SARS-CoV-2 spike D614G change enhances replication and transmission.

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.

Red knots in Europe: a dead end host species or a new niche for highly pathogenic avian influenza?

The 2020/2021 epidemic in Europe of highly pathogenic avian influenza virus (HPAIV) of subtype H5 surpassed all previously recorded European outbreaks in size, genotype constellations and reassortment frequency and continued into 2022 and 2023. The causative 2.3.4.4b viral lineage proved to be highly proficient with respect to reassortment with cocirculating low pathogenic avian influenza viruses and seems to establish an endemic status in northern Europe. A specific HPAIV reassortant of the subtype H5N3 was detected almost exclusively in red knots (Calidris canutus islandica) in December 2020. It caused systemic and rapidly fatal disease leading to a singular and self-limiting mass mortality affecting about 3500 birds in the German Wadden Sea, roughly 1 % of the entire flyway population of islandica red knots. Phylogenetic analyses revealed that the H5N3 reassortant very likely had formed in red knots and remained confined to this species. While mechanisms of virus circulation in potential reservoir species, dynamics of spill-over and reassortment events and the roles of environmental virus sources remain to be identified, the year-round infection pressure poses severe threats to endangered avian species and prompts adaptation of habitat and species conservation practices.

Detection of Novel Poxvirus from Gray Seal (Halichoerus grypus), Germany.

We detected a novel poxvirus from a gray seal (Halichoerus grypus) from the North Sea, Germany. The juvenile animal showed pox-like lesions and deteriorating overall health condition and was finally euthanized. Histology, electron microscopy, sequencing, and PCR confirmed a previously undescribed poxvirus of the Chordopoxvirinae subfamily, tentatively named Wadden Sea poxvirus.

Mass mortality among colony-breeding seabirds in the German Wadden Sea in 2022 due to distinct genotypes of HPAIV H5N1 clade 2.3.4.4b.

Mass mortality was observed among colony-breeding seabirds in the German Wadden Sea area of the North Sea during the summer months of 2022. Several species' colonies were affected, most notably sandwich terns (Thalasseus sandvicensis), common terns (Sterna hirundo) and Germany's only northern gannet (Morus bassanus) colony on the island of Heligoland. Mortality in some tern colonies reached 40%, while other colonies were almost spared. In all cases, infections with the high-pathogenicity avian influenza virus (HPAIV) subtype H5N1 of clade 2.3.4.4b were identified to have caused the epidemic. Phylogenetic analysis of whole-genome sequences revealed that the outbreaks were dominated by two genotypes, Ger-10-21 N1.2 and Ger-10-21 N1.5, previously identified in Germany. Spatiotemporal analyses of phylogenetic data suggested that these viruses could have entered the continental North Sea coastal region via the British Isles. A close linkage of viruses from tern colonies in the German Wadden Sea was evident with further connections to breeding colonies in Belgium and the Netherlands, and further spread to Denmark and Poland. Several of the affected species are endangered, such that negative effects of epizootic HPAIV infections on populations are feared, with uncertain long-term consequences.

Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1.

Repeated outbreaks due to H3N1 low pathogenicity avian influenza viruses (LPAIV) in Belgium were associated with unusually high mortality in chicken in 2019. Those events caused considerable economic losses and prompted restriction measures normally implemented for eradicating high pathogenicity avian influenza viruses (HPAIV). Initial pathology investigations and infection studies suggested this virus to be able to replicate systemically, being very atypical for H3 LPAIV. Here, we investigate the pathogenesis of this H3N1 virus and propose a mechanism explaining its unusual systemic replication capability. By intravenous and intracerebral inoculation in chicken, we demonstrate systemic spread of this virus, extending to the central nervous system. Endoproteolytic viral hemagglutinin (HA) protein activation by either tissue-restricted serine peptidases or ubiquitous subtilisin-like proteases is the functional hallmark distinguishing (H5 or H7) LPAIV from HPAIV. However, luciferase reporter assays show that HA cleavage in case of the H3N1 strain in contrast to the HPAIV is not processed by intracellular proteases. Yet the H3N1 virus replicates efficiently in cell culture without trypsin, unlike LPAIVs. Moreover, this trypsin-independent virus replication is inhibited by 6-aminohexanoic acid, a plasmin inhibitor. Correspondingly, in silico analysis indicates that plasminogen is recruitable by the viral neuraminidase for proteolytic activation due to the loss of a strongly conserved N-glycosylation site at position 130. This mutation was shown responsible for plasminogen recruitment and neurovirulence of the mouse brain-passaged laboratory strain A/WSN/33 (H1N1). In conclusion, our findings provide good evidence in natural chicken strains for N1 neuraminidase-operated recruitment of plasminogen, enabling systemic replication leading to an unusual high pathogenicity phenotype. Such a gain of function in naturally occurring AIVs representing an established human influenza HA-subtype raises concerns over potential zoonotic threats.

