ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

The SARS-CoV-2 virus emerged in December 2019 and has caused a worldwide pandemic due to the lack of any pre-existing immunity. Accurate serology testing is urgently needed to help diagnose infection, determine past exposure of populations and assess the response to a future vaccine. The landscape of antibody responses to SARS-CoV-2 is unknown. In this study, we utilized the luciferase immunoprecipitation system to assess the antibody responses to 15 different SARS-CoV-2 antigens in patients with COVID-19. We identified new targets of the immune response to SARS-CoV-2 and show that nucleocapsid, open reading frame (ORF)8 and ORF3b elicit the strongest specific antibody responses. ORF8 and ORF3b antibodies, taken together as a cluster of points, identified 96.5% of COVID-19 samples at early and late time points of disease with 99.5% specificity. Our findings could be used to develop second-generation diagnostic tests to improve serological assays for COVID-19 and are important in understanding pathogenicity.

Author Correction: ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Infection of dogs with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in Wuhan in December 2019 and caused coronavirus disease 2019 (COVID-19)1,2. In 2003, the closely related SARS-CoV had been detected in domestic cats and a dog3. However, little is known about the susceptibility of domestic pet mammals to SARS-CoV-2. Here, using PCR with reverse transcription, serology, sequencing the viral genome and virus isolation, we show that 2 out of 15 dogs from households with confirmed human cases of COVID-19 in Hong Kong were found to be infected with SARS-CoV-2. SARS-CoV-2 RNA was detected in five nasal swabs collected over a 13-day period from a 17-year-old neutered male Pomeranian. A 2.5-year-old male German shepherd was positive for SARS-CoV-2 RNA on two occasions and virus was isolated from nasal and oral swabs. Antibody responses were detected in both dogs using plaque-reduction-neutralization assays. Viral genetic sequences of viruses from the two dogs were identical to the virus detected in the respective human cases. The dogs remained asymptomatic during quarantine. The evidence suggests that these are instances of human-to-animal transmission of SARS-CoV-2. It is unclear whether infected dogs can transmit the virus to other animals or back to humans.

Amino acid substitution L232F in non-structural protein 6 identified as a possible human-adaptive mutation in clade B MERS coronaviruses.

IMPORTANCE: Viral host adaptation plays an important role in inter-species transmission of coronaviruses and influenza viruses. Multiple human-adaptive mutations have been identified in influenza viruses but not so far in MERS-CoV that circulates widely in dromedary camels in the Arabian Peninsula leading to zoonotic transmission. Here, we analyzed clade B MERS-CoV sequences and identified an amino acid substitution L232F in nsp6 that repeatedly occurs in human MERS-CoV. Using a loss-of-function reverse genetics approach, we found the nsp6 L232F conferred increased viral replication competence in vitro, in cultures of the upper human respiratory tract ex vivo, and in lungs of mice infected in vivo. Our results showed that nsp6 L232F may be an adaptive mutation associated with zoonotic transmission of MERS-CoV. This study highlighted the capacity of MERS-CoV to adapt to transmission to humans and also the need for continued surveillance of MERS-CoV in camels.

Decreased risk of non-influenza respiratory infection after influenza B virus infection in children.

Previous studies suggest that influenza virus infection may provide temporary non-specific immunity and hence lower the risk of non-influenza respiratory virus infection. In a randomized controlled trial of influenza vaccination, 1 330 children were followed-up in 2009-2011. Respiratory swabs were collected when they reported acute respiratory illness and tested against influenza and other respiratory viruses. We used Poisson regression to compare the incidence of non-influenza respiratory virus infection before and after influenza virus infection. Based on 52 children with influenza B virus infection, the incidence rate ratio (IRR) of non-influenza respiratory virus infection after influenza virus infection was 0.47 (95% confidence interval: 0.27-0.82) compared with before infection. Simulation suggested that this IRR was 0.87 if the temporary protection did not exist. We identified a decreased risk of non-influenza respiratory virus infection after influenza B virus infection in children. Further investigation is needed to determine if this decreased risk could be attributed to temporary non-specific immunity acquired from influenza virus infection.

Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Monitoring International Travelers Arriving in Hong Kong for Genomic Surveillance of SARS-CoV-2.

We sequenced ≈50% of coronavirus disease cases imported to Hong Kong during March-July 2021 and identified 70 cases caused by Delta variants of severe acute respiratory syndrome coronavirus 2. The genomic diversity detected in Hong Kong was similar to global diversity, suggesting travel hubs can play a substantial role in surveillance.

Viral RNA and Infectious Influenza Virus on Mobile Phones of Patients With Influenza in Hong Kong and the United States.

Mobile phones are among the most highly touched personal objects. As part of a broader study on the contribution of fomites to influenza transmission, between 2017 and 2019, we swabbed mobile phones from 138 patients with influenza in 2 locations. Influenza viral RNA detection rates were 23% (23 of 99 phones) and 36% (14 of 39) in Hong Kong and Maryland, respectively. In Hong Kong, infectious influenza virus was recovered from 3 of 23 mobile phones which had influenza viral RNA detected. Mobile phone influenza contamination was positively associated with upper respiratory tract viral load and negatively associated with age. Cleaning personal objects of patients with influenza should be recommended, and individuals should avoid sharing objects with these patients.

SARS-CoV-2 Superspread in Fitness Center, Hong Kong, China, March 2021.

To investigate a superspreading event at a fitness center in Hong Kong, China, we used genomic sequencing to analyze 102 reverse transcription PCR-confirmed cases of severe acute respiratory syndrome coronavirus 2 infection. Our finding highlights the risk for virus transmission in confined spaces with poor ventilation and limited public health interventions.

