Virus Isolation Research Articles

Genetic evolutionary analysis of a strain of Senecavirus A in Anhui and the establishment of its detection method

BackgroundSenecavirus A (SVA) is the only member of the genus Senecavirus in the family Picornaviridae, and is one of the pathogens of porcine blistering disease. SVA has been reported in the United States, Canada, China, Thailand, and Colombia. MethodsIn this study, positive SVA infection was detected by RT-PCR in sick materials collected from pig farms of different sizes in Anhui Province. ResultsIn this study, a virulent strain of SVA was successfully obtained by viral isolation on BHK21 cells and named SVA-CH-AHAU-1. Meanwhile, a simple, rapid and accurate nano-PCR method for the detection of SVA infection was established in this study, using the recombinant plasmid pClone-SVA-3D as a template. ConclusionsThe complete genome of SVA-CH-AHAU-1 is 7286 bp, including a 5′ non-coding region (UTR), an open reading frame (ORF) of 6546 nucleotides, encoding 2182 amino acids (aa), and a 3’ UTR with Poly(A) features, and phylogenetic analysis showed that this isolate had the highest nucleotide homology (97.9 %) with the US isolate US-15-41901SD. In this study, the virulent strain SVA-CH-AHAU-1 was found to recombine in the ORF region with isolates SVA-CH-SDGT-2017 and SVA/Canada/ON/FMA-2015-0024 T2/2015. The complete genome has been submitted to GeneBank with the accession number OM654411. In addition, our results suggest that the established nano-PCR assay can be used as an economical, reliable and sensitive method for the field diagnosis of SVA method, especially in resource-limited areas.

Clinical and etiological characteristics of severe hemorrhagic fever caused by coinfection of hantaan orthohantavirus and severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) and hemorrhagic fever with renal syndrome (HFRS) usually have different infection routes, and coinfection is relatively rare. This study examines the clinical and etiological characteristics of coinfection by these two pathogens to provide important references for clinical diagnosis and treatment. Blood samples from 22 clinically diagnosed patients with HFRS were collected for molecular detection of HFRS and common tick and mouse borne diseases. Inoculate the blood of six severe and critically patients into cells to isolate and proliferate potential viruses, and retest the cell culture to determine the pathogen. In addition, complete data were collected from these 22 HFRS and concurrent SFTS patients, and white blood cells (WBCs), platelet (PLT), blood urea nitrogen (BUN), creatinine (Cr) and other data were compared and analyzed. A total of 31 febrile patients, including 22 HFRS patients and 9 SFTS patients, were collected from September 2021 to October 2022. Among these HFRS patients, 11 were severe or critical. Severe and critical HFRS patients were characterized by rodent exposure history, pharyngeal and conjunctival hyperemia, abnormal WBC and PLT counts, and elevated BUN and Cr values. Virus isolation and molecular detection on blood samples from 6 patients showed that three of the six severe patients were positive for hantaan virus (HTNV), and two of the three HTNV positives were also positive for SFTS bunyavirus (SFTSV). The two coinfected patients exhibited different clinical and laboratory characteristics compared to those infected by either virus alone. Coinfection of HTNV and SFTSV leads to severe and complex hemorrhagic fever. Laboratory characteristics, such as the indicators of WBC, PLT, BUN, and Cr, may differ between HFRS and SFTS. These findings have implications and provide references for the diagnosis and treatment of coinfected cases.

Febrile young infants and the association with enterovirus infection

BackgroundEnterovirus is a common pediatric infectious disease, but the epidemiological data in young infants were lacking. This study aims to evaluate the role of enterovirus in febrile young infants and identify risk factors for severe infections. MethodsWe enrolled febrile infants younger than 90 days admitted to National Taiwan University Hospital from January 2010 to June 2021. Enterovirus infection was confirmed via viral isolation or pan-enterovirus PCR. Central nervous system involvement was defined by positive culture or PCR in cerebrospinal fluid. Severe complications included sepsis, hepatic failure, myocarditis, shock, encephalitis, acute kidney injury, respiratory failure, and multiorgan failure. ResultsOut of 840 febrile infants, 17.4% (n = 146) had enterovirus infection. Among these, 46% (n = 67) presented with meningitis and/or encephalitis. Early-onset enterovirus infection within the first two weeks of life was significantly linked to increased risks of anemia (hemoglobin <9 g/dL), ICU admission, central nervous system involvement, shock, hepatic failure, and mortality. Multivariable logistic regression identified high-risk serotypes (aOR 17.4, [95% CI 1.58, 191.5], p = 0.019) and hemoglobin <9 g/dL (aOR 44.9, [95% CI 5.6, 357.6], p < 0.001) as significant risk factors for severe complications. ConclusionsEnterovirus accounted for 17.4% of the etiology in febrile young infants and the case-fatality rate was 2%. Febrile young infants who had risk factors of enterovirus infection should consider viral culture or PCR examination for confirmation.