Chitranjan S. Ranawat Award: Tourniquet Use Does Not Impact Trajectory of Total Knee Arthroplasty Early Recovery: A Prospective, Randomized Controlled Trial.

BACKGROUND: The impact of tourniquet use on recovery after total knee arthroplasty (TKA) remains controversial. The purpose of this prospective, single blinded, randomized controlled trial was to investigate the effect of tourniquet use on early recovery after TKA using a smartphone app-based patient engagement platform (PEP) with a wrist-based activity monitor to obtain more robust data on early recovery. METHODS: There were 107 patients undergoing primary TKA for osteoarthritis who were enrolled (54 tourniquet [TQ+]; 53 no tourniquet [TQ-]). All patients utilized a PEP and wrist-based activity sensor for 2 weeks preoperatively and 90 days postoperatively to collect Visual Analog Scale (VAS) pain scores and opioid consumption, as well as weekly Oxford Knee Score (OKS) and monthly Forgotten Joint Score (FJS). There was no difference in demographics between groups. Formal physical therapy assessments were performed preoperatively and 3 months postoperatively. Independent sample t-tests were used for continuous data and Chi-square and Fisher's exact tests were used for discrete data. RESULTS: Tourniquet use did not have a statistically significant impact on daily VAS pain or opioid consumption during the first 30 days postoperatively (P > .05). Tourniquet use did not have a significant impact on OKS or FJS at 30 or 90 days postoperatively (P > .05), or on performance of formal physical therapy testing at 3 months postoperatively (P > .05). CONCLUSION: Using a digital technology to collect daily patient data, we found that tourniquet use has no clinically significant negative impact on pain and function in the first 90 days after primary TKA.

Iceland as Stepping Stone for Spread of Highly Pathogenic Avian Influenza Virus between Europe and North America.

Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin type H5 and clade 2.3.4.4b have widely spread within the northern hemisphere since 2020 and threaten wild bird populations, as well as poultry production. We present phylogeographic evidence that Iceland has been used as a stepping stone for HPAIV translocation from northern Europe to North America by infected but mobile wild birds. At least 2 independent incursions of HPAIV H5N1 clade 2.3.4.4b assigned to 2 hemagglutinin clusters, B1 and B2, are documented for summer‒autumn 2021 and spring 2022. Spread of HPAIV H5N1 to and among colony-breeding pelagic avian species in Iceland is ongoing. Potentially devastating effects (i.e., local losses >25%) on these species caused by extended HPAIV circulation in space and time are being observed at several affected breeding sites throughout the North Atlantic.

Human Infection with Eurasian Avian-Like Swine Influenza A(H1N1) Virus, the Netherlands, September 2019.

We report a zoonotic infection of a pig farmer in the Netherlands with a Eurasian avian-like swine influenza A(H1N1) virus that was also detected in the farmed pigs. Both viruses were antigenically and genetically characterized. Continued surveillance of swine influenza A viruses is needed for risk assessment in humans and swine.

Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples.

BACKGROUND: As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5' untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. RESULTS: To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19-32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. CONCLUSIONS: Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

Novel Reassortant Highly Pathogenic Avian Influenza A(H5N2) Virus in Broiler Chickens, Egypt.

We detected a novel reassortant highly pathogenic avian influenza A(H5N2) virus in 3 poultry farms in Egypt. The virus carried genome segments of a pigeon H9N2 influenza virus detected in 2014, a nucleoprotein segment of contemporary chicken H9N2 viruses from Egypt, and hemagglutinin derived from the 2.3.4.4b H5N8 virus clade.

Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses.

