Longitudinal Screening of Retail Milk from Canadian Provinces Reveals No Detections of Influenza A Virus RNA (April - July 2024): Leveraging a Newly-Established Pan-Canadian Network for Responding to Emerging Viruses.

Highly pathogenic avian influenza (HPAI) H5N1 has been associated with significant morbidity and mortality. Given recent detections of HPAI H5N1 in dairy cattle and RNA detections in pasteurized retail milk in the United States, we established the Pan-Canadian Milk Network in April 2024. Through this, retail milk was procured longitudinally and sent to a central laboratory to test for the presence of influenza A virus RNA. We tested 109 retail milk samples from all ten Canadian provinces and all samples tested negative. Our independent testing results have aligned with reporting from federal retail milk testing initiatives. Despite no known HPAI infections of dairy cattle in Canada to date, H5N1 poses a significant threat to the health of both humans and other animals. By performing routine surveillance of retail milk on a national scale, we have shown that academic networks and initiatives can rapidly establish nationwide emerging infectious disease surveillance that is cost-effective, standardized, scalable, and easily accessible. Our network can serve as an early detection system to help inform containment and mitigation activities if positive samples are identified and can be readily reactivated should H5N1 or other emerging zoonotic viruses be identified in agricultural or livestock settings.

California Serogroup Viruses in a Changing Canadian Arctic: A Review.

The Arctic is warming at four times the global rate, changing the diversity, activity and distribution of vectors and associated pathogens. While the Arctic is not often considered a hotbed of vector-borne diseases, Jamestown Canyon virus (JCV) and Snowshoe Hare virus (SSHV) are mosquito-borne zoonotic viruses of the California serogroup endemic to the Canadian North. The viruses are maintained by transovarial transmission in vectors and circulate among vertebrate hosts, both of which are not well characterized in Arctic regions. While most human infections are subclinical or mild, serious cases occur, and both JCV and SSHV have recently been identified as leading causes of arbovirus-associated neurological diseases in North America. Consequently, both viruses are currently recognised as neglected and emerging viruses of public health concern. This review aims to summarise previous findings in the region regarding the enzootic transmission cycle of both viruses. We identify key gaps and approaches needed to critically evaluate, detect, and model the effects of climate change on these uniquely northern viruses. Based on limited data, we predict that (1) these northern adapted viruses will increase their range northwards, but not lose range at their southern limits, (2) undergo more rapid amplification and amplified transmission in endemic regions for longer vector-biting seasons, (3) take advantage of northward shifts of hosts and vectors, and (4) increase bite rates following an increase in the availability of breeding sites, along with phenological synchrony between the reproduction cycle of theorized reservoirs (such as caribou calving) and mosquito emergence.

Association Between Human Immunodeficiency Virus Viremia and Compromised Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may be associated with worse clinical outcomes in people with human immunodeficiency virus (HIV) (PWH). We report anti-SARS-CoV-2 antibody responses in patients hospitalized with coronavirus disease 2019 in Durban, South Africa, during the second SARS-CoV-2 infection wave dominated by the Beta (B.1.351) variant. METHODS: Thirty-four participants with confirmed SARS-CoV-2 infection were followed up with weekly blood sampling to examine antibody levels and neutralization potency against SARS-CoV-2 variants. Participants included 18 PWH, of whom 11 were HIV viremic. RESULTS: SARS-CoV-2-specific antibody concentrations were generally lower in viremic PWH than in virologically suppressed PWH and HIV-negative participants, and neutralization of the Beta variant was 4.9-fold lower in viremic PWH. Most HIV-negative participants and antiretroviral therapy-suppressed PWH also neutralized the Delta (B.1.617.2) variant, whereas the majority of viremic PWH did not. CD4 cell counts <500/µL were associated with lower frequencies of immunoglobulin G and A seroconversion. In addition, there was a high correlation between a surrogate virus neutralization test and live virus neutralization against ancestral SARS-CoV-2 virus in both PWH and HIV-negative individuals, but correlation decreased for the Beta variant neutralization in PWH. CONCLUSIONS: HIV viremia was associated with reduced Beta variant neutralization. This highlights the importance of HIV suppression in maintaining an effective SARS-CoV-2 neutralization response.

HIV skews the SARS-CoV-2 B cell response towards an extrafollicular maturation pathway.

