Journal of Clinical Virology Plus

We detected H5N1 high pathogenicity avian influenza in captive Greater Rhea (Rhea americana). Viral genetic analysis revealed the mammalian associated PB2-E627K mutation, indicating selection of mammalian-relevant mutations in ratites. Pathologic investigation of available tissues demonstrated severe multifocal necrotising inflammation, and a strong vasculotropism.

A commercially available antibody, Cox mAB 31A2, raised against the VP1 protein of coxsackievirus B3 (CVB3) has been reported as suitable for the detection of CVB3 in diagnostic samples (Ettischer-Schmid 2016). The authors compared this antibody with the widely used, multi-specific, monoclonal anti-VP1 antibody marketed by Dako (clone 5D8/1) and concluded that clone 5D8/1 should not be used to identify enterovirus infections in diagnostic samples. Rather they suggested that Cox mAB 31A2 is preferable for this purpose. Here we address these issues and show that Cox mAB 31A2 can be used successfully to diagnose CVB3 infection in various cell and tissue samples but we demonstrate that it fails to detect many clinically relevant enterovirus types, thereby limiting its use as a general diagnostic reagent for clinical specimens. Rather, we propose that, when used under carefully controlled conditions, clone 5D8/1 should remain the reagent of choice for such purposes.

Highly pathogenic avian influenza (HPAI) H5N1 has been a global concern since its emergence in 1997, causing widespread outbreaks in birds and sporadic human infections. The clade 2.3.4.4b H5N1 virus has rapidly expanded across continents, infecting numerous mammalian species. In 2024, it was detected in dairy cattle for the first time in the U.S., along with human cases following exposure. In Canada, the first human case of this avian influenza was reported in a critically ill adolescent in late 2024. No human-to-human transmission has been documented, but concerns persist regarding mutations associated with enhanced virulence and human adaptation. Although seasonal influenza vaccines are not directed against H5N1, studies suggest that pre-existing immunity from prior infections or vaccinations may provide partial protection against severe H5N1 infections through cross-reactive immune response. Given the ongoing circulation of avian influenza and the rise in human infections, this study evaluated the effectiveness of neutralizing antibodies developed against seasonal influenza viruses and their cross-reactivity with recent H5N1 strains. Serum samples from 194 retail sector workers in Quebec, collected between late 2021 and 2022, were analyzed using a microneutralization assay. While strong neutralizing activity was found against seasonal influenza viruses, no neutralizing antibodies were detected against H5N1 strains in either vaccinated or unvaccinated individuals. These findings emphasize the need to evaluate cross-reactive antibodies against the neuraminidase protein of H5N1, assess cellular immune responses potentially linked to protection against severe HPAI H5N1 infections and targeted vaccine strategies against recently emerged H5N1 influenza viruses.

IntroductionConcerns have been raised regarding the accuracy of diagnostic antigen testing for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant. We compared the performance of the LumiraDx SARS-CoV-2 Antigen Test between symptomatic participants recruited prospectively during the Delta to Omicron variant transition in the USA. MethodsTwo paired anterior nasal swabs were collected from each participant (adults and children) within 12 days of symptom onset between November 24th, 2021 and February 1st, 2022, during which time Omicron replaced Delta as the dominant variant in the sample population. Swabs were tested by the LumiraDx SARS-CoV-2 Antigen Test and compared using real-time polymerase chain reaction (RT-PCR) reference testing. Reference samples identified as positive were sequenced to identify the SARS-CoV-2 variant. Positive percent agreement (PPA) was calculated, with results stratified by RT-PCR cycle threshold (Ct). ResultsOf the 38 participants for whom LumiraDx SARS-CoV-2 Antigen Test results were available, 36 were confirmed positive by RT-PCR. Overall, PPA of the LumiraDx SARS-CoV-2 Antigen Test was 94.7% (95% confidence interval: 82.3%, 99.4%) and PPA was 100% for samples with a Ct <33. Sufficient viral load for sequencing was present in nine samples (six Delta, three Omicron), all of which returned a positive result using the LumiraDx SARS-CoV-2 Antigen Test. There were no performance differences observed between participants with the Delta and Omicron variants. ConclusionsSARS-CoV-2 differences between Delta and Omicron variant mutations did not affect the performance of the LumiraDx SARS-CoV-2 Antigen Test which detects the nucleocapsid protein antigen. The LumiraDx SARS-CoV-2 Antigen Test can be a useful antigen test to diagnose emerging variants of coronavirus disease 2019.

