Virus In Iran Research Articles

Dynamics of the population structure and genetic variability within Iranian isolates of grapevine fanleaf virus: evidence for polyphyletic origin.

To determine the genetic diversity and population structure of grapevine fanleaf virus (GFLV), the complete nucleotide sequence of the coat protein gene of 41 isolates from different regions in Iran was determined. Phylogenetic analyses of these isolates together with those available in the GenBank revealed two evolutionary divergent lineages, designated GFLV-G and GFLV-Ir that reflect origin of the isolates. Analysis of the genetic variability in the coat protein of these isolates revealed 37 genotype groups in GFLV population. Analyses indicate that GFLV-G and GFLV-Ir clades are significantly differentiated populations of GFLV. Also, geographical subpopulations of the virus in Iran were completely distinct from each other. Examination of nonsynonymous/synonymous nucleotide diversity showed that the CP gene has been under purifying selection. The neutrality tests indicate balancing selection operating within isolates of the northwest of Iran and purifying selection within the other populations.

Epidemiology of Rotavirus-Norovirus Co-Infection and Determination of Norovirus Genogrouping among Children with Acute Gastroenteritis in Tehran, Iran.

Background:Enteric viruses, particularly human rotavirus and norovirus, have been shown to replace bacteria and parasites, as the most common pathogens responsible for acute diarrhea. However, there are still few epidemiological data on the simultaneous occurrence of these viruses in Iran. In this regard, the aim of this study was to assess the useful epidemiological data on the gastroenteritis associated with rotavirus-norovirus mixed infection and to examine the prevalence of norovirus genogrouping among children aged less than five years old in Iran.Methods:A total of 170 stool samples were collected from children under five years of age with the clinical signs and symptoms of acute gastroenteritis, from May 2013 to May 2014. For the detection of both rotavirus and norovirus, total RNA was extracted from all samples, followed by reverse transcription polymerase chain reaction (RT-PCR). For both detected rotaviruses and noroviruses, genogrouping was performed.Results:Of 170 samples, 49 (28.8%) and 15 (8.8%) samples were found to be positive for rotavirus and norovirus infections by RT-PCR. Interestingly, 6 (3.5%) patients were positive for both infections. Among the 15 norovirus-positive patients, 13 (86.6%) and 2 (13.3%) belonged to genogroups GII and GI.Conclusion:The norovirus genogroup GII and rotavirus lead to the serious infections in children with acute gastroenteritis. However, more well-designed studies are needed to further elucidate the role of other enteric viruses in acute gastroenteritis

Evaluation of sugar beet lines for resistance to beet curly top viruses

Curly top disease caused by beet curly top viruses (BCTVs) has been a problem in sugar beet fields for a long time. In recent years, climate change has increased the spread of virus vectors and disease damage in infected regions. To find a natural resistance source to curly top viruses in Iran (Beet curly top virus-C, BCTV-C and Beet curly top Iran virus, BCTIV), the reaction of 50 sugar beet lines to the above-mentioned viruses was evaluated in a completely randomized design experiment in 2014. Sugar beet lines were agroinoculated at the 4–6 leaf stage by virus infectious clones under greenhouse condition and then the symptom severity and virus presence was monitored by enzyme-linked immunosorbent assay and polymerase chain reaction methods 8 weeks after inoculation. Five out of 50 lines were found to be resistant to both viruses. Given that both viruses were detected in most sugar beet cultivation areas in Iran, these lines could be suggested for cultivation in Iran. In addition, some lines displayed completely different reactions to the viruses indicating different effects of the viruses on sugar beet lines.

Prevalence and genotypic characterization of Human Parvovirus B19 in children with measles- and rubella-like illness in Iran.

