said, adding that the man was in a stable condition and his close contacts had reported no “abnormalities.”
The NHC said there had been no human cases of H10N3 previously reported in the world.
The last human epidemic of bird flu in China occurred in late 2016 to 2017, with the H7N9 virus.
Following recent avian flu outbreaks in Africa and Eurasia, the head of China’s Centre for Disease Control and Prevention last week urged stricter surveillance in poultry farms, markets and wild birds.
H10 subtype influenza viruses have been isolated in various species of waterfowl across worldwide geographic areas for more than 50 years1,2,3. The viruses remain avian receptor binding, however, some strains are highly pathogenic to chickens, even though they lack multiple basic amino acids at the hemagglutinin cleavage site4,5,6,7.
H10 viruses occasionally infect humans. An H10N3 virus was isolated in Hong Kong in 19798, and in a live-bird market in Thailand in 20119. However, pathogenicity in mammals due to H10N3 viruses remains largely unclear. The first H10N7 isolate was identified in chickens in Germany10. In 2010, an H10N7 strain caused disease in a chicken farm in Australia11. Recently, an H10N7 virus was isolated from dead harbor seals in Denmark12.
A novel reassortant H10N7 AIV was found in chickens in Eastern China11,12,13,14,15,16,17,18,19,20,21,22,23. Additionally, an H10N4 isolate caused an outbreak of respiratory disease in mink in Sweden15. H10N5 virus was detected in pigs in 200824.
Human infections with H10N8 subtype avian influenza virus (AIV) were reported in Jiangxi province, China, in 2013–201425. Sequencing these viruses showed that all six internal segments were from the H9N2 subtype G57 genotype26. Transmission of this subtype from avian species to humans increases the risk of adaptive point mutations or reassortment events with H7N9, H9N2 subtype AIV, or human seasonal viruses, which could be the source of a highly pandemic virus27,28.
The H10N8 virus also showed high pathogenicity in mice29,30. A subsequent surveillance study also showed the presence of H10N8 in waterfowls, feral dogs, and live poultry markets (LPMs)26,27,31,32. While multiple H10 genotype viruses (e.g. H10N8, H10N3, and H10N7) are circulating in LPMs in China, their potential to infect mammals remains largely unknown.
To address this question, three H10N8, H10N7, and H10N3 subtype influenza viruses circulating in domestic ducks were characterized in this study. We found that their complex reassortments and pathobiology patterns in chickens, ducks, and mice indicates a potential threat to humans.
Complex reassortment patterns of the three H10 subtype influenza viruses
Three strains of H10 subtype avian influenza virus were isolated from healthy domestic ducks in different provinces of China (Table 1). The isolates were designated as A/duck/Shanghai/602/2009 (H10N8) (thereafter SH602/H10N8), A/duck/Fujian/1761/2010 (H10N3) (thereafter FJ1761/H10N3), and A/duck/Shanxi/3180/2010 (H10N7) (thereafter SX3180/H10N7).
H10 subtype AIV isolates.
|Isolates||Subtype||Abbr.||Viral titers (EID50/ml)||IVPI*||Genomic Accession Number|
|A/duck/Shanghai/602/2009||H10N8||SH602||1.00 × 108||0.39||KU921391 (PB2); KU921394 (PB1); KU921397 (PA); KU921400 (HA); KU921403 (NP); KU921406 (NA); KU921409 (M); KU921412 (NS)|
|A/duck/Fujian/1761/2010||H10N3||FJ1761||5.62 × 108||1.60||KU921392 (PB2); KU921395 (PB1); KU921398 (PA); KU921401 (HA); KU921404 (NP); KU921407 (NA); KU921410 (M); KU921413 (NS)|
|A/duck/Shanxi/3180/2010||H10N7||SX3180||3.16 × 108||1.27*||KU921393 (PB2); KU921396 (PB1); KU921399 (PA); KU921402 (HA); KU921405 (NP); KU921408 (NA); KU921411 (M); KU921414 (NS)|
*Note: Exceptionally viruses of H10 subtype have given IVPI’s marginally in excess of 1.20 and would, according to the European Union definition, be classified as highly pathogenic irrespective of the amino acid motif at the cleavage site39.
To characterize the molecular evolution of the three H10 viruses, the full-length genomes of the serially purified H10 viruses were sequenced and analyzed by using RT-PCR (Table 1).