The SARS-CoV-2 outbreak and subsequent COVID-19 pandemic have highlighted the urgent need to determine what cells are susceptible to infection and for assays to detect and quantify SARS-CoV-2. Furthermore, the ongoing efforts for vaccine development have necessitated the development of rapid, high-throughput methods of quantifying infectious SARS-CoV-2, as well as the ability to screen human polyclonal sera samples for neutralizing antibodies against SARS-CoV-2. To this end, our lab has adapted focus forming assays for SARS-CoV-2 using Vero CCL-81 cells, referred to in this text as Vero WHO. Using the focus forming assay as the basis for screening cell susceptibility and to develop a focus reduction neutralization test. We have shown that this assay is a sensitive tool for determining SARS-CoV-2 neutralizing antibody titer in human, non-human primate, and mouse polyclonal sera following SARS-CoV-2 exposure. Additionally, we describe the viral growth kinetics of SARS-CoV-2 in a variety of different immortalized cell lines and demonstrate via human ACE2 and viral spike protein expression that these cell lines can support viral entry and replication.

Antibody dependent enhancement has been well described between Zika and dengue viruses, but is poorly characterized between West Nile and dengue viruses. We demonstrate that neuroinvasive West Nile virus infection leads to the development of non-neutralizing, cross-reactive IgG antibodies to dengue and Zika viruses capable of causing antibody dependent enhancement in vitro of dengue virus and leads to the formation of flavivirus cross-reactive memory B cells in some patients.

Numbers of human encephalitis cases caused by infection with Borna disease virus 1 (BoDV1) increase continuously in endemic areas. The reservoir host of BoDV1 is the bicoloured white-toothed shrew, albeit few naturally infected individuals of other shrew species have been detected. To establish a reliable experimental reservoir model, 15 greater white-toothed shrews were infected with a shrew-derived BoDV1 isolate by different inoculation routes (intracerebral, intranasal, oral, subcutaneous, intraperitoneal) and monitored up to 41 days. Except for the oral route all other animals (12/15) were successfully infected, and the majority of them displayed temporary reduced feed intake and loss of body weight but no inflammatory lesions. Infectious virus was isolated from 11/12 infected animals. Viral RNA was demonstrated by RT-qPCR in the central nervous system (CNS) and the majority of organs. Immunohistochemistry demonstrated BoDV1 antigen in neurons and astrocytes in the CNS and peripheral nerves. High viral loads in the CNS and the spinal cord points towards spread from periphery to the CNS to enhance viral replication, and subsequent centrifugal spread to organs capable of secretion and excretions. In general, successful experimental BoDV1 infection of shrews proves their usefulness as animal model, enabling further studies on maintenance, transmission, pathogenesis, and risk assessment for human spill-over infections.

The COVID-19 Pandemic has prompted innovation and research to further understand not only SARS-CoV-2, but other respiratory viruses as well. Since the start of the pandemic there has been a lack in influenza collection and surveillance. In October 2021 the Life Sciences Testing Center at Northeastern University implemented the TaqPath COVID-19, Flu A, Flu B combo kit to test for multiple respiratory diseases among the Universitys population. During this time the SARS-CoV-2 variant of concern, Omicron B.1.1.529, became the dominant strain in the greater Boston area. During this time an inverse correlation in the detection of positive SARS-CoV-2 and Influenza A was observed. More data is needed to determine if this observed inverse correlation on positivity rate is linked to public health measures or biological mechanism within the immune system.

