Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic

Abstract

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and b-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

Manipulation of Spray-Drying Conditions to Develop an Inhalable Ivermectin Dry Powder

“…The susceptibility of the SARS-CoV-2 isolate hCoV-19/New Zealand/NZ1_patient/2020 [ 39 ] to (i) ivermectin, (ii) the ivermectin dry powder with L-leucine (F8) and (iii) L-leucine was evaluated in Calu-3 cells. Serial dilutions spanning empirically determined ranges of each compound were added in triplicate to 96-well plates containing Calu-3 cells (20,000 cells/well) and incubated at 37 °C, 5% CO 2 for two hours.…”

“…Virus inoculum was removed, cells washed twice and the complete medium with the corresponding agent dilution replenished. SARS-CoV-2 replication was quantified 72 h post-infection by CPE and a cell protection assay based on the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), as described [ 39 ]. Concentrations of the compounds required to inhibit SARS-CoV-2 replication by 50% (EC 50 ) were calculated by plotting the percent inhibition of virus replication versus log 10 drug concentration and fitting the inhibition curves to the data using nonlinear regression analysis (GraphPad Prism v.9.3.1, GraphPad Software).…”

Ultraviolet-C Irradiation, Heat, and Storage as Potential Methods of Inactivating SARS-CoV-2 and Bacterial Pathogens on Filtering Facepiece Respirators

“…PPE was in short supply across the globe, affecting the availability of this critical safety equipment in many countries, including New Zealand. The country was able to organize an good response to the COVID-19 pandemic, starting with a series of public health-measures [48,49], establishing molecular diagnostic assays [2][3][4], monitoring the SARS-CoV-2 variants in the region [6,50], and isolating the virus circulating in New Zealand [51]. However, New Zealand experienced the same PPE shortages as numerous other countries, and the NZ Office of the Auditor-General reported gaps in planning of PPE procurement and distribution, with insufficient national stock reserves (https: //oag.parliament.nz/, accessed on 26 October 2021).…”

“…All three cell lines were maintained in a humidified incubator with 5% CO 2 at 37 • C. The influenza A virus (IAV) A/Mallard/Alberta/287/2012 (H1N1) strain was donated by Dr. Richard J. Webby (St. Jude Children's Research Hospital, Memphis, TN, USA). The New Zealand SARS-CoV-2 NZ/Queenstown/01 strain was originally isolated by our group [51]. Virus stocks were prepared by growing IAV and SARS-CoV-2 in MDCK and Vero cells, respectively.…”

“…SARS-CoV-2 FFR coupons were inverted so that the inoculated face (dark-colored thread facing downwards) was in contact with the cell culture media of a matching 24-well plate containing 50,000 VeroE6/TMPRSS2 cells/well. Cells were incubated at 37 • C, 5% CO 2 for 72 h and SARS-CoV-2 replication quantified by determining cytopathic effect (CPE), as described [51]. 4.10.2.…”