Background: HIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. Methods: We compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing, and regulatory features. Results: This revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal centre (GC) activity, homing capacity, and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated naïve B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2-specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. Conclusions: Despite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge. Funding: This work was supported by a grant from the Wellcome Trust to the Africa Health Research Institute (Wellcome Trust Strategic Core Award [grant number 201433/Z/16/Z]). Additional funding was received from the South African Department of Science and Innovation through the National Research Foundation (South African Research Chairs Initiative [grant number 64809]), and the Victor Daitz Foundation.

Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014-2018).

Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014-2018 were investigated. In total, 69/1084 (6.36%) and 11/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data.

SARS-CoV-2 prolonged infection during advanced HIV disease evolves extensive immune escape.

Characterizing SARS-CoV-2 evolution in specific geographies may help predict properties of the variants that come from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from ancestral virus in a person with advanced HIV disease in South Africa; this person was infected prior to emergence of the Beta and Delta variants. We longitudinally tracked the evolved virus and tested it against self-plasma and convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, but it evolved a multitude of mutations found in Omicron and other variants. It showed substantial but incomplete Pfizer BNT162b2 escape, weak neutralization by self-plasma, and despite pre-dating Delta, it also showed extensive escape of Delta infection-elicited neutralization. This example is consistent with the notion that SARS-CoV-2 evolving in individual immune-compromised hosts, including those with advanced HIV disease, may gain immune escape of vaccines and enhanced escape of Delta immunity, and this has implications for vaccine breakthrough and reinfections.

Similar Antibody Responses Against Severe Acute Respiratory Syndrome Coronavirus 2 in Individuals Living Without and With Human Immunodeficiency Virus on Antiretroviral Therapy During the First South African Infection Wave.

BACKGROUND: There is limited understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis in African populations with a high burden of infectious disease comorbidities such as human immunodeficiency virus (HIV). The kinetics, magnitude, and duration of virus-specific antibodies and B-cell responses in people living with HIV (PLWH) in sub-Saharan Africa have not been fully characterized. METHODS: We longitudinally followed SARS-CoV-2-infected individuals in Durban, KwaZulu-Natal, South Africa, and characterized SARS-CoV-2 receptor-binding domain-specific immunoglobulin (Ig) M, IgG, and IgA weekly for 1 month and at 3 months post-diagnosis. Thirty of 72 (41.7%) were PLWH, 25/30 (83%) of whom were on antiretroviral therapy (ART) with full HIV suppression. Plasma neutralization was determined using a live virus neutralization assay, and antibody-secreting cell population frequencies were determined by flow cytometry. RESULTS: Similar seroconversion rates, time to peak antibody titer, peak magnitude, and durability of anti-SARS-CoV-2 IgM, IgG, and IgA were observed in people not living with HIV and PLWH with complete HIV suppression on ART. In addition, similar potency in a live virus neutralization assay was observed in both groups. Loss of IgA was significantly associated with age (P = .023) and a previous diagnosis of tuberculosis (P = .018). CONCLUSIONS: Similar antibody responses and neutralization potency in people not living with HIV and PLWH on stable ART in an African setting suggest that coronavirus disease 2019 (COVID-19) natural infections may confer comparable antibody immunity in these groups. This provides hope that COVID-19 vaccines will be effective in PLWH on stable ART.

SARS-CoV-2 evolved during advanced HIV disease immunosuppression has Beta-like escape of vaccine and Delta infection elicited immunity.

Characterizing SARS-CoV-2 evolution in specific geographies may help predict the properties of variants coming from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from the ancestral virus in a person with advanced HIV disease. Infection was before the emergence of the Beta variant first identified in South Africa, and the Delta variant. We compared early and late evolved virus to the ancestral, Beta, Alpha, and Delta viruses and tested against convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral, whereas late virus was similar to Beta, exhibiting vaccine escape and, despite pre-dating Delta, strong escape of Delta-elicited neutralization. This example is consistent with the notion that variants arising in immune-compromised hosts, including those with advanced HIV disease, may evolve immune escape of vaccines and enhanced escape of Delta immunity, with implications for vaccine breakthrough and reinfections. HIGHLIGHTS: A prolonged ancestral SARS-CoV-2 infection pre-dating the emergence of Beta and Delta resulted in evolution of a Beta-like serological phenotypeSerological phenotype includes strong escape from Delta infection elicited immunity, intermediate escape from ancestral virus immunity, and weak escape from Beta immunityEvolved virus showed substantial but incomplete escape from antibodies elicited by BNT162b2 vaccination.

