Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.

The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

Influenza A(H1N1)pdm09 Virus Infection in a Captive Giant Panda, Hong Kong.

We report influenza A(H1N1)pdm09 virus infection in a captive giant panda in Hong Kong. The viral load peaked on day 1 and became undetectable on day 5, and an antibody response developed. Genome analysis showed 99.3%-99.9% nucleotide identity between the virus and influenza A(H1N1)pdm09 virus circulating in Hong Kong.

First Isolation and Rapid Identification of Newcastle Disease Virus from Aborted Fetus of Dromedary Camel Using Next-Generation Sequencing.

Newcastle disease virus (NDV) causes morbidities and mortalities in wild and domestic birds globally. For humans, exposure to infected birds can cause conjunctivitis and influenza-like symptoms. NDV infections in mammals are rarely reported. In this study, using next-generation sequencing, an NDV was identified and isolated from Vero cells inoculated with the nasal swab of an aborted dromedary fetus in Dubai, during the time when an NDV outbreak occurred in a pigeon farm located in close proximity to the dairy camel farm where the mother of the aborted dromedary fetus resided, and there were a lot of pigeons in the camel farm. Genome analysis revealed that the structurally and functionally important features of other NDVs were also present in this dromedary NDV genome. Phylogenetic analysis based on the nucleotide sequences of fusion protein (F), hemagglutinin-neuraminidase protein (HN) and complete polyprotein showed that the virus belonged to sub-genotype VIg of class II NDV and is most closely related to pigeon NDVs in Egypt in the same year. The present study is the first that demonstrated isolation of NDV in dromedaries. Further study is warranted to investigate the relationship between NDV infection and abortion.

Metagenomic analysis of Sichuan takin fecal sample viromes reveals novel enterovirus and astrovirus.

The Sichuan takin inhabits the bamboo forests in the Eastern Himalayas and is considered as a national treasure of China with the highest legal protection and conservation status considered as vulnerable according to The IUCN Red List of Threatened Species. In this study, fecal samples of 71 Sichuan takins were pooled and deep sequenced. Among the 103,553 viral sequences, 21,961 were assigned to mammalian viruses. De novo assembly revealed genomes of an enterovirus and an astrovirus and contigs of circoviruses and genogroup I picobirnaviruses. Complete genome sequencing and phylogenetic analysis showed that Sichuan takin enterovirus is a novel serotype/genotype of the species Enterovirus G, with evidence of recombination. Sichuan takin astrovirus is a new subtype of bovine astrovirus, probably belonging to a new genogroup in the genus Mamastrovirus. Further studies will reveal whether these viruses can also be found in Mishmi takin and Shaanxi takin and their pathogenic potentials.