Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.

Mass Die-Off of Saiga Antelopes, Kazakhstan, 2015.

In 2015, a mass die-off of ≈200,000 saiga antelopes in central Kazakhstan was caused by hemorrhagic septicemia attributable to the bacterium Pasteurella multocida serotype B. Previous analyses have indicated that environmental triggers associated with weather conditions, specifically air moisture and temperature in the region of the saiga antelope calving during the 10-day period running up to the event, were critical to the proliferation of latent bacteria and were comparable to conditions accompanying historically similar die-offs in the same areas. We investigated whether additional viral or bacterial pathogens could be detected in samples from affected animals using 3 different high-throughput sequencing approaches. We did not identify pathogens associated with commensal bacterial opportunisms in blood, kidney, or lung samples and thus concluded that P. multocida serotype B was the primary cause of the disease.

Monitoring of influenza A viruses in wild bird populations in Kazakhstan in 2002-2009.

A comprehensive influenza virus monitoring study of wild birds was carried out at important flyway resting places and wintering sites in Kazakhstan over eight years. More than 3200 birds belonging to 155 species were sampled. Nearly three-fourths of the birds belonged to the orders Anseriformes and Charadriiformes. In total, 118 hemagglutinating agents were isolated, and 95 of them were identified as influenza A viruses. The influenza viruses comprised eight different subtypes with a high prevalence of H13 and H3 viruses and also included low-pathogenic H5 viruses. The vast majority of the H13 viruses were isolated from members of the family Laridae, whereas the H3 viruses mostly originated from members of the family Anatidae, both in concordance with other monitoring studies. All virus isolates were recovered from cloacal swabs or fecal samples only. The influenza viruses were identified mainly in wetlands north of the Caspian Sea. These findings should be integrated in the design of further wild-bird-monitoring activities.

Circulation of avian paramyxoviruses in wild birds of Kazakhstan in 2002-2013.

BACKGROUND: Screening wild birds for avian paramyxoviruses is of increasing importance. 6913 samples of tracheal and cloacal swabs were collected during 2002-2013 and tested to study the prevalence of APMVs in wild avifauna of Kazakhstan. As a result, 45 isolates were obtained during this period and their ecological niches and genetic relationships were defined. METHODS: Tracheal and cloacal samples from wild birds were collected using sterile swabs placed in viral transport medium and kept in liquid nitrogen until delivery to the laboratory. Samples were inoculated into 10-day-old embryonated chicken eggs and reverse transcription PCR (RT-PCR) assays were performed via a one-step protocol. The PCR products were sequenced and phylogenetic trees were constructed using the 'Neighbour Joining' method. RESULTS: Six thousand nine hundred thirteen samples from 183 bird species were investigated and 45 isolates belonging to four different serotypes APMV-1, APMV-4, APMV-6 and APMV-8 were identified. All APMVs were isolated predominantly from birds belonging to Anatidae family (ducks and geese) and only one APMV-4 isolate was obtained from shorebird (Curlew) on the Caspian seashore. Genetic studies showed that the recovered APMV-1 strains had highest homology with European isolates. APMV-4 strains isolated in 2003, and APMV-6 and APMV-8 isolated in 2013 were 99 % identical to isolates from Far East. CONCLUSION: This is the first reported characterization of avian paramyxoviruses from wild birds isolated in Kazakhstan. These data confirm the wide distribution of APMV-1, APMV-4 and APMV-6 in the Asian subcontinent. The obtained data contribute to the accumulation of knowledge on the genetic diversity and prevalence of APMVs in wild bird populations.

Isolation and Genetic Characterization of a Novel Adenovirus Associated with Mass Mortality in Great Cormorants (Phalacrocorax carbo).

High mortality in great cormorants (Phalacrocorax carbo) was registered on the Alakol Lake in eastern Kazakhstan in 2021 when about 20% of juveniles died. High-throughput sequencing revealed the presence of a putative novel cormorant adenovirus significantly divergent from known aviadenoviruses. We suggest that this cormorant adenovirus can be considered an emerging threat to the health and conservation of this species.