Characteristics and predictors of outcome in children with severe acute bronchiolitis: A 10-yearexperience

BackgroundSevere acute bronchiolitis (SAB) can be life-threatening for infants and may be responsible for the congestion of intensive care units (ICU) during epidemics. We aimed to study the clinical and paraclinical characteristics of patients with SAB requiring a transfer to the ICU in order to examine their outcomes and to identify the predictors of a stay of ≥7 days and/or death. MethodsThis was a cross-sectional retrospective study including infants aged ≤12 months transferred to the ICU for their first episode of SAB between 1 January 2010 and 31 December 2019. ResultsWe collected data on 380 patients with a median age of 1.75 months. They had a history of prematurity (20.53 %), low birth weight (18.68 %), parental atopy (12.89 %), and comorbidity (7.37 %, mainly congenital heart disease [5 %]). The leading cause of transfer was hypoxemia and increased oxygen requirements (49.73 %). The patients required mechanical ventilation (MV) in 63.42 % of the cases and noninvasive ventilation (NIV) in 67.63 %. NIV has supplanted MV over the years. Its use has increased from 40.4 % in 2010 to 96 % in 2019 compared with 83.84 % and 42 % for MV. A total of 14 (3.68 %) patients died. The independent predictors of a stay of ≥7 days and/or death were young age ≤2 months (p = 0.002), failure to thrive (p = 0.006), apnea (p = 0.045), dehydration (p = 0.018), the presence of biological inflammatory reaction (p = 0.002), isolation of respiratory syncytial virus (p < 0.001), and bacterial coinfection (p = 0.013).NIV was a protective factor (p < 0.001). A severity score ranging from 0 to 17 was established with an optimal cut-off value of 5 points. ConclusionSpecific caution is needed in patients with these severity predictors. The generalization ofNIV in general pediatrics departments would improve SAB management and reduce transfers to the ICU.

Thrip transmission patterns of peanut bud necrosis virus (PBNV) and Tobacco streak virus (TSV) isolates of blackgram and greengram of Andhra Pradesh

Thrip transmission patterns of peanut bud necrosis virus (PBNV) and Tobacco streak virus (TSV) isolates of blackgram and greengram of Andhra Pradesh

Symptomatology and particle morphology studies of necrosis causing Peanut bud necrosis virus (PBNV) and Tobacco streak virus (TSV) isolates of blackgram and greengram of Andhra Pradesh

Symptomatology and particle morphology studies of necrosis causing Peanut bud necrosis virus (PBNV) and Tobacco streak virus (TSV) isolates of blackgram and greengram of Andhra Pradesh

Experimental and natural host range studies of Tobacco streak virus (TSV) isolates of Blackgram and Greengram of Telangana using DAC-ELISA

Experimental and natural host range studies of Tobacco streak virus (TSV) isolates of Blackgram and Greengram of Telangana using DAC-ELISA

Introduction and temporospatial tracing of piscine orthoreovirus-1 (PRV-1) in Norwegian farmed Atlantic salmon (Salmo salar) after local fallowing.

Piscine orthoreovirus-1 (PRV-1) is a prevalent agent in Atlantic salmon (Salmo salar) and the causative agent of heart and skeletal muscle inflammation (HSMI), an important disease in farmed Atlantic salmon. Investigations into the introduction and dissemination routes of PRV-1 in a field setting have been limited. This study aimed to better understand PRV-1 infections and HSMI-associated mortality under field conditions. We tracked introduction and spread of PRV-1 over one production cycle in a geographically isolated region in Norwegian aquaculture. From five sites, a total of 32 virus isolates were sequenced and genogrouped. The results indicated multiple introductions of PRV-1 to the area, but also revealed a high level of genetic homogeneity among the virus variants. The variants differed from that of the previous production cycle at two out of three sites investigated, suggesting that synchronized fallowing can be a useful tool for preventing dissemination of PRV-1 between generations of fish. Exposure to PRV-1 at the freshwater stage was identified as a potential source of introduction. A low level of HSMI-associated mortality was observed at all sites, with the onset of mortality showing some variation across PRV-1 genogroups. However, the study highlighted the complexity of associating viral genogroups with mortality in a field setting. Overall, this study contributes valuable insights into PRV-1 dynamics in a real-world aquaculture setting, offering potential strategies for disease management and prevention.