In 2023, an unprecedented outbreak of highly pathogenic avian influenza (HPAI) H5N1 resulted in the death of thousands of pinnipeds along the Argentinean coast, raising concerns about its ecological and epidemiological impact. Here, we present clinical, pathological, and molecular findings associated with HPAI H5N1 infection in pinnipeds from Chubut, Argentina. Necropsies were conducted on three South American Sea Lions (SASLs) (Otaria flavescens) and one Southern Elephant Seal (SES) (Mirounga leonina), followed by histopathological, immunohistochemical and RT-sqPCR analyses. Neurological clinical signs were observed in two SASLs, with one also exhibiting respiratory distress. Neuropathological findings included lymphoneutrophilic meningoencephalomyelitis and choroiditis, neuronal necrosis, gliosis, hemorrhages, and perivascular cuffing. Viral antigen was localized in neurons, glial cells, choroid plexus epithelial cells, ependymal cells, and the neuropil. Systemic manifestations included HPAI-related necrotizing myocarditis in the elephant seal and placental necrosis in a sea lion, with fetal tissues testing positive for HPAIV. Pulmonary lesions were minimal, limited to bronchial glands in one individual. RT-sqPCR confirmed HPAI H5 in all tested animals. Our findings highlight the neurotropism of HPAI H5N1 in pinnipeds, and expand the known systemic effects of the virus, revealing new tissue tropism and vertical transmission.

We describe an unusual mortality event caused by a highly pathogenic avian influenza virus (HPAI) H5N1 clade 2.3.4.4b involving harbor (Phoca vitulina) and grey (Halichoerus grypus) seals in the St. Lawrence Estuary, Quebec, Canada. Fifteen (56%) of the seals submitted for necropsy were considered to be fatally infected by H5N1 containing fully Eurasian (EA) or Eurasian/North American genome constellation. Concurrently, presence of large numbers of bird carcasses infected with H5N1 at haul-out sites most likely contributed to the spill-over of infection to the seals. Histologic changes included meningoencephalitis (100%), fibrinosuppurative alveolitis, and multi-organ acute necrotizing inflammation. This is the first report of fatal H5N1 infection in pinnipeds in Canada, raising concerns about the expanding host of this virus, potential for establishment of a marine mammal reservoir, and the public health risks associated with spillover to mammals.

Monodelphis domestica, also known as the laboratory opossum, is a marsupial native to South America. At birth, these animals are developmentally equivalent to human embryos at approximately 5 weeks of gestation which, when coupled with other characteristics including the size of the animals, the development of a robust immune system during juvenile development, and the relative ease of experimental manipulation, have made M. domestica a valuable model in many areas of biomedical research. However, their suitability as models for infectious diseases, especially diseases caused by viruses such as Zika virus (ZIKV), is currently unknown. Here, we describe the replicative effects of ZIKV using a fetal intra-cerebral model of inoculation. Using immunohistochemistry and in situ hybridization, we found that opossum embryos and fetuses are susceptible to infection by ZIKV administered intra-cerebrally, that the infection persists long term, and that the infection and viral replication consistently results in neural pathology and may occasionally result in global growth restriction. These results demonstrate the utility of M. domestica as a new animal model for investigating ZIKV infection in vivo. This new model will facilitate further inquiry into viral pathogenesis, particularly for those viruses that are neurotropic, that may require a host with the ability to support sustained viral infection, and/or that may require intra-cerebral inoculations of large numbers of embryos or fetuses.\n\nAUTHOR SUMMARYHere we show that the laboratory opossum (Monodelphis domestica) is a valuable new model for studying Zika virus pathogenesis. Newborns are at the developmental stage of 5-week human embryos. Zika virus inoculated on a single occasion into the brains of pups at the human developmental stages of 8-20 weeks post conception replicated in neuronal cells and persisted as a chronic infection until the experimental endpoint at 74-days post infection. In addition, we observed global growth restriction in one of 16 inoculated animals; global growth restriction has been observed in humans and other animal models infected with Zika virus. The results illustrate great potential for this new animal model for high throughput research on the neurological effects of Zika virus infection of embryos and fetuses.

This report includes a 2015 epizootic of highly pathogenic H7N3 avian influenza virus among captive and wild birds at "El Zapotal" ecologic reserve, located in the state of Chiapas, Mexico. Epidemiological control measures were implemented to prevent virus dissemination. The infection with the highly pathogenic H7N3 virus was detected predominantly among Plain Chachalaca (Ortalis vetula), with occasional detections in a White-fronted Parrot (Amazona albifrons) and a single Clay-colored Thrush (Turdus grayi). Here, we describe the characteristics of the outbreak environment, the surveillance strategy, the biosecurity measures, and the evaluation of the site, including external farms. These actions, timely implemented by the veterinary authorities, helped to contain the outbreak beyond the ecologic reserve. This contingency showed the importance of developing a more complete analysis of the existing risks and the challenges to implement minimal biosecurity measures in these facilities.