Aislamiento y caracterización genética de un nuevo adenovirus asociado con la mortalidad masiva en cormoranes grandes (Phalacrocorax carbo). En 2021 se registró una alta mortalidad de cormoranes grandes (Phalacrocorax carbo) en el lago Alakol, en el este de Kazajstán, cuando murieron alrededor del 20% de las aves jóvenes. La secuenciación de alto rendimiento reveló la presencia de un supuesto nuevo adenovirus de cormorán significativamente divergente de los aviadenovirus conocidos. Sugerimos que este adenovirus de cormorán puede considerarse una amenaza emergente para la salud y conservación de esta especie.

Has avian influenza virus H9 originated from a bat source?

Influenza A viruses are important pathogens that can cause diseases with high mortality in humans, animals, and birds; and wild birds are considered the primary reservoir of all subtypes in nature. After discovering the H9 influenza A viruses in bats, questions arose about their potential to serve as an additional natural reservoir and about the priority of the viral origin: Did the virus initially circulate in bats and then transmit to birds or vice versa? Influenza A viruses of the H9 subtype are of particular interest because fatal infections of humans caused by H5, H7, and H10 influenza viruses contained RNA segments from H9 viruses. Recently, a novel subtype of influenza A virus (H19) was reported and it was closely related to the H9 bat influenza A virus by its hemagglutinin structure. The genome of novel H19 has revealed a mixed characteristic genomic signature of both avian and bat influenza viruses. The time to most recent common ancestor (TMRCA) estimates have shown that the divergence time between the bat and avian H9-similar influenza virus occurred approximately at the end of the XVIII century. This article discusses the evolution and possible origin of influenza viruses of the H9 subtype isolated from bats and birds. The obtained data, along with the known data, suggest that the primary reservoir of the H9 influenza virus is wild birds, from which the virus was transmitted to bats. We hypothesize that the novel H19 could be a descendant of an intermediate influenza virus that was in the transition stage of spillover from avian to bat hosts.

Genome Sequence of the Attenuated Equid Alphaherpesvirus 1 Strain AK-2011, Isolated in Kazakhstan.

In 2011, there was an outbreak of a disease with mass abortions among horses in southeastern Kazakhstan. The AK-2011 strain was isolated from an aborted fetus and subsequently identified as equid alphaherpesvirus 1. Here, we describe the nearly complete genome sequence of the AK-2011 strain, attenuated for vaccine development.

Detection and Phylogenetic Characterization of a Novel Adenovirus Found in Lesser Mouse-Eared Bat (Myotis blythii) in South Kazakhstan.

Bats are an important natural reservoir of various pathogenic microorganisms, and regular monitoring is necessary to track the situation of zoonotic infections. When examining samples from bats in South Kazakhstan, nucleotide sequences of putative novel bat adenovirus (AdV) species were found. Estimates of amino acid identities of the hexon protein have shown that potentially novel Bat mastadenovirus BatAdV-KZ01 shared higher similarity with monkey Rhesus adenovirus 59 (74.29%) than with Bat AdVs E and H (74.00%). Phylogenetically, BatAdV-KZ01 formed a separate clade, distant from Bat AdVs and other mammalian AdVs. Since adenoviruses are essential pathogens for many mammals, including humans and bats, this finding is of interest from both scientific and epidemiological points of view.

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.

Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses.

ABSTRACTAvian influenza viruses (AIV) have been classified on the basis of 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase. Here we describe genomic evidence for a new candidate HA subtype, nominally H19, with a large genetic distance to all previously described AIV subtypes, derived from a cloacal swab sample of a Common Pochard (Aythya ferina) in Kazakhstan, in 2008. Avian influenza monitoring in wild birds especially in migratory hotspots such as central Asia is an important approach to gain information about the circulation of known and novel influenza viruses. Genetically, the novel HA coding sequence exhibits only 68.2% nucleotide and 68.5% amino acid identity with its nearest relation in the H9 (N2) subtype. The new HA sequence should be considered in current genomic diagnostic AI assays to facilitate its detection and eventual isolation enabling further study and antigenic classification.