In the phylogenetic tree of HA sequences, these viruses comprise different sublineages of the Eurasian lineage. H10N3 fell in the Europe sublineage, and H10N7 and H10N8 were located in the JX346-like (Asian) sublineage, which also contains H10N8 viruses (Fig. 1A). The three H10 isolates shared the amino acid sequence (PEIMQGRGLFG) at the cleavage site between HA1 and HA2, indicating they are low pathogenic strains. The amino acids 95Y, 151W, 183H, 190E, 191K, 194L, 226Q, 227S, 228G, and 229R were observed at the receptor-binding pocket area of all 3 viruses. None of these residues have been reported to be involved in the recognition of human-type receptors, suggesting that all the isolates likely bind to avian-like receptors30.
All the isolates are likely susceptible to neuraminidase inhibitors (Oseltamivir, Zanamivir, and Peramivir) based upon their NA amino acid sequences33. In the phylogenetic trees of NA genes, evolution of the three strains showed significant differences (Fig. 1B). SH602/H10N8 reassorted with a strain from an American lineage, closely related to A/duck/Beijing/33/04 (H3N8)25. FJ1761/H10N3 reassorted with A/duck/Zhejiang/12/2011 (H7N3), which has been classified in the Eurasian lineage34. SX3180/H10N7 reassorted with A/mallard/Netherlands/2/2009 (H7N7) in the Eurasian lineage.
The PB2 segment of FJ1761/H10N3 seems to be derived from a highly pathogenic H5N1 strain (Fig. 1C). However, the PB2 segments of SH602/H10N8 and SX3180/H10N7 viruses might be derived from different H4N6-like strains isolated from Mongolia or China, respectively (Fig. 1C).
For PB1 and PA, FJ1761/H10N3 virus showed a unique reassortment pattern, in that the PB1 and PA segments were not from H4N6 subtype viruses (Supplementary Fig. 1A,B), but were instead derived from an H7N3 subtype AIV in Eastern China, very closely related to A/duck/Zhejiang/12/2011 (H7N3), which is also a donor for H7N9 AIV in humans34. For the NP segment, SX3180/H10N7 and FJ171/H10N3 viruses fell into an H7N3-like group, but only NP of SH602/H10N8 was from H4N6 subtype AIV (Supplementary Fig. 1C). The M and NS segments of all the three viruses appear to originate from a Korean H4N6-like subtype AIV isolated from wild ducks (Supplementary Fig. 1D,E)35.
Amino acids E627 and D701 were found in PB2 of all three H10 isolates, which suggests that the 3 isolates are poorly adapted to mammals29. Amino acids L26, V27, A30, and S31 in the M2 protein confer no resistance to M2 ion channel drugs36,37. The three H10 viruses bear an ESEV motif in their NS1 carboxy termini,. indicating an H5N1-like PDZ domain related to virulence38.
The pathogenicity of H10 viruses vary in chickens
To determine the pathogenicity of the H10 viruses in chickens, the virus stocks were purified three times by end-point infection. All three H10 viruses replicated to high titers in eggs (Table 1). The viruses were injected into the veins of chickens and 10 days later the intravenous pathogenicity indices were calculated. The H10 viruses varied in pathogenicity to chickens. SH602/H10N8 is a lentogenic strain with an IVPI value of 0.39, and FJ1761/H10N3 and SX3180/H10N7 are highly pathogenic to chickens with IVPI values of 1.60 and 1.27 respectively39 (Table 1).
To characterize further the virulence and transmissibility of the H10 viruses in chickens, 4-week-old SPF chickens (10/group) were intranasally inoculated with 100 μl virus stocks at a dose of 106 EID50. The titers of all the oralpharyngeal and the cloaca swabs in the SH602/H10N8 virus groups were below the detection limit at 3 dpi. However, a sight difference was observed between the FJ1761/H10N3 and SX3180/H10N7 viruses. In FJ1761/H10N3 virus – infected chickens, 3/10 oralpharyngeal swabs were positive with low titers of 10, 178, and 316 EID50/ml. In the cloaca swabs for this virus, only one was positive with a low titer of 316 EID50/ml. A low transmission capability of FJ1761/H10N3 was observed found in one of six oralpharyngeal swabs in the contact group (FJ1761-C group) with a low titer (10 EID50/ml). No virus was found in the cloaca swabs of the FJ1761-C group. For SX3180/H10N7-infected chickens, one was positive with a high titer of 1.78 × 103 EID50/ml and another was positive without dilution (10 EID50/mL). No virus was found in the contact group (SX3180-C) (Fig. 2A).