New information is emerging about SARS-CoV-2 epidemiology and immunity, but little of this information comes from low- and middle-income countries or from patients receiving care in the outpatient setting. The current study investigated the SARS-CoV-2 infection status and antibody responses in 157 patients seeking care for a respiratory disease suggestive of COVID-19 in private healthcare clinics during the first wave (June-October 2020) of infections in Nicaragua. We examined nasal swabs for the presence of viral RNA via RT-PCR and longitudinally collected sera for the changes in SARS-CoV-2 Spike antibody levels over six months. Among patients with confirmed SARS-CoV-2 infections, we evaluated if clinical symptoms were associated with age, hematological parameters and co-morbidities. The combination of PCR and paired serology identified 60 (38%) of the 157 outpatients as acute COVID-19. While both PCR and serology identified the majority (n = 38, 64%) of the acute infections, a notable number of outpatients were identified by RT-qPCR (n = 13, 22%) or by serology (n = 9, 14%) only. During the longitudinal study, we identified 6 new infections by serology among the 97 non-COVID-19 subjects. In conclusion, this study report that more than one third of the outpatients seeking care for acute respiratory disease during the first epidemic wave of SARS-CoV-2 in Nicaragua had an acute mild COVID-19 infection that correlate with prolonged humoral response. This immune response to the RBD antigen, more likely IgG dependent, significantly increased between the acute to convalescent and decay in the late convalescent but still remained seropositive.

Determining when individuals should be released from quarantine is critical for successfully managing a COVID-19 outbreak and local protocols frequently call for testing during the quarantine period, generally after a reasonable incubation period, which raises a question about the interpretation of test results during the quarantine period. We report the negative predictive value of SARS-CoV-2 qPCR tests based on a retrospective longitudinal analysis of 5349 qPCR tests collected from 1227 US service members infected with COVID-19 aboard the USS Theodore Roosevelt (CVN-71) aircraft carrier. In our retrospective evaluation of recovering qPCR-positive quarantined crew members undergoing repeated testing, the negative predictive value is 80% for tests occurring as late as seven weeks following an initial positive qPCR test result. Repeated qPCR testing is necessary to ensure that a once-infected person is no longer shedding viral RNA. When deciding the stringency of exit criteria, we recommend considering local operational and community risk factors.

Non-pharmaceutical interventions (NPIs) to contain the SARS-CoV-2 pandemic drastically reduced human-to-human interactions, decreasing the circulation of other respiratory viruses as well. As a consequence, influenza virus circulation - normally responsible for 3-5 million hospitalizations per year globally - was significantly reduced. With downscaling the NPI countermeasures, there is a concern for increased influenza disease, particularly in individuals suffering from post-acute effects of SARS-CoV-2 infection. To investigate this possibility, we performed a sequential influenza H1N1 infection 4 weeks after an initial SARS-CoV-2 infection in the ferret model. Upon H1N1 infection, ferrets that were previously infected with SARS-CoV-2 showed an increased tendency to develop clinical symptoms compared to the control H1N1 infected animals. Histopathological analysis indicated only a slight increase for type II pneumocyte hyperplasia and bronchitis. The effects of the sequential infection thus appeared minor. However, ferrets were infected with B.1.351-SARS-CoV-2, the beta variant of concern, which replicated poorly in our model. The histopathology of the respiratory organs was mostly resolved 4 weeks after SARS-CoV-2 infection, with only reminiscent histopathological features in the upper respiratory tract. Nevertheless, SARS-CoV-2 specific cellular and humoral responses were observed, confirming an established infection. Thus, there may likely be a SARS-CoV-2 variant-dependent effect on the severity of disease upon a sequential influenza infection as we observed mild effects upon a mild infection. It, however, remains to be determined what the impact is of more virulent SARS-CoV-2 variants. ImportanceDuring the COVID-19 pandemic, the use of face masks, social distancing and isolation were not only effective in decreasing the circulation of SARS-CoV-2, but also in reducing other respiratory viruses such as influenza. With less restrictions, influenza is slowly returning. In the meantime, people still suffering from long-COVID, could be more vulnerable to an influenza virus infection and develop more severe influenza disease. This study provides directions to the effect of a previous SARS-CoV-2 exposure on influenza disease severity in the ferret model. This model is highly valuable to test sequential infections under controlled settings for translation to humans. We could not induce clear long-term COVID-19 effects as SARS-CoV-2 infection in ferrets was mild. However, we still observed a slight increase in influenza disease severity compared to ferrets that had not encountered SARS-CoV-2 before. It may therefore be advisable to include long-COVID patients as a risk group for influenza vaccination.