Risk Assessment for Highly Pathogenic Avian Influenza A(H5N6/H5N8) Clade 2.3.4.4 Viruses.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Genetic Diversity of SARS-CoV-2 among Travelers Arriving in Hong Kong.

We sequenced 10% of imported severe acute respiratory syndrome coronavirus 2 infections detected in travelers to Hong Kong and revealed the genomic diversity of regions of origin, including lineages not previously reported from those countries. Our results suggest that international or regional travel hubs might be useful surveillance sites to monitor sequence diversity.

Introduction of ORF3a-Q57H SARS-CoV-2 Variant Causing Fourth Epidemic Wave of COVID-19, Hong Kong, China.

We describe an introduction of clade GH severe acute respiratory syndrome coronavirus 2 causing a fourth wave of coronavirus disease in Hong Kong. The virus has an ORF3a-Q57H mutation, causing truncation of ORF3b. This virus evades induction of cytokine, chemokine, and interferon-stimulated gene expression in primary human respiratory cells.

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface.

Presence of Influenza Virus on Touch Surfaces in Kindergartens and Primary Schools.

BACKGROUNDS: Influenza virus can survive on some surfaces, facilitating indirect person-to-person transmission. METHODS: We collected swab samples weekly from commonly touched surfaces in 7 kindergartens and primary schools during the 2017/2018 winter influenza season in Hong Kong. RESULTS: We detected influenza virus ribonucleic acid (RNA) in 12 of 1352 samples (<1%) collected from 7 of 11 classrooms (5 to 2 × 106 RNA copies/mL). Viral RNA was more frequently recovered from communal items inside classrooms such as bookshelves and doorknobs. CONCLUSIONS: Surface contamination indicates the potential role of fomites in influenza virus transmission in schools. Communal items inside classrooms may cause greater potential risks of transmission during influenza epidemics.

Maternal Antibodies Against Influenza in Cord Blood and Protection Against Laboratory-Confirmed Influenza in Infants.

BACKGROUND: Studies that correlate maternal antibodies with protection from influenza A or B virus infection in young infants in areas with prolonged influenza circulation are lacking. METHODS: We conducted a prospective, observational study to evaluate the effects of maternally transferred antibodies against influenza A and B viruses against laboratory-confirmed influenza in a cohort born over 24 months. Cord blood samples were retrieved at birth and infants were actively followed for the first 6 months of life. Nasal swabs were collected and tested for influenza A and B by reverse transcriptase-polymerase chain reaction whenever an illness episode was identified. Cord blood samples were tested by the hemagglutination inhibition (HAI) assay to viruses that circulated during the follow-up period. RESULTS: 1162 infants were born to 1140 recruited women: 1092 (94%) infants completed 6 months of follow-up. Proportions of cord blood with HAI antibody titers ≥40 against A(H1N1), A(H3N2), B/Victoria, and B/Yamagata were 31%, 24%, 31%, and 54%, respectively. Only 4% of women had maternal influenza vaccination. Cord blood antigen-specific HAI titers ≥40 were found to correlate with protection from infection only for influenza B/Yamagata. No influenza B virus infection occurred in infants ≤60 days old. Proportional hazards analysis showed that a cord blood HAI titer of 40 was associated with 83% (95% confidence interval, 44-95%) reduction in the risk of influenza B/Yamagata infections compared with a cord blood titer <10. CONCLUSIONS: We documented that maternal immunity against influenza B/Yamagata was conferred to infants within the first 6 months of life.

Phylogenetic Analysis of MERS-CoV in a Camel Abattoir, Saudi Arabia, 2016-2018.

We detected Middle East respiratory syndrome coronavirus (MERS-CoV) RNA in 305/1,131 (27%) camels tested at an abattoir in Al Hasa, Eastern Province, Saudi Arabia, during January 2016-March 2018. We characterized 48 full-length MERS-CoV genomes and noted the viruses clustered in MERS-CoV lineage 5 clade B.

In-Flight Transmission of SARS-CoV-2.

Four persons with severe acute respiratory syndrome coronavirus 2 infection had traveled on the same flight from Boston, Massachusetts, USA, to Hong Kong, China. Their virus genetic sequences are identical, unique, and belong to a clade not previously identified in Hong Kong, which strongly suggests that the virus can be transmitted during air travel.

SARS-CoV-2 in Quarantined Domestic Cats from COVID-19 Households or Close Contacts, Hong Kong, China.

We tested 50 cats from coronavirus disease households or close contacts in Hong Kong, China, for severe acute respiratory syndrome coronavirus 2 RNA in respiratory and fecal samples. We found 6 cases of apparent human-to-feline transmission involving healthy cats. Virus genomes sequenced from 1 cat and its owner were identical.

SARS-CoV-2 Virus Culture and Subgenomic RNA for Respiratory Specimens from Patients with Mild Coronavirus Disease.

We investigated 68 respiratory specimens from 35 coronavirus disease patients in Hong Kong, of whom 32 had mild disease. We found that severe acute respiratory syndrome coronavirus 2 and subgenomic RNA were rarely detectable beyond 8 days after onset of illness. However, virus RNA was detectable for many weeks by reverse transcription PCR.

Influenza A Virus Infections in Dromedary Camels, Nigeria and Ethiopia, 2015-2017.

We examined nasal swabs and serum samples acquired from dromedary camels in Nigeria and Ethiopia during 2015-2017 for evidence of influenza virus infection. We detected antibodies against influenza A(H1N1) and A(H3N2) viruses and isolated an influenza A(H1N1)pdm09-like virus from a camel in Nigeria. Influenza surveillance in dromedary camels is needed.