Genome sequence of equine influenza virus isolated from horses in southeast Kazakhstan in 2020.

An influenza virus strain, A/equine/Almaty/268/2020, was isolated from horses in southeast Kazakhstan in 2020. Here, we present the nearly complete genome sequence of this epidemic strain. This study was aimed at obtaining the complete genome sequence of the isolate.

Genome Sequence of Influenza B Epidemic Strain B/Almaty/8/2018.

An influenza virus strain, B/Almaty/8/2018, was isolated in Almaty (in southeastern Kazakhstan) during a human population surveillance study in 2018. Here, we present the nearly complete genome sequence of this epidemic strain, compared to the Yamagata-like and Victoria-like variants of the influenza B virus.

AK-2011 strain for the development of a vaccine against equine rhinopneumonitis.

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The purpose of the studies presented in this paper is to develop a technology for the manufacture of a cell-derived equine rhinopneumonitis vaccine, as well as to assess the safety and immunogenicity of the newly developed vaccine in laboratory animals model. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0 lg TCID50/cm 3 . Sequencing and polymerase chain reaction analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPEs) induced by the AK-2011 virus stain (dilution 101 ) in calf trachea and horse kidney cell cultures were stable from the first to tenth passages, with biological activity of 5.75-6.00 lg TCID50/cm 3 . CPEs caused by the virus were apparent on days 2-3, further developed intensively and extended to 60-80% of the cell monolayer on days 5-7. The vaccine results can be used to immunize horses on farms against rhinopneumonia, and horses should be immunized twice with an interval of 2-3 months.

Serological Screening for Middle East Respiratory Syndrome Coronavirus and Hepatitis E Virus in Camels in Kazakhstan.

After the recent Middle East Respiratory Syndrome coronavirus (MERS-CoV) pandemic in 2013, more attention has been paid to the camel as an important source of zoonotic viral infections. Almost simultaneously, in 2013, new genotypes 7 and 8 of the hepatitis E virus (HEV) were discovered in dromedary and Bactrian camels, respectively. HEV 7 was further shown to be associated with chronic viral hepatitis in a transplant recipient. In this study, serological screening for antibodies to MERS-CoV and hepatitis E virus was carried out on large camel farms in the south and west of Kazakhstan. 6.42% of the tested camels were found to be positive for antibodies to the hepatitis E virus, which indicates its circulation in local camel population. For the first time, antibodies to the hepatitis E virus were found in Bactrians, which have been little studied to date. Antibodies to MERS-CoV were not found in the camel sera.

Phylogenetic analysis of avian influenza viruses of H11 subtype isolated in Kazakhstan.

Avian influenza viruses A/turkey/Almaty/535/04 (H11N9) and A/herring gull/Atyrau/2186/07 (H11N2) isolated in Kazakhstan were characterized as low pathogenic in biological and genetic studies. Putative glycosylation sites were identical to the putative sites in published H11, N2, and N9 isolates sequences. Compared with published data no additional basic amino acid residues were found in the hemagglutinin (HA) cleavage site of these Kazakhstan strains. Phylogenetic analysis revealed a rare case of Eurasian-American reassortment in the HA gene of A/herring gull/Atyrau/2186/07 (H11N2) virus and significant sequence difference of the HA and the neuraminidase genes of the virus A/turkey/Almaty/535/04 (H11N9) from the previously published GenBank viruses.

Cormorants as a Potentially Important Reservoir and Carrier of Newcastle Disease Virus on the Asian Continent.