Human Parvovirus B19 (B19V) is a prototype of the Erythroparvovirus genus in Parvoviridae family. B19V infections are often associated with fever and rash, and can be mistakenly reported as measles or rubella. Differential diagnosis of B19V illness is necessary for case management and also for public health control activities, particularly in outbreak situations in which measles or rubella is suspected. To investigate the causative role of B19V infection in children with measles- and rubella-like illness, a total of 583 sera from children with exanthema were tested for presence of B19V by determining anti-B19V IgG and IgM antibodies by ELISA as well as B19V DNA detection by nested PCR. DNA positive samples were assessed further for determination of viral load and sequence analysis by Real-Time PCR and Sanger sequencing method, respectively. Out of 583 patients, 112 (19.21%) patients were positive for B19V-IgM antibody, 110 (18.87%) were positive for B19V-IgG antibody, and 63 (10.81%) were positive for B19V viral DNA. The frequency of B19V-IgG antibodies were increased with age; that is children under 6 year old showed 7.11% seroprevalence for B19V-IgG as compared to 18.39% and 28.91% for age groups 6 to >11 and 11-14 years old, respectively. Phylogenetic analysis of the NS1-VPu1 overlapping region revealed that all sequenced B19V-DNA belonged to genotype 1. The results of this study may aid the surveillance programs aiming at eradicating measles/rubella virus in Iran, as infections with B19V can be mistakenly reported as measles or rubella if laboratory testing is not conducted.

Analysis of Amino Acid Changes in NS Protein of Influenza A/(H3N2) Virus in Iranian Isolates

Aim: Roles of NS gene of influenza A virus in virulence and replication are well established but extent of its variation in seasonal influenza A (H3N2) viruses in Iran is not well known. Materials & methods: NS gene of 37 (A/H3N2) virus isolates were sequenced and analyzed for information on genetic changes. Results: Data analysis of NS1 protein revealed two amino acid substitutions E26K and Q193R in almost all strains. Substitutions in T58P in 27.0%, A86S in 13.5% and each of V11G, M81I and P85T in 2.7% Iranian strains were also observed. Mutations in NS2/NEP protein were observed in K36E, Q101L and F107S. Conclusion: Many mutations were observed for the first time in Iranian strains. Their function remains to be determined.

Assessment of Recombination in the S-segment Genome of Crimean-Congo Hemorrhagic Fever Virus in Iran.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) belongs to genus Nairovirus and family Bunyaviridae. The main aim of this study was to investigate the extent of recombination in S-segment genome of CCHFV in Iran. Samples were isolated from Iranian patients and those available in GenBank, and analyzed by phylogenetic and bootscan methods. Through comparison of the phylogenetic trees based on full length sequences and partial fragments in the S-segment genome of CCHFV, genetic switch was evident, due to recombination event. Moreover, evidence of multiple recombination events was detected in query isolates when bootscan analysis was used by SimPlot software. Switch of different genomic regions between different strains by recombination could contribute to CCHFV diversification and evolution. The occurrence of recombination in CCHFV has a critical impact on epidemiological investigations and vaccine design.

Molecular and serological findings in suspected patients with Crimean-Congo hemorrhagic fever virus in Iran.

Crimean-Congo hemorrhagic fever (CCHF) is an arthropod-borne disease of humans associated with a severe clinical picture, including hemorrhagic syndrome and a high mortality rate. CCHF virus is widely distributed throughout large areas of the world. To characterize the serological status in CCHF patients, paired clinical samples were collected from suspected CCHF patients and analyzed by microbiological and other laboratory analyses with the aim of: determining the presence of neutralizing antibodies against CCHF virus; investigating the cross-reactivity of these neutralizing antibodies against virus isolated from the same outbreak and against other available laboratory strain; and studying the relationship between the isolated virus with other virus by whole genome sequencing. Patients at Boo-Ali Hospital, Zahedan, Iran, with clinical symptoms ranging from mild to severe hemorrhagic fever were included in the study. Two serum samples were taken from each patient, the first as soon as the patient matched the criteria for CCHF notification and the second when the patient was discharged from hospital (2 weeks later). Commercial and in-house assays revealed a positive IgM signal in acute serum samples from six patients. A novel finding was that CCHF patients develop neutralizing antibodies soon after infection. Interestingly these antibodies were able to neutralize other CCHF virus strains too. The complete sequence of the Zahedan 2007 isolate, including the hitherto unknown first L-segment sequence, was identified using an original clinical sample from one patient with confirmed CCHF infection.