BACKGROUND: Due to the frequent reassortment and zoonotic potential of influenza A viruses, rapid gain of sequence information is crucial. Alongside established next-generation sequencing protocols, the MinION sequencing device (Oxford Nanopore Technologies) has become a serious competitor for routine whole-genome sequencing. Here, we established a novel, rapid and high-throughput MinION multiplexing workflow based on a universal RT-PCR. METHODS: Twelve representative influenza A virus samples of multiple subtypes were universally amplified in a one-step RT-PCR and subsequently sequenced on the MinION instrument in conjunction with a barcoding library preparation kit from the rapid family and the MinIT performing live base-calling. The identical PCR products were sequenced on an IonTorrent platform and, after final consensus assembly, all data was compared for validation. To prove the practicability of the MinION-MinIT method in human and veterinary diagnostics, we sequenced recent and historical influenza strains for further benchmarking. RESULTS: The MinION-MinIT combination generated over two million reads for twelve samples in a six-hour sequencing run, from which a total of 72% classified as quality screened, trimmed and mapped influenza reads to produce full genome sequences. Identities between the datasets of > 99.9% were achieved, with 100% coverage of all segments alongside a sufficient confidence and 4492fold mean depth. From RNA extraction to finished sequences, only 14 h were required. CONCLUSIONS: Overall, we developed and validated a novel and rapid multiplex workflow for influenza A virus sequencing. This protocol suits both clinical and academic settings, aiding in real time diagnostics and passive surveillance.

Genetic Characterization and Zoonotic Potential of Highly Pathogenic Avian Influenza Virus A(H5N6/H5N5), Germany, 2017-2018.

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade 2.3.4.4b isolates found in Germany in 2017-2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.

Validation of the TAPS-1: A Four-Item Screening Tool to Identify Unhealthy Substance Use in Primary Care.

BACKGROUND: The Tobacco, Alcohol, Prescription Medication, and Other Substance use (TAPS) tool is a combined two-part screening and brief assessment developed for adult primary care patients. The tool's first-stage screening component (TAPS-1) consists of four items asking about past 12-month use for four substance categories, with response options of never, less than monthly, monthly, weekly, and daily or almost daily. OBJECTIVE: To validate the TAPS-1 in primary care patients. DESIGN: Participants completed the TAPS tool in self- and interviewer-administered formats, in random order. In this secondary analysis, the TAPS-1 was evaluated against DSM-5 substance use disorder (SUD) criteria to determine optimal cut-points for identifying unhealthy substance use at three severity levels (problem use, mild SUD, and moderate-to-severe SUD). PARTICIPANTS: Two thousand adult patients at five primary care sites. MAIN MEASURES: DSM-5 SUD criteria were determined via the modified Composite International Diagnostic Interview. Oral fluid was used as a biomarker of recent drug use. KEY RESULTS: Optimal frequency-of-use cut-points on the self-administered TAPS-1 for identifying SUDs were ≥ monthly use for tobacco and alcohol (sensitivity = 0.92 and 0.71, specificity = 0.80 and 0.85, AUC = 0.86 and 0.78, respectively) and any reported use for illicit drugs and prescription medication misuse (sensitivity = 0.93 and 0.89, specificity = 0.85 and 0.91, AUC = 0.89 and 0.90, respectively). The performance of the interviewer-administered format was similar. When administered first, the self-administered format yielded higher disclosure rates for past 12-month alcohol use, illicit drug use, and prescription medication misuse. Frequency of use alone did not provide sufficient information to discriminate between gradations of substance use problem severity. Among those who denied drug use on the TAPS-1, less than 4% had a drug-positive biomarker. CONCLUSIONS: The TAPS-1 can identify unhealthy substance use in primary care patients with a high level of accuracy, and may have utility in primary care for rapid triage.

Performance of the Tobacco, Alcohol, Prescription Medication, and Other Substance Use (TAPS) Tool for Substance Use Screening in Primary Care Patients.

