Detection and genomic characterization of an avian influenza virus A/mute swan/Mangystau/1-S24R-2/2024 (H5N1; clade 2.3.4.4b) strain isolated from the lung of a dead swan in Kazakhstan.

The influenza virus strain A/mute swan/Mangystau/1-S24R-2/2024 (H5N1; clade 2.3.4.4b) was isolated in embryonated chicken eggs from the lung of a dead swan found around Lake Karakol (Kazakhstan) during a highly pathogenic avian influenza outbreak in 2024. The aim of this study was to characterize the genetic profile of the isolated strain.

Metagenomic profiling of nasopharyngeal samples from adults with acute respiratory infection.

Diagnosis of acute respiratory infections (ARIs) is challenging due to the broad diversity of potential microbial causes. We used metagenomic next-generation sequencing (mNGS) to analyze the nasopharyngeal virome of ARI patients, who had undergone testing with a clinical multiplex PCR panel (Amplisens ARVI-screen-FRT). We collected nasopharyngeal swabs from 49 outpatient adults, 32 of whom had ARI symptoms and were PCR-positive, and 4 asymptomatic controls in Kazakhstan during Spring 2021. We assessed the biodiversity of the mNGS-derived virome and concordance with PCR results. PCR identified common ARI viruses in 65% of the symptomatic cases. mNGS revealed viral taxa consisting of human, non-human eukaryotic and bacteriophage groups, comprising 15, 11 and 28 genera, respectively. Notable ARI-associated human viruses included rhinovirus (16.3%), betaherpesvirus 7 (14.3%) and Epstein-Barr virus (8.16%). The primary phage hosts were Streptococcus spp. (32.7%), Pseudomonas aeruginosa (24.5%) and Burkholderia spp. (20.4%). In total, 47% of ARIs were linked solely to bacterial pathogens, a third to viral-bacterial co-infections, and less than 10% to only viral infections by mNGS. PCR showed low concordance with mNGS, except for rhinovirus. These results underscore the importance of broad diagnostic methods and question the effectiveness of commonly used PCR panels in ARI diagnosis.

Full genome sequence of a human rhinovirus A1B, obtained in Kazakhstan.

Here, we report the full nucleotide sequence of the RvA1B/KZ/2021/87 rhinovirus, identified through metagenomic sequencing of nasopharyngeal swabs collected from patients exhibiting respiratory symptoms in Kazakhstan during 2021.

Evaluation of a novel real-time polymerase chain reaction assay for identifying H3 equine influenza virus in Kazakhstan.

Background and Aim: Equine influenza (EI) is a highly contagious disease that causes fever and upper respiratory tract inflammation. It is caused by influenza virus A, belonging to the Orthomyxoviridae family, with subtypes H3N8 and H7N7. This study presents data on the development of a real-time polymerase chain reaction (RT-PCR) assay using TaqMan probes to detect the H3 subtype of EI virus (EIV). Materials and Methods: The evaluation of the developed RT-PCR assay involved five strains of EIV as positive controls and ten nasopharyngeal swab samples collected from horses. RNA was isolated using the GeneJet Viral DNA and RNA Purification Kit, and primers and probes were designed using the Integrated DNA Technology PrimerQuest Tool. The assay was optimized by investigating the annealing temperature, primer and probes concentrations, sensitivity, and specificity. Sequencing was performed using the Thermo Fisher 3130 Genetic Analyzer, and the evolutionary history was inferred using the Neighbor-Joining method. Results: The designed primers and probes, targeting the H3 gene, were found to be specific to the EIV. The RT-PCR assay was capable of detecting as low as 50 femtogram (f) or 3 × 103 copies of genomic RNA. No cross-reactions were observed with other respiratory viral and bacterial pathogens, indicating the high specificity of the assay. To evaluate its effectiveness, ten nasopharyngeal swab samples collected from farms in North Kazakhstan regions during disease monitoring were analyzed. The accuracy of the analysis was confirmed by comparing the results with those obtained from a commercial RT-PCR assay for EI identification. The developed RT-PCR assay exhibited high sensitivity and specificity for detecting the EIV. Conclusion: The results demonstrate that the developed RT-PCR assay is suitable for diagnosing EI. This simple, highly sensitive, and specific assay for detecting H3 EIV can be a reliable tool for diagnosing and surveilling EI. Implementing this RT-PCR assay in veterinary practice will enhance and expedite the timely response to potential outbreaks of EI, thus positively impacting the overall epizootic well-being of EI in Kazakhstan.

Characterization of viral pathogens associated with symptomatic upper respiratory tract infection in adults during a low COVID-19 transmission period.