BackgroundAfter infection with severe acute respiratory syndrome coronavirus (SARS-CoV-2), Immunoglobulin G (IgG) antibodies and virus-specific neutralizing antibodies (nAbs) develop. This study describes antibody responses in a cohort of recovered COVID-19 patients to identify predictors. MethodsWe recruited patients with confirmed SARS-CoV-2 infection from Heidelberg, Germany. Blood samples were collected three weeks after COVID-19 symptoms ended. Participants with high antibody titers were invited for follow-up visits. IgG titers were measured by the Euroimmun Assay, and nAbs titers in a SARS-CoV-2 infection-based assay. Results281 participants were enrolled between April and August 2020 with IgG testing, 145 (51.6%) had nAbs, and 35 (12.5%) had follow-up. The median IgG optical density (OD) ratio was 3.1 (Interquartile range (IQR) 1.6-5.1), and 24.1% (35/145) had a nAb titer>1:80. Higher IgG titers were associated with increased age and more severe disease, and higher nAbs were associated with male gender and CT-value of 25-30 on RT-PCR at diagnosis. The median IgG OD ratio on follow-up was 3.7 (IQR 2.9-5.9), a median increase of 0.5 (IQR -0.3-1.7). Six participants with follow-up nAbs all had titers [&le;] 1:80. ConclusionsWhile age and disease severity were correlated with IgG responses, predictive factors for nAbs in convalescent patients remain unclear.

To estimate the effectiveness of vaccines in development, a robust mechanism is required to understand immunity, risks of reinfection and measure the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and how this may change over time. This study is a longitudinal analysis of COVID-19 infection rates using PCR, membrane immunoassay and chemiluminescent microparticle immunoassay (CMIA) diagnostic tests. Our data confirm that antibody levels wane in the three months after symptom onset. Comparison of the three methods used suggests that quantitative CMIA testing may exaggerate numbers of COVID-19 negative individuals.

The relative isolation of many island communities provides some protection from the COVID-19 pandemic, as imported cases can be limited and traced effectively. Until recently, this was true for the population of the French overseas department, Reunion Island, where only limited numbers of autochthonous cases were observed prior to August 2020. Since the report of the first case of COVID-19, contact tracing has been carried out for each new case identified in Reunion Island to identify transmission and clusters. To contribute to the public health response and understand the diffusion of SARS-Cov-2 strains in Reunion Island, we established in-house genome sequencing capability in Reunion using Oxford nanopore technology (MinION) as an inexpensive option for genomic typing of SARS-CoV-2 lineages on the island, and cross-validated typing results between viral isolation methods and different sequencing technologies. The results of our work during the early phase of the epidemics are presented herein. Article Summary LineThe COVID-19 pandemic has had an unprecedented impact on the global community. Here we provide epidemiological and genomic details of the early stages of the pandemic on Reunion Island.

Hemagglutination inhibition (HI) and neuraminidase inhibition (NI) antibodies to a clade 2.3.4.4b A(H5N1) highly pathogenic avian influenza virus were measured in 63 age-stratified healthy adults in Hong Kong. No HI antibody was detected; 61 subjects had detectable NI antibodies to A(H5N1). NI titers to A(H5N1) and A(H1N1)pdm09 viruses were correlated.

We report a downward trend in the initial SARS-CoV-2 viral load in nasopharyngeal swab samples of hospitalized patients with COVID-19 in Detroit, Michigan, coinciding with a decrease in the number of deaths during April-June 2020. A gradual decrease in the initial viral load reflected the downward progression of the pandemic.