BACKGROUND: Substance use, a leading cause of illness and death, is underidentified in medical practice. OBJECTIVE: The Tobacco, Alcohol, Prescription medication, and other Substance use (TAPS) tool was developed to address the need for a brief screening and assessment instrument that includes all commonly used substances and fits into clinical workflows. The goal of this study was to assess the performance of the TAPS tool in primary care patients. DESIGN: Multisite study, conducted within the National Drug Abuse Treatment Clinical Trials Network, comparing the TAPS tool with a reference standard measure. (ClinicalTrials.gov: NCT02110693). SETTING: 5 adult primary care clinics. PARTICIPANTS: 2000 adult patients consecutively recruited from clinic waiting areas. MEASUREMENTS: Interviewer- and self-administered versions of the TAPS tool were compared with a reference standard, the modified World Mental Health Composite International Diagnostic Interview (CIDI), which measures problem use and substance use disorder (SUD). RESULTS: Interviewer- and self-administered versions of the TAPS tool had similar diagnostic characteristics. For identifying problem use (at a cutoff of 1+), the TAPS tool had a sensitivity of 0.93 (95% CI, 0.90 to 0.95) and specificity of 0.87 (CI, 0.85 to 0.89) for tobacco and a sensitivity of 0.74 (CI, 0.70 to 0.78) and specificity of 0.79 (CI, 0.76 to 0.81) for alcohol. For problem use of illicit and prescription drugs, sensitivity ranged from 0.82 (CI, 0.76 to 0.87) for marijuana to 0.63 (CI, 0.47 to 0.78) for sedatives; specificity was 0.93 or higher. For identifying any SUD (at a cutoff of 2+), sensitivity was lower. LIMITATIONS: The low prevalence of some drug classes led to poor precision in some estimates. Research assistants were not blinded to participants' TAPS tool responses when they administered the CIDI. CONCLUSION: In a diverse population of adult primary care patients, the TAPS tool detected clinically relevant problem substance use. Although it also may detect tobacco, alcohol, and marijuana use disorders, further refinement is needed before it can be recommended broadly for SUD screening. PRIMARY FUNDING SOURCE: National Institute on Drug Abuse.

Effect of Bevacizumab vs Aflibercept on Visual Acuity Among Patients With Macular Edema Due to Central Retinal Vein Occlusion: The SCORE2 Randomized Clinical Trial.

IMPORTANCE: Studies have established the efficacy and safety of aflibercept for the treatment of macular edema due to central retinal vein occlusion. Bevacizumab is used off-label to treat this condition despite the absence of supporting data. OBJECTIVE: To investigate whether bevacizumab is noninferior to aflibercept for the treatment of macular edema secondary to central retinal or hemiretinal vein occlusion. DESIGN, SETTING, AND PARTICIPANTS: The SCORE2 randomized noninferiority clinical trial was conducted at 66 private practice or academic centers in the United States, and included 362 patients with macular edema due to central retinal or hemiretinal vein occlusion who were randomized 1:1 to receive aflibercept or bevacizumab. The first participant was randomized on September 17, 2014, and the last month 6 visit occurred on May 6, 2016. Analyses included data available as of December 30, 2016. INTERVENTIONS: Eyes were randomized to receive intravitreal injection of bevacizumab (1.25 mg; n = 182) or aflibercept (2.0 mg; n = 180) every 4 weeks through month 6. MAIN OUTCOMES AND MEASURES: The primary outcome was mean change in visual acuity (VA) letter score (VALS) from the randomization visit to the 6-month follow-up visit, based on the best-corrected electronic Early Treatment Diabetic Retinopathy Study VALS (scores range from 0-100; higher scores indicate better VA). The noninferiority margin was 5 letters, and statistical testing for noninferiority was based on a 1-sided 97.5% confidence interval. RESULTS: Among 362 randomized participants (mean [SD] age, 69 [12] years; 157 [43.4%] women; mean [SD] VALS at baseline, 50.3 [15.2] [approximate Snellen VA 20/100]), 348 (96.1%) completed the month 6 follow-up visit. At month 6, the mean VALS was 69.3 (a mean increase from baseline of 18.6) in the bevacizumab group and 69.3 (a mean increase from baseline of 18.9) in the aflibercept group (model-based estimate of between-group difference, -0.14; 97.5% CI, -3.07 to ∞; P = .001 for noninferiority), meeting criteria for noninferiority. Ocular adverse events in the aflibercept group included 4 participants with intraocular pressure (IOP) more than 10 mm Hg greater than baseline; ocular adverse events in the bevacizumab group included 1 participant with endophthalmitis (culture negative), 9 with IOP more than 10 mm Hg greater than baseline, 2 with IOP higher than 35 mm Hg, and 1 with angle-closure glaucoma not attributed to the study drug or procedure. CONCLUSIONS AND RELEVANCE: Among patients with macular edema due to central retinal or hemiretinal vein occlusion, intravitreal bevacizumab was noninferior to aflibercept with respect to visual acuity after 6 months of treatment.