Background: The epidemiology of respiratory tract infections (RTI) has dramatically changed over the course of the COVID-19 pandemic. A major effort in the clinical management of RTI has been directed toward diagnosing COVID-19, while the causes of other, common community RTI often remain enigmatic. To shed light on the etiological causes of RTI during a low COVID-19 transmission period in 2021, we did a pilot study using molecular testing for virologic causes of upper RTI among adults with respiratory symptoms from Almaty, Kazakhstan. Methods: Adults presenting at two public hospitals with respiratory symptoms were screened using SARS-CoV-2 PCR on nasopharyngeal swabs. A subset of RTI+, COVID-19-negative adults (n = 50) was then tested for the presence of common RTI viruses and influenza A virus (IAV). Next generation virome sequencing was used to further characterize the PCR-detected RTI pathogens. Results: Of 1,812 symptomatic adults, 21 (1.2%) tested SARS-CoV-2-positive. Within the COVID-19 negative outpatient subset, 33/50 subjects (66%) had a positive PCR result for a common community RTI virus, consisting of human parainfluenza virus 3-4 (hPIV 3-4) in 25/50 (50%), rhinovirus (hRV) in 2 (4%), hPIV4-hRV co-infection in four (8%) and adenovirus or the OCR43/HKU-1 coronavirus in two (4%) cases; no IAV was detected. Virome sequencing allowed to reconstruct sequences of most PCR-identified rhinoviruses and hPIV-3/human respirovirus-3. Conclusions: COVID-19 was cause to a low proportion of symptomatic RTI among adults. Among COVID-negative participants, symptomatic RTI was predominantly associated with hPIV and hRV. Therefore, respiratory viruses other than SARS-CoV-2 should be considered in the clinical management and prevention of adult RTI in the post-pandemic era.

Development and evaluation of a multiplex polymerase chain reaction in real-time for differential diagnosis of Moraxella-induced keratoconjunctivitis in livestock.

Background and Aim: Infectious bovine keratoconjunctivitis (IBK) is a prevalent ocular disease that affects livestock, leading to substantial economic losses due to reduced production and culling of infected animals. Moraxella spp. is common bacterial pathogens that can cause keratoconjunctivitis in livestock. Therefore, rapid and accurate diagnosis is crucial for effective treatment and disease control. This study aimed to develop a multiplex real-time polymerase chain reaction (mRT-PCR) assay for the detection and differentiation of Moraxella bovoculi, Moraxella ovis, and Moraxella bovis. Materials and Methods: Three reference strains of Moraxella as positive controls and 36 lacrimal swab samples collected from cattle were used to evaluate the developed mRT-PCR assay DNA extraction that was performed using the RIBO-sorb DNA/RNA extraction kit. Primers and probes were designed using the SpeciesPrimer pipeline. The annealing temperature, primer and probe concentrations, and sensitivity and specificity of the assay were optimized. Results: An mRT-PCR assay was developed to detect pathogens associated with IBK in cattle on the basis of optimized parameters. The specificity and sensitivity of this assay were confirmed using samples containing individual pathogens (O - M. ovis, B - M. bovis, and BO - M. bovoculi), combinations of two pathogens (O-B, B-BO, and O-BO), and when the DNA of all three pathogens was present in a single reaction (O-B-BO). The analytical sensitivity of mRT-PCR for detecting M. ovis and M. bovoculi DNA was 21 copies or 50 fg per reaction, whereas that for M. bovis was 210 copies or 500 fg per reaction. In addition, this assay has been tested on samples isolated from the affected eyes of cattle in the Akmola region of the Republic of Kazakhstan. Conclusion: For the first time in the Republic of Kazakhstan, the proposed mRT-PCR assay for the simultaneous detection of three Moraxella spp. pathogens has been developed. This assay exhibits the required specificity and high sensitivity for m RT-PCR, facilitating the timely implementation of effective measures for disease control and the prevention of economic losses. These losses are linked to a reduction in livestock breeding value, a reduction in meat and milk production, a reduction in the reproductive performance of heifers, resulting in fewer offspring, as well as costs related to the treatment of affected animals.

Next Generation Sequencing Approaches to Characterize the Respiratory Tract Virome.

The COVID-19 pandemic and heightened perception of the risk of emerging viral infections have boosted the efforts to better understand the virome or complete repertoire of viruses in health and disease, with a focus on infectious respiratory diseases. Next-generation sequencing (NGS) is widely used to study microorganisms, allowing the elucidation of bacteria and viruses inhabiting different body systems and identifying new pathogens. However, NGS studies suffer from a lack of standardization, in particular, due to various methodological approaches and no single format for processing the results. Here, we review the main methodological approaches and key stages for studies of the human virome, with an emphasis on virome changes during acute respiratory viral infection, with applications for clinical diagnostics and epidemiologic analyses.

Complete Coding Genome Sequence of an Influenza A/H3N8 Equine Virus Isolated in Kazakhstan in 2007.

Here, we reported the complete coding sequence of the influenza A/equine/Otar/3/2007 (H3N8) equine virus, first isolated in Kazakhstan in 2007. The hemagglutinin (HA) sequences of the Kazakhstan isolates appeared to be closely related to viruses isolated in early 2000 in Asia. Phylogenetic analysis characterized the Kazakhstan isolates as a member of the Florida sublineage clade 2 by the HA protein sequence.

Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens.

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

Near-Complete Genome Sequence of an H5N1 Avian Influenza Virus Strain Isolated from a Swan in Southwest Kazakhstan in 2006.

We report the near-complete genome sequence of an influenza H5N1 virus strain isolated from a dead swan on the southeastern Caspian seashore in 2006. The results of the surface protein HA phylogenetic analysis showed that the A/swan/Mangystau/3/2006 virus belongs to the EA-nonGsGD clade.