Wastewater Surveillance of SARS-CoV-2: A Comparison of Two Concentration Methods.

Wastewater surveillance is crucial for the epidemiological monitoring of SARS-CoV-2. Various concentration techniques, such as skimmed milk flocculation (SMF) and polyethylene glycol (PEG) precipitation, are employed to isolate the virus effectively. This study aims to compare these two methods and determine the one with the superior recovery rates. From February to December 2021, 24-h wastewater samples were collected from the Ioannina Wastewater Treatment Plant's inlet and processed using both techniques. Subsequent viral genome isolation and a real-time RT-qPCR detection of SARS-CoV-2 were performed. The quantitative analysis demonstrated a higher detection sensitivity with a PEG-based concentration than SMF. Moreover, when the samples were positive by both methods, PEG consistently yielded higher viral loads. These findings underscore the need for further research into concentration methodologies and the development of precise protocols to enhance epidemiological surveillance through wastewater analysis.

Circulating serotypes and genotypes of dengue virus during the 2023 outbreak in Eastern Nepal

Dengue virus (DENV) is one of the most significant mosquito-borne diseases in Nepal. In 2023, DENV outbreaks began in Eastern Nepal, near the border with India, and rapidly spread nationwide. The study aims to describe the outbreak's epidemiological pattern, laboratory characteristics, DENV serotypes, and genotypes. A hospital-based cross-sectional study was conducted in four hospitals in Jhapa, Eastern Nepal, in 2023. Acute serum samples were obtained from dengue suspected patients within 7 days of illness and subjected to virus isolation, conventional and real-time polymerase chain reaction (RT-PCR), and phylogenetic analysis. Out of 60 samples, 42 (70 %), 11 (18.3 %) and 7 (11.7 %) were primary, secondary and non-dengue infection, respectively. Among 53 dengue confirmed patients, 46 (86.7 %) were positive for NS1 and 12 (22.6 %) were positive for both NS1 and IgM. Out of 42 dengue isolates, a new clade of the cosmopolitan genotype of DENV-2 was the most prevalent (28, 66.7 %), followed by genotype III of DENV-3 (11, 26.2 %) and genotype V of DENV-1 (3, 7.1 %). Genotype III of DENV-3 was first introduced in 2022–2023 in Nepal. Phylogenetic analysis of the E gene revealed the DENV-2 isolates from Nepal had 98 % homologous nucleotide similarity with the strains from India and Bangladesh. To our knowledge, this is the first report of circulating serotypes and genotypes of DENV in Jhapa. Integrating molecular findings into the dengue control plan can enhance surveillance efforts, monitor disease trends, and implement proactive measures to reduce the burden of dengue and prevent fatalities in future outbreaks.

Evaluation of a qualified MDCK cell line for virus isolation to develop cell-based influenza vaccine viruses with appropriate antigenicity

We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.

Genome sequencing of canine distemper virus isolates from unvaccinated dogs in Mongolia

Canine distemper virus (CDV) triggers a severe, often fatal disease in dogs and wildlife known as canine distemper (CD). Prior research has noted significant genetic diversity and recombination among CDV isolates from different geographical regions, potentially contributing to vaccine failures. Despite this, no genetic characterization of Mongolian CDVs has been conducted. This study, isolated CDVs from three unvaccinated dogs: two 10-month-old mixed-breeds and an 18-month-old Samoyed. All exhibited CD symptoms and subsequently died. Virus isolation was conducted using Vero/dog SLAM cells, with genome sequencing performed via nanopore technology. The mixed-breed dogs were infected with non-recombinant CDV isolates, forming a sister clade to the Asia-1 lineage prevalent in Asia. The Samoyed was infected with a non-recombinant CDV isolate, classifying as Asia-4 lineage sporadically reported in some Asian countries. This sequencing data offers foundational information on genetic diversity, aiding CD control measure development and benefiting future Eurasia and Asian studies.

