Characterization of a pangolin SARS-CoV-2-related virus isolate that uses the human ACE2 receptor.

Various SARS-CoV-2-related coronaviruses have been increasingly identified in pangolins, showing a potential threat to humans. Here we report the infectivity and pathogenicity of the SARS-CoV-2-related virus, PCoV-GX/P2V, which was isolated from a Malayan pangolin (Manis javanica). PCoV-GX/P2V could grow in human hepatoma, colorectal adenocarcinoma cells, and human primary nasal epithelial cells. It replicated more efficiently in cells expressing human angiotensin-converting enzyme 2 (hACE2) as SARS-CoV-2 did. After intranasal inoculation to the hACE2-transgenic mice, PCoV-GX/P2V not only replicated in nasal turbinate and lungs, but also caused interstitial pneumonia, characterized by infiltration of mixed inflammatory cells and multifocal alveolar hemorrhage. Existing population immunity established by SARS-CoV-2 infection and vaccination may not protect people from PCoV-GX/P2V infection. These findings further verify the hACE2 utility of PCoV-GX/P2V by in vivo experiments using authentic viruses and highlight the importance for intensive surveillance to prevent possible cross-species transmission.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.

Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

[Coverage on cataract surgery among adults aged 40 or above in a multiethnic rural district of high altitude area in Gongshan county, Yunnan province, China].

OBJECTIVE: To estimate the prevalence of cataract and its surgical coverage rate together with the burden related to bilateral cataract-blindness, among adults aged 40 or above in Gongshan county of Yunnan province and to evaluate the current cataract status and the efficacy of local cataract prevention program. METHODS: Cluster sampling was used. The protocol consisted of personal interview, pilot study, visual acuity checking, measuring the intraocular pressure; slit lamp microscopy and the fundus of the eye examination etc. Cataract was graded clinically using the Lens Opacity Classification System (LOCS) III. Bilateral cataract-blindness burden, bilateral cataract-blindness burden and cataract surgical coverage rate were calculated respectively, using two different criteria. Odds ratios (OR) were compared among different groups regarding age, gender, education, ethnic group and altitude of living area. RESULTS: Among the 1236 eligible residents, 1116 (90.3%) were enrolled in the present study. The prevalence of cataract was 23.8% among adults aged 40 or order. When the bilateral best refractive vision<3/60 was defined as the blindness criterion, the bilateral cataract-blindness burden showed as 1.3%, and cataract blindness surgical coverage rate was 50.0%. When the bilateral presenting vision<6/60 was defined as the blindness criterion, the bilateral cataract-blindness burden was 25.0%, and cataract blindness surgical coverage rate was 12.9%. The cataract surgical coverage rates were much lower and the bilateral cataract-blindness burden much higher in women, illiterates, living in high altitude areas and those who were aged 70 or above. CONCLUSION: Cataract blindness was a serious public health problem in aged individuals and illiteracy in the residents of the studied areas. Poor prevention programs on cataract called for urgent action to be taken.

[Study of anterior chamber configuration with panoramic ultrasound biomicroscopy after cataract surgery with phacoemulsification and foldable intraocular lenses implantation].

OBJECTIVE: To study the changes in anterior chamber configuration after phacoemulsification and foldable intraocular lenses (IOL) implantation with panoramic ultrasound biomicroscopy (UBM). METHODS: A novel panoramic UBM was developed for anterior segment scanning. Small-incision phacoemulsification and foldable IOL implantation were performed in 102 eyes of 81 patients with age-related cataract. The anterior chamber diameter in horizontal meridian and vertical meridian and the anterior chamber depth (ACD) were measured by computer calipers before and two months after the surgery. The changes of the anterior chamber angle were measured in 36 eyes. RESULTS: Diameter of anterior chamber in vertical meridian was greater than that in horizontal meridian in most cases. In all patients, horizontal anterior chamber diameter was widened significantly two months after surgery (t = 7.10, P < 0.01), a significantly negative correlation existed between preoperative and postoperative data(r = 0.801, P < 0.01). The ACD was deepened ( t = 39.97, P < 0.01) . In 36 eyes, the anterior chamber angle was widened. CONCLUSIONS: Diameter of the anterior chamber in vertical meridian is greater than that in horizontal meridian in most cases. After phacoemulsification and foldable IOL implantation, UBM revealed that the anterior chamber diameter is increased and postoperative chamber diameter can be estimated by preoperative one. After the operation, the iris diaphragm shifts backward, the anterior chamber is deepened and anterior chamber angle is widened.