Discovery and Characterization of Novel Bat Coronavirus Lineages from Kazakhstan.

Coronaviruses are positive-stranded RNA viruses that infect a variety of hosts, resulting in a range of symptoms from gastrointestinal illness to respiratory distress. Bats are reservoirs for a high diversity of coronaviruses, and focused surveillance detected several strains genetically similar to MERS-coronavirus, SARS-coronavirus, and the human coronaviruses 229E and NL63. The bat fauna of central Asia, which link China to eastern Europe, are relatively less studied than other regions of the world. Kazakhstan is the world's ninth largest country; however, little is understood about the prevalence and diversity of bat-borne viruses. In this study, bat guano was collected from bat caves in three different sites of southern Kazakhstan that tested positive for coronaviruses. Our phylogenetic reconstruction indicates these are novel bat coronaviruses that belong to the genus Alphacoronavirus. In addition, two distinct lineages of Kazakhstan bat coronaviruses were detected. Both lineages are closely related to bat coronaviruses from China, France, Spain, and South Africa, suggesting that co-circulation of coronaviruses is common in multiple bat species with overlapping geographical distributions. Our study highlights the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases.

Complete Genome Sequencing of Two Equine Influenza A(H3N8) Virus Strains Isolated in Kazakhstan.

Here, we report the complete genome sequencing of strains A/equine/Kostanay/9/2012(H3N8) and A/equine/LKZ/9/2012(H3N8) of the equine influenza virus belonging to Florida sublineage, clade 2. The strains were isolated in 2012 in the northern and southern regions of Kazakhstan, respectively.

Genetic diversity of avian avulavirus 1 (Newcastle disease virus genotypes VIg and VIIb) circulating in wild birds in Kazakhstan.

In order to improve current understanding of the molecular epidemiology of avian avulavirus 1 (AAvV-1, formerly avian paramyxovirus 1) in wild birds in Kazakhstan, 860 cloacal swab samples were evaluated. Samples were collected from 37 families of wild birds in nine different regions in the years 2011 and 2014. Overall, 54 positive samples (4.2%) were detected from 17 different families of wild birds, and 16 AAvV-1 isolates were characterized. Three of the isolates contained the fusion protein cleavage site motif RRQKR, and 13 contained KRQKR, which is typical for pathogenic strains of AAvV-1. The AAvV-1 isolates were found to belong to the genotypes VIg and VIIb.

New oligonucleotide microarray for rapid diagnosis of avian viral diseases.

BACKGROUND: We developed a new oligonucleotide microarray comprising 16 identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious bursal disease virus (IBDV) in single- and mixed-virus infections. The objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. METHODS AND RESULTS: The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry. Sensitivity and specificity of the developed microarray was evaluated with use of 122 specimens of biological material: 44 cloacal swabs from sick birds and 78 tissue specimens from dead wild and domestic birds, as well as with use of 15 AIV, NDV, IBV and IBDV strains, different in their origin, epidemiological and biological characteristics (RIBSP Microbial Collection). This microarray demonstrates high diagnostic sensitivity (99.16% within 95% CI limits 97.36-100%) and specificity (100%). Specificity of the developed technique was confirmed by direct sequencing of NP and M (AIV), VP2 (IBDV), S1 (IBV), NP (NDV) gene fragments. CONCLUSION: Diagnostic effectiveness of the developed DNA microarray is 99.18% and therefore it can be used in mass survey for specific detection of AIV, NDV, IBV and IBDV circulating in the region in the course of epidemiological surveillance. Rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic methods is proposed.

Retrospective Analysis of the Equine Influenza Virus A/Equine/Kirgizia/26/1974 (H7N7) Isolated in Central Asia.

A retrospective phylogenetic characterization of the hemagglutinin, neuraminidase and nucleoprotein genes of equine influenza virus A/equine/Kirgizia/26/1974 (H7N7) which caused an outbreak in Kirgizia (a former Soviet Union republic, now Kyrgyzstan) in 1977 was conducted. It was defined that it was closely related to the strain London/1973 isolated in Europe and it shared a maximum nucleotide sequence identity at 99% with it. This Central Asian equine influenza virus isolate did not have any specific genetic signatures and can be considered as an epizootic strain of 1974 that spread in Europe. The absence of antibodies to this subtype EI virus (EIV) in recent research confirms its disappearance as of the 1990s when the antibodies were last found in unvaccinated horses.

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

Molecular and genetic analysis of NS gene from high pathogenic strains of the avian influenza (H5N1) virus isolated in Kazakhstan.

The high pathogenic strains of the avian influenza H5N1 virus isolated in Kazakhstan have NS of different genotypes. The influenza virus strains isolated in 2005 is of NS1E Qinghai genotype. A/swan/Mangystau/3/2006 strain is of NS2A genotype that is typical for Gs/Gd-like strains. The results of the analysis allow assuming that A/swan/Mangystau/3/2006 strain has been brought onto the territory of Kazakhstan from the European part of the continent along the Black Sea-Mediterranean flyway.