Molecular characteristic, evolution, and pathogenicity analysis of avian infectious bronchitis virus isolates associated with QX type in China

Infectious bronchitis virus (IBV) is one of the major avian pathogens plaguing the global poultry industry. Although vaccination is the primary preventive measure for IBV infection, the emergence of virus variants with mutations and recombination has resulted in IBV circulating globally, presenting a challenge for IB control. Here, we isolated 3 IBV strains (CZ200515, CZ210840, and CZ211063) from suspected sick chickens vaccinated with IBV live attenuated vaccines (H120, 4/91, or QXL87). Phylogenetic analysis of the S1 gene sequence of the spike (S) revealed that the 3 isolates belonged to the QX-type (GI-19 lineage). Whole genome sequencing and recombination analysis indicated that CZ200515 and CZ210840 contained genetic material from 4/91 and Scyz3 (QX-type), possibly due to recombination between the circulating strain and the 4/91 vaccine strain, while no evidence of recombination was found in CZ211063. Pathogenicity analysis in 1-day-old specific pathogen-free (SPF) chickens demonstrated that all 3 isolates caused severe tissue damage and varying degrees of mortality. Virus cross-neutralization assay revealed decreased antigen relatedness between the isolates and the QX-type vaccine strain (QXL87). Amino acid sequence homology analysis of S1 revealed 5%-6.5% variances between the isolates and QXL87. Analysis of the S1 subunit structure revealed that mutations of amino acid residues in the hypervariable region (HVR) and the neutralizing epitope region resulted in antigenic variation in isolates by changing the antigen conformation. Our data indicate antigenicity variances between QX isolates and QXL87 vaccine strains, potentially resulting in immune evasion occurrences. Overall, these results offer crucial insights into the epidemiology and pathogenicity of QX-type IBV, facilitating improved selection and formulation of vaccines for disease management.

Isolation, Identification and Molecular Characterization of Lumpy Skin Disease Virus for Development of Vaccine

The highly contagious Lumpy skin disease (LSD) virus has been rapidly spreading across South and East Asia, posing a grave threat to cattle populations in the region. LSD initially emerged in Bangladesh during July 2019, starting in the Chattogram district and rapidly spreading across the entire country. To investigate cases of LSD in cattle, a comprehensive study was conducted in various districts of Bangladesh, including Natore, Kurigram, Dinajpur, Dhaka, and Mymensingh. A total of 58 samples comprising blood, skin scraping, buccal swabs, and nasal swabs were collected for analysis. In order to identify LSDV, the DNA was isolated from the processed samples, and a conentional PCR method was employed utilizing the specific primer designed for the 'P-32 Gene' gene. Out of the blood samples tested, only 29.17 percent yielded a positive result for LSDV, whereas a substantial 80 percent of the skin samples tested positive. This suggests that skin scrapings are a viable specimen for LSD diagnosis using PCR. Five positive samples were partially sequenced utilizing the "ANK Gene" for molecular characterization. Bangladeshi isolates have a strong association with LSDV Kenyan (KSGP 0240, NI-2490) strains and Indian isolates (Ranchi, 2019) strains, based on multiple sequence alignment and phylogenetic analysis. Additionally, the sequencing of all the samples from the five research sites were almost identical, suggesting that a single strain is probably spreading throughout the nation. These findings emphasize the need for continuous monitoring of the genetic makeup and molecular epidemiology of LSDV, which will be useful in the future for the development of vaccines specific to the strains that are then in circulation. J. of Sci. and Tech. Res. 5(1): 31-36, 2023

Antiviral effect of the viroporin inhibitors against Taiwan isolates of infectious bronchitis virus (IBV)

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy.In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4’-diisothiocyano stilbene-2,2’-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

Emergence of Two Different Genotypes of Bagaza Virus (BAGV) Affecting Red-Legged Partridges in Spain, in 2019 and 2021.

Bagaza virus (BAGV) is a flavivirus that affects avian species. In Europe, it was detected for the first time in Spain in 2010, exhibiting high genetic relatedness to Israel turkey meningoencephalomyelitis virus (ITMV) isolates from Israel. After a period of epidemiological silence, BAGV re-emerged, causing important outbreaks in 2019 and 2021. This study aims to characterize the newly detected strains and to elucidate if these recent outbreaks were caused by single or different virus introductions into the country. Hence, Spanish BAGV isolates from 2019 (n = 3) and 2021 (n = 1) outbreaks, obtained from red-legged partridges in Cádiz, were sequenced and further characterized. The phylogenetic analyses showed that they belong to two different genotypes: BAGV-Genotypes 1 and 2. Isolates from 2019 belong to BAGV-Genotype 1, closely related to isolates from Senegal, where BAGV has been circulating for decades. In turn, the 2021 isolates belong to BAGV-Genotype 2, closely related to those detected in Spain in 2010. Additionally, the comparison of the viral polyproteins of several BAGV isolates from both genotypes supports and confirms the phylogenetic findings. To conclude, BAGV has been introduced into Spain on at least three independent occasions, with alternating genetic clades, thus confirming that BAGV is able to sporadically reach Southern Europe.