An Investigation of Culicoides (Diptera: Ceratopogonidae) as Potential Vectors of Medically and Veterinary Important Arboviruses in South Africa.

Culicoides-borne viruses such as bluetongue, African horse sickness, and Schmallenberg virus cause major economic burdens due to animal outbreaks in Africa and their emergence in Europe and Asia. However, little is known about the role of Culicoides as vectors for zoonotic arboviruses. In this study, we identify both veterinary and zoonotic arboviruses in pools of Culicoides biting midges in South Africa, during 2012-2017. Midges were collected at six surveillance sites in three provinces and screened for Alphavirs, Flavivirus, Orthobunyavirus, and Phlebovirus genera; equine encephalosis virus (EEV); and Rhaboviridae, by reverse transcription polymerase chain reaction. In total, 66/331 (minimum infection rate (MIR) = 0.4) pools tested positive for one or more arbovirus. Orthobunyaviruses, including Shuni virus (MIR = 0.1) and EEV (MIR = 0.2) were more readily detected, while only 2/66 (MIR = 0.1) Middelburg virus and 4/66 unknown Rhabdoviridae viruses (MIR = 0.0) were detected. This study suggests Culicoides as potential vectors of both veterinary and zoonotic arboviruses detected in disease outbreaks in Africa, which may contribute to the emergence of these viruses to new regions.

The utilisation of CytB and COI barcodes for the identification of bloodmeals and Culicoides species (Diptera: Ceratopogonidae) reveals a variety of novel wildlife hosts in South Africa.

Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) are vectors of pathogens that can cause diseases of major economic importance in humans and animals. Identifying host ranges of these biting midges might aid in understanding the complex epidemiology of such diseases, often involving reservoir hosts and multiple species. In this study, we aim to identify bloodmeal origin from engorged female Culicoides biting midges. All bloodfed females were opportunistically collected as part of an ongoing surveillance programme using Onderstepoort light traps in two provinces in South Africa. DNA of individuals was extracted and subjected to PCR targeting the cytochrome B (CytB) gene region of mammals and avians as well as cytochrome oxidase I (COI) for species identification. In total, 21 new reference barcodes were generated for C. bedfordi, C imicola, C. leucosticus, C. magnus, and C. pycnostictus. Seventy-four blood meals were identified, originating from 12 mammal and three avian species. COI sequence data performed well for species delimitation and 54 Culicoides specimens were identified with C. imicola the predominant species identified (41.8%). Generally, Culicoides species feed on a variety of hosts and host availability might be an important factor when selecting a host. Culicoides species thus appear to be opportunistic feeders rather than specialists. This implicates Culicoides as transfer vectors and demonstrates possible transmission routes of arboviruses and other pathogens from wildlife onwards to domestic animals and humans.

Epidemiology and Genomic Analysis of Equine Encephalosis Virus Detected in Horses with Clinical Signs in South Africa, 2010-2017.

Equine encephalosis virus (EEV) is a neglected virus endemic to South Africa and is considered to generally result in mild disease in equines. Specimens were analyzed from live horses that presented with undefined neurological, febrile, or respiratory signs, or sudden and unexpected death. Between 2010 and 2017, 111 of 1523 (7.3%) horse samples tested positive for EEV using a nested real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Clinical signs were reported in 106 (7.2%) EEV positive and 1360 negative horses and included pyrexia (77/106, 72.6%), icterus (20/106, 18.9%) and dyspnea (12/106, 11.3%). Neurological signs were inversely associated with EEV infection (OR < 1, p < 0.05) relative to EEV negative cases despite a high percentage of animals presenting with neurological abnormalities (51/106, 48.1%). Seventeen of the EEV positive horses also had coinfections with either West Nile (5/106, 4.7%), Middelburg (4/106, 3.8%) or African Horse sickness virus (8/106, 7.6%). To investigate a possible genetic link between EEV strains causing the observed clinical signs in horses, the full genomes of six isolates were compared to the reference strains. Based on the outer capsid protein (VP2), serotype 1 and 4 were identified as the predominant serotypes with widespread reassortment between the seven different serotypes.