At 3 and 5 dpi, three chickens from each group were euthanized. No lesions were observed. No viruses were found in the chicken lungs by either titration or RT-PCR analysis. No significant pathology was observed in the lungs after H & E staining. The sera of the remaining chickens were collected for hemagglutination inhibition (HI) assays at 3, 5, and 14 dpi. Except for the titers under the detection limit at 3 dpi and 5 dpi, the sera were positive at 14 dpi. Three of four serum samples were positive with titers of 64, 128, and 128 in the SH602-I group, but the sera of the SH602-C group were under the detection limit. In the FJ1761-I group, all the HI titers were positive with titers of 32, 64, 128, and 256. In the FJ1761-C group, three sera samples were positive with titers of 128. In the SX3180-I group, the HI titers were also positive with HI titers of 32, 64, 128, and 256. In the SX3180-C group, three sera samples were positive with titers of 32, 64, and 128. Thus, FJ1761/H10N3 and SX3180/H10N7 viruses infect and are transmitted between chickens, whereas SH602/H10N8 virus does not (Fig. 2B).
The H10 viruses were avirulent but transmissible in ducks
At 3 dpi of oralpharyngeal swabs, 70% of samples in SH602/H10N8-infected ducks (SH602-I group) were positive but with low titers (<20 EID50/ml), in which the highest titer was 316 EID50/ml. Four out of six oralpharyngeal swabs in the SH602-C group were positive. For the FJ1761-I group at 3 dpi, 50% oralpharyngeal swabs were positive, but with titers less than 50 EID50/ml, in which the highest titer was 316 EID50/ml. However, the virus was not detected in the oralpharyngeal swabs of the FJ1761-C group. For SX3180/H10N7, only one sample was positive with a titer of 178 EID50/ml. No virus was detected in the oralpharyngeal swabs of SX3180-C group (Fig. 3A).
A greater percentage of cloacal swabs were positive at 3 dpi, suggesting that the viruses may be transmitted by the fecal-oral route. In the SH602-I group, 100% of samples were positive but at titers less than 100 EID50/ml, in which the highest titer was 1.78 × 103 EID50/ml. All samples in the SH602-C group were positive (<100 EID50/ml), in which the highest titer was 562 EID50/ml. The titers of the FJ1761-I group at 3 dpi were higher than in the SH602-I group; 100% of oralpharyngeal swabs were positive with a mean titer of 282 EID50/ml, in which the highest titer was 1.78 × 104 EID50/ml. All swabs of the FJ1761-C group were positive with a mean titer of 102.13 EID50/ml, in which the highest titer was 3.16 × 103 EID50/ml. For SX3180/H10N7, 50% of samples were positive but at titers less than 100 EID50/ml. Two out of six samples were positive in the oralpharyngeal swabs of the SX3180-C group (Fig. 3A).
The titers of the H10 viruses were lower in oralpharyngeal swabs at 5 dpi. Only one of seven samples was positive in the SH602-I and FJ1761-I groups, two of six samples were positive in the SH602-C group, and four of six samples were positive in the FJ1761-C group. Two of seven samples were positive in the SX3180-I group and three of four samples were positive in SX3180-C group (Fig. 3B).
However, the virus titers of cloaca swabs increased at 5 dpi. Five of seven samples were positive in the SH602-I, FJ1761-I, and SX3180-I groups with mean titers of 501, 112, and 79 EID50/ml, respectively. Five of six samples were positive in the SH602-C group with the higher titer of 1.12 × 103 EID50/ml, and 100% samples were positive with titers of 135 and 380 EID50/ml for FJ1761-C and SX3180 group, respectively (Fig. 3B).
The remaining seven ducks, including four inoculated and three contact ducks in each group were monitored daily for clinical signs. All survived the 14-day observation period. The ducks were euthanized at 3, 5 and 14 dpi and serum was collected for HI test. HI titers at 14 dpi were less than 32 in all groups (Fig. 3C), No positive HI reactions were observed for sera collected at 3 and 5 dpi.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039634/