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

Presumptive West Nile Virus infection in a Komodo Dragon (Varanus komodoensis)

Abstract A 3-yr-old, male Komodo dragon (Varanus komodoensis) presented acutely in early October 2021 with an abnormal posture and asymmetric tetraparesis. The subadult lizard had spent the previous 4 months either in an outdoor space or in an indoor space with open windows. Physical examination, radiographs, and complete blood count and chemistry panel failed to reveal the cause of the neurologic signs. West Nile virus (WNV) plaque reduction serum neutralization on presentation was negative (1:20), as was WNV polymerase chain reaction (PCR) testing and viral isolation on whole blood. Empirical treatment with a daily corticosteroid, course of antibiotics, and encouragement of mild exercise was initiated. Magnetic resonance imaging of the brain and spinal cord was unremarkable, and a lumbar cerebrospinal fluid (CSF) tap revealed a mononuclear pleocytosis and protein elevation. Follow-up WNV serology revealed rising titer and seroconversion 13 days after presentation (1:80) and 32 days after presentation (1:640); however, WNV PCR testing of CSF fluid was negative. Thi Komodo dragon gradually improved over six weeks before being put back on public display 74 days after presentation. The WNV titer remained high (1:640) through the winter of 2021-22, and an anamnestic response was observed following a vaccination series with a killed equine vaccine in the spring of 2022. There is a previous brief report of WNV infection in a captive crocodile monitor (Varanus salvadorii), and an unpublished case in the same species at the first author’s institution. This is the first report of neurologic disease from presumptive WNV infection in a Komodo dragon. We recommend that WNV infection be a differential diagnosis if a varanid lizard present with acute neurologic signs with a history of arthropod vector exposure, particularly in the summer or fall.

The First Isolation of Insect-Specific Alphavirus (Agua Salud alphavirus) in Culex (Melanoconion) Mosquitoes in the Brazilian Amazon.

Advances in diagnostic techniques coupled with ongoing environmental changes have resulted in intensified surveillance and monitoring of arbovirus circulation in the Amazon. This increased effort has resulted in increased detection of insect-specific viruses among hematophagous arthropods collected in the field. This study aimed to document the first isolation of Agua Salud alphavirus in mosquitoes collected within the Brazilian Amazon. Arthropods belonging to the family Culicidae were collected within a forest fragment located in the Environmental Protection Area of the metropolitan region of Belem. Subsequently, these specimens were meticulously identified to the species level. Afterward, the collected batches were macerated, and the resulting supernatant was then inoculated into C6/36 and Vero cell cultures to facilitate viral isolation. The presence of arboviruses within the inoculated cell cultures was determined through indirect immunofluorescence analysis. Furthermore, positive supernatant samples underwent nucleotide sequencing to precisely identify the viral strains present. Notably, a batch containing Culex (Melanoconion) mosquitoes was identified to be positive for the genus Alphavirus via indirect immunofluorescence. This study is the first report on insect-specific alphavirus isolation in Brazil and the first-ever description of Agua Salud alphavirus isolation within Amazon Forest remnants.

Surveillance of avian influenza viruses in Hebei Province of China from 2021 to 2023: Identification of a novel reassortant H3N3

Avian influenza remains a global public health concern for its well-known point mutation and genomic segment reassortment, through which plenty of serum serotypes are generated to escape existing immune protection in animal and human populations. Some occasional cases of human infection of avian influenza viruses (AIVs) since 2020 posed a potential pandemic risk through human-to-human transmission. Both east-west and north-south migratory birds fly through and linger in the Hebei Province of China as a stopover habitat, providing an opportunity for imported AIVs to infect the local poultry and for viral gene reassortment to generate novel stains. In this study, we collected more than 6000 environmental samples (mostly feces) in Hebei Province from 2021 to 2023. Samples were screened using real-time RT-PCR, and virus isolation was performed using the chick embryo culture method. We identified 10 AIV isolates, including a novel reassortant H3N3 isolate. Sequencing analysis revealed these AIVs are highly homologous to those isolated in the Yellow River Basin. Our findings supported that AIVs keep evolving to generate new isolates, necessitating a continuous risk assessment of local avian influenza in wild waterfowl in Hebei, China.