Despite numerous disease prevention measures and control programs, Newcastle disease (ND) remains one of the most significant infections in poultry worldwide, especially in developing countries. It is known that wild birds, mainly of the Anseriformes order, are the main carrier of lentogenic (non-pathogenic) variants of Newcastle disease virus (NDV) in nature. But the question of the reservoir of velogenic (highly pathogenic) NDV in nature still remains open. In the 1970s, 1990s, and 2000s in North America during epizootics among cormorants, velogenic NDV strains were isolated. It was later concluded that cormorants play an important role in the maintenance and circulation of NDV in North America. New data have been obtained on the circulation of velogenic NDV strains in wild birds in Central Asia: VIIb and XIII genotype strains were isolated from cormorants for the first time in Kazakhstan. Interestingly, outbreaks of NDV registered in poultry in Central and Southern Asia were phylogenetically close to the viruses from cormorants that support the idea that cormorants can serve as the potential reservoir of velogenic NDV in developing countries of Asia. The seasonal migrations of cormorants may contribute to the distribution of the virus throughout Asia but more evidence must be obtained to confirm this hypothesis. There is increasing evidence of the introduction of NDV into the poultry farms from wild nature worldwide. This article continues the discussion on the likelihood of cormorants to serve as a reservoir and carrier of NDV on the Asian continent.

High Mortality in Terns and Gulls Associated with Infection with the Novel Gull Adenovirus.

High mortality in Caspian Terns (Hydroprogne caspia) and Great Black-headed Gulls (Larus ichthyaetus), was recorded on the northeastern shores of the Caspian Sea in June 2013. Retrospective high throughput sequencing of archived tissue samples conducted in 2018 revealed the presence of the recently identified novel gull adenovirus similar to that associated with mortality in gulls in the Netherlands in 2001. We suggest that that this gull adenovirus specifically can be considered as an emerging threat to the health and conservation of gulls and terns.

A Highly Pathogenic H5N1 Influenza A Virus Isolated from a Flamingo on the Caspian Sea Shore.

In 2015, in the Kazakh part of the northern Caspian Sea region, during the monitoring of wild birds for avian influenza viruses, a highly pathogenic A/flamingo/Mangistau/6570/2015(H5N1) influenza virus was isolated from a dead flamingo. This study aimed to obtain the complete genome sequence of the isolate.

Evolution of Avian orthoavulavirus 16 in wild avifauna of Central Asia.

In 2014, a novel Avian orthoavulavirus 16 species was described among wild birds in Korea. In 2018, after massive parallel sequencing of archival strains of Avian orthoavulaviruses, isolated in 2006 in Central Kazakhstan, isolates belonging to this serotype were detected. The obtained data allowed to trace the evolution of this serotype in Asia and to reveal its evolutionary relationships with other Avulavirinae subfamily species. It was determined that Avian orthoavulavirus 16 is phylogenetically very close to Avian orthoavulavirus 1 (Newcastle disease virus) in its genomic characteristics. It is known that Avian orthoavulavirus 1 is divided into two phylogenetically distant Classes I and II. Avian orthoavulavirus 16 turned out to be very close to lentogenic Class I, which circulates mainly among wild birds. It was suggested that Avian orthoavulaviruses 1 and 16 may have common evolutionary origin and in ecological terms, both serotypes are circulating among wild birds of the order Anseriformes (ducks and geese), but Avian orthoavulavirus 1 has gradually replaced Avian orthoavulavirus 16 from active circulation.

Near-Complete Genome Sequence of Avian Influenza Virus Strain A/mallard/Balkhash/6304/2014 (H1N1) from Kazakhstan.

An avian influenza virus strain, A/mallard/Balkhash/6304/2014 (H1N1), was isolated during a wild bird monitoring study in Kazakhstan in 2014. The virus was isolated from a wild mallard duck (Anas platyrhynchos) in eastern Kazakhstan. Here, we present the near-complete genome sequence of the virus.