Mitochondrial DNA haplogroups confer differences in risk for age-related macular degeneration: a case control study.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly, Caucasian populations. There is strong evidence that mitochondrial dysfunction and oxidative stress play a role in the cell death found in AMD retinas. The purpose of this study was to examine the association of the Caucasian mitochondrial JTU haplogroup cluster with AMD. We also assessed for gender bias and additive risk with known high risk nuclear gene SNPs, ARMS2/LOC387715 (G > T; Ala69Ser, rs10490924) and CFH (T > C; Try402His, rs1061170). METHODS: Total DNA was isolated from 162 AMD subjects and 164 age-matched control subjects located in Los Angeles, California, USA. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the J, U, T, and H mitochondrial haplogroups and the ARMS2-rs10490924 and CFH-rs1061170 SNPs. PCR amplified products were sequenced to verify the nucleotide substitutions for the haplogroups and ARMS2 gene. RESULTS: The JTU haplogroup cluster occurred in 34% (55/162) of AMD subjects versus 15% (24/164) of normal (OR = 2.99; p = 0.0001). This association was slightly greater in males (OR = 3.98, p = 0.005) than the female population (OR = 3.02, p = 0.001). Assuming a dominant effect, the risk alleles for the ARMS2 (rs10490924; p = 0.00001) and CFH (rs1061170; p = 0.027) SNPs were significantly associated with total AMD populations. We found there was no additive risk for the ARMS2 (rs10490924) or CFH (rs1061170) SNPs on the JTU haplogroup background. CONCLUSIONS: There is a strong association of the JTU haplogroup cluster with AMD. In our Southern California population, the ARMS2 (rs10490924) and CFH (rs1061170) genes were significantly but independently associated with AMD. SNPs defining the JTU mitochondrial haplogroup cluster may change the retinal bioenergetics and play a significant role in the pathogenesis of AMD.

Experimental Pathogenicity of H9N2 Avian Influenza Viruses Harboring a Tri-Basic Hemagglutinin Cleavage Site in Sonali and Broiler Chickens.

Low-pathogenic avian influenza (LPAI) H9N2 virus is endemic in Bangladesh, causing huge economic losses in the poultry industry. Although a considerable number of Bangladeshi LPAI H9N2 viruses have been molecularly characterized, there is inadequate information on the pathogenicity of H9N2 viruses in commercial poultry. In this study, circulating LPAI H9N2 viruses from recent field outbreaks were characterized, and their pathogenicity in commercial Sonali (crossbred) and broiler chickens was assessed. Phylogenetic analysis of currently circulating field viruses based on the hemagglutinin (HA) and neuraminidase (NA) gene sequences revealed continuous circulation of G1 lineages containing the tri-basic hemagglutinin cleavage site (HACS) motif (PAKSKR*GLF) at the HA protein. Both the LPAI susceptible Sonali and broiler chickens were infected with selected H9N2 isolates A/chicken/Bangladesh/2458-LT2/2020 or A/chicken/Bangladesh/2465-LT56/2021 using intranasal (100 µL) and intraocular (100 µL) routes with a dose of 106 EID50/mL. Infected groups (LT_2-So1 and LT_56-So2; LT_2-Br1 and LT_56-Br2) revealed no mortality or clinical signs. However, at gross and histopathological investigation, the trachea, lungs, and intestine of the LT_2-So1 and LT_56-So2 groups displayed mild to moderate hemorrhages, congestion, and inflammation at different dpi. The LT 2-Br1 and LT 56-Br2 broiler groups showed nearly identical changes in the trachea, lungs, and intestine at various dpi, indicating no influence on pathogenicity in the two commercial bird species under study. Overall, the prominent lesions were observed up to 7 dpi and started to disappear at 10 dpi. The H9N2 viruses predominantly replicated in the respiratory tract, and higher titers of virus were shed through the oropharyngeal route than the cloacal route. Finally, this study demonstrated the continuous evolution of tri-basic HACS containing H9N2 viruses in Bangladesh with a low-pathogenic phenotype causing mild to moderate tracheitis, pneumonia, and enteritis in Sonali and commercial broiler chickens.

Characterization of a Novel African Swine Fever Virus p72 Genotype II from Nigeria.

