Pigs are important intermediate hosts for generating novel influenza viruses. The Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses (SIVs) have circulated in pigs since 1979, and human cases associated with EAH1N1 SIVs have been reported in several countries. However, the biologic properties of EAH1N1 SIVs are largely unknown. Here, we performed extensive influenza surveillance in pigs in China and isolated 228 influenza viruses from 36,417 pigs. We found that 139 of the 228 strains from pigs in 10 provinces in China belong to the EAH1N1 lineage. These viruses formed five genotypes, with two distinct antigenic groups, represented by A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013, both of which are antigenically and genetically distinct from the current human H1N1 viruses. Importantly, the EAH1N1 SIVs preferentially bound to human-type receptors, and 9 of the 10 tested viruses transmitted in ferrets by respiratory droplet. We found that 3.6% of children (≤10 y old), 0% of adults, and 13.4% of elderly adults (≥60 y old) had neutralization antibodies (titers ≥40 in children and ≥80 in adults) against the EAH1N1 A/swine/Guangxi/18/2011 virus, but none of them had such neutralization antibodies against the EAH1N1 A/swine/Guangdong/104/2013 virus. Our study shows the potential of EAH1N1 SIVs to transmit efficiently in humans and suggests that immediate action is needed to prevent the efficient transmission of EAH1N1 SIVs to humans.
Fig. 4. Opposing effects of TCDD and EGF on differentiation of NHEKs. (A)NHEKs were grown to confluence, and basal medium, or medium with ␣-naphthoflavone (NF, 1 M) was added 24 h before treatment. CEs were isolated after treatment with either 0.1% DMSO or TCDD (10 nM) for 5 days. (B) NHEKs were grown to confluence, and basal medium with or without EGF (10 ng/mL) was added 24 h before treatment. CEs were isolated after treatment with either 0.1% DMSO or TCDD (10 nM) for 5 days. (C) NHEKs were grown to confluence, and basal medium, or medium with EGF (10 ng/mL), was added 24 h before treatment. CEs were isolated after treatment with either 0.1% DMSO or TCDD (10 nM) in the presence or absence of PD153035 (300 nM) for 5 days. (A-C) The values for CEs are a mean of triplicate samples Ϯ SD. (D-G) NHEKs were grown to a cell density of either 50% or 100% confluence before basal medium, or medium with EGF (10 ng/mL), was added for 24 h before treatment. Total mRNA was isolated after treatment with either control vehicle (0.1% DMSO) or TCDD (10 nM) for 24 h. Real-time PCR was used to determine the relative expression of each indicated gene (y axis). Levels of mRNA [mean (n ϭ 3) Ϯ SD] are expressed in units relative to the minimum, given a value of 1. (A-G) The a indicates that the value from treatment with TCDD is significantly different from the DMSO control; the b indicates that the value from cotreatment with TCDD and NF is significantly different from TCDD alone; the c indicates that the value from cotreatment with TCDD and EGF is significantly different from with TCDD alone; the d indicates that the value from treatment with TCDD, EGF, and PD153035 is significantly different from treatment with TCDD and EGF; and the e indicates that treatment with EGF is significantly different from DMSO control treatment. For each of these comparisons, P Յ 0.01 by Student's t test. Comparisons made in D-G are within the group grown to a cell density of 100% confluence.
We have studied the occurrence of enterovirus (EV)-RNA at the onset of childhood type 1 diabetes in all 24 new cases of childhood type 1 diabetes during 1 year in Uppsala county, Sweden. We also studied 24 matched control subjects and 20 siblings of the patients. RNA was isolated from peripheral blood mononuclear cells and EV-RNA detected by RT-PCR. Primers (groups A and B) corresponding to conserved regions in the 5 noncoding region (NCR) of EV were used in the PCRs, and the amplicons were sequenced. By the use of group A primers, EV-RNA was found in 12 (50%) of the 24 type 1 diabetic children, 5 (26%) of 19 siblings, and none of the control subjects. Both patients and siblings showed a higher frequency of EV-RNA compared with the control subjects. The group B primers detected EV-RNA in all three groups but did not show statistically significant differences between the groups. The EV-RNA positivity with the group B primers was 11 (46%) of 24 in the type 1 diabetic children, 11 (58%) of 19 in the siblings, and 7 (29%) of 24 in the control subjects. The significant difference between groups seen with the group A primers but not with the group B primers might indicate the existence of diabetogenic EV strains. The phylogenetic analysis of the PCR products revealed clustering of the sequences from patients and siblings into five major branches when the group A PCR primers were used. With the group B primers, the sequences from patients, siblings, and control subjects formed three major branches in the phylogenetic tree, where 6 of the 7 control subjects clustered together in a subbranch of CBV-4/VD2921. Seven of the type 1 diabetic children clustered together in another sub-branch of CBV-4/VD2921. Five of the type 1 diabetic children formed a branch together with the CBV-4/E2 strain, four clustered together with CBV-5, and one formed a branch with echovirus serotype. The presence of EV-RNA in the blood cells of most newly diagnosed type 1 diabetic children supports the hypothesis that a viral infection acts as an exogenous factor. In addition, sequencing of the PCR amplicons from the type 1 diabetic children, their siblings, and matched control subjects might reveal differences related to diabetogenic properties of such a virus.
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