African swine fever (ASF) is a high-consequence transboundary hemorrhagic fever of swine. It continues to spread across the globe causing socio-economic issues and threatening food security and biodiversity. In 2020, Nigeria reported a major ASF outbreak, killing close to half a million pigs. Based on the partial sequences of the genes B646L (p72) and E183L (p54), the virus responsible for the outbreak was identified as an African swine fever virus (ASFV) p72 genotype II. Here, we report further characterization of ASFV RV502, one of the isolates obtained during the outbreak. The whole genome sequence of this virus revealed a deletion of 6535 bp between the nucleotide positions 11,760-18,295 of the genome, and an apparent reverse complement duplication of the 5' end of the genome at the 3' end. Phylogenetically, ASFV RV502 clustered together with ASFV MAL/19/Karonga and ASFV Tanzania/Rukwa/2017/1 suggesting that the virus responsible for the 2020 outbreak in Nigeria has a South-eastern African origin.

Neighbourhood watch: genomic epidemiology of SARS-CoV-2 variants circulating in a German federal state, Mecklenburg-Western Pomerania, in 2020-2022.

ABSTRACTGlobal and even national genome surveillance approaches do not provide the resolution necessary for rapid and accurate direct response by local public health authorities. Hence, a regional network of microbiological laboratories in collaboration with the health departments of all districts of the German federal state of Mecklenburg-Western Pomerania (M-V) was formed to investigate the regional molecular epidemiology of circulating SARS-CoV-2 lineages between 11/2020 and 03/2022. More than 4750 samples from all M-V counties were sequenced using Illumina and Nanopore technologies. Overall, 3493 (73.5%) sequences fulfilled quality criteria for time-resolved and/or spatially-resolved maximum likelihood phylogenic analyses and k-mean/ median clustering (KMC). We identified 116 different Pangolin virus lineages that can be assigned to 16 Nextstrain clades. The ten most frequently detected virus lineages belonged to B.1.1.7, AY.122, AY.43, BA.1, B.1.617.2, BA.1.1, AY.9.2, AY.4, P.1 and AY.126. Time-resolved phylogenetic analyses showed the occurrence of virus clades as determined worldwide, but with a substantial delay of one to two months. Further spatio-temporal phylogenetic analyses revealed a regional outbreak of a Gamma variant limited to western M-V counties. Finally, KMC elucidated a successive introduction of the various virus lineages into M-V, possibly triggered by vacation periods with increased (inter-) national travel activities. The COVID-19 pandemic in M-V was shaped by a combination of several SARS-CoV-2 introductions, lockdown measures, restrictive quarantine of patients and the lineage specific replication rate. Complementing global and national surveillance, regional surveillance adds value by providing a higher level of surveillance resolution tailored to local health authorities.

Genuine lethal infection of a wood pigeon (Columba palumbus) with high pathogenicity avian influenza H5N1, clade 2.3.4.4b, in Germany, 2022.

Despite the increasing frequency of avian influenza (AI) cases in wild birds in Europe during the last decade, doves and pigeons were not recognized to be part of the AI epidemiology. Here we describe a natural, lethal HPAIV infection of subtype H5N1, clade 2.3.4.4b, in a wood pigeon (Columba palumbus) in Germany, 2022. The animal was housed in a bird sanctuary that suffered an HPAI-outbreak with multiple bird species affected. The pigeon's post mortem findings were dominated by an acute lymphohistiocytic meningoencephalitis as well as neuronal necrosis in the grey matter of the cerebral hemispheres and in the brain stem. Influenza A viral antigen was associated to these alterations with a striking ependymal and periventricular distribution most probably indicating intraventicular liquorogenic spread of the virus. In addition, typical severe multifocal to coalescing necrotizing pancreatitis was evident. Occasionally, vascular endothelial cells showed an intense viral antigen staining. Examination of oropharyngeal and cloacal swabs and of various tissues by real-time RT-PCR corroborated systemic infection with highly pathogenic (HP) AIV of subtype H5N1. Viral loads soared in brain and pancreas samples. Full genome sequencing directly from brain tissue confirmed the assignment of the virus to clade 2.3.4.4b of the goose/Guangdong lineage bearing a polybasic hemagglutinin cleavage site. Our results demonstrate the principal susceptibility of wood pigeons to HPAIV H5N1 resulting in a sporadic infection. Considering the wide distribution and roaming of wild, feral, and racing pigeons with often close contact to humans, thorough investigation of suspected cases including pathological manifestation is advisable.