Phylogenetic study based on the phosphoprotein gene of Iranian Newcastle disease viruses (NDV) isolates, 2010 -2012

BACKGROUND: Newcastle disease virus (NDV) is the causativeagent of the Newcastle disease (ND), a highly contagiousdisease in birds that causes significant economic losses to thepoultry industry worldwide. ND is endemic in Iran and outbreaksare reported regularly in commercial poultry flocks and differentspecies of birds. OBJECTIVES:The current study was carried outto characterize NDV based on phosphorprotein (P) gene fromrecent outbreaks in Iran, 2010-2012. METHODS: The P genefragment of NDV isolates of five chickens, 1 ostrich, and 1Pigeon paramyxovirus-1 was obtained by RT-PCR and sequenced.RESULTS: Phylogenetic analysis of sequences revealed thatchicken and ostrich NDV isolates were closely related andplaced in the genotype VII and Pigeon Paramyxovirus-1 waslocated in the genotype V. CONCLUSIONS:This is the first reportof Phosphoprotein gene sequences of NDV strains isolated inIran. This study will help us to understand the epidemiology andmolecular characteristics of Newcastle disease virus in Iran.

Molecular investigation and cultivation of camelpox virus in Iran

Camelpox virus (genus Orthopoxvirus, family Poxviridae) is the etiologic agent of camel pox. The clinical manifestations of this virus range from inapparent infection to mild, moderate and, less commonly, severe systemic infection and death. Following an outbreak of camelpox, samples that were collected from camel flocks suspected to have camelpox in Qom Province in central Iran and Khash city, Sistan and Baluchestan Province and South Khorasan Province in eastern Iran were sent to Razi Vaccine and Serum Research Institute in Mashhad. DNA extraction was performed primarily by the phenol-chloroform method, and PCR was carried out using a Bioneer kit. Using the primer pair 5'-AAT-ACA-AGG-AGG-ATC-T-3' and 5'-CTT-AAC-TTT-TTC-TTT-CTC-3', the gene sequence encoding the A-type inclusion protein (ATIP) was amplified. The size of the PCR product, specific for camelpox virus, was 881 bp. The PCR product was purified, and to confirm its sequence, it was sent to the reference laboratory. The sequence was subjected to a BLAST search and then phylogenetically analyzed using CLC software. The results showed that all samples were nearly 100 % identical to each other and to strains CMS and M-96. These isolates also had 99 % and 95 % similarity to the CP-1 strain and isolate FIN/T2000, respectively. In Vero cell culture, inoculation with this virus caused a cytopathic effect (CPE), which appeared 2-5 days post-inoculation. Characteristic CPE showing foci of rounded cells, ballooning, giant-cell formation and syncytia with degenerative changes appeared.

The status of Cucumber vein yellowing virus in Iran

Yellowing symptoms of greenhouse- and open field-grown cucurbit crops are becoming increasingly important in many cucurbit growing regions of the world, and particularly in Iran. A survey was conducted from 2011 to 2012 in eight major cucurbit growing regions in Iran. Yellowing and specifically vein clearing symptoms were observed in many cucumber plants grown in greenhouses and open fields, suggesting the presence of Cucumber vein yellowing virus (CVYV, genus Ipomovirus , family Potyviridae ). The identification of CVYV was carried out with a specific triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and reverse transcription (RT)-PCR. CVYV was detected in 42% of the collected samples, in all surveyed provinces, except Bushehr. CVYV was also detected in melon and cucumber crops grown in open fields. These results indicate that CVYV is widely distributed on these two cucurbit species in the major cucumber growing areas of Iran. CVYV positive samples were also tested, using DAS-ELISA, for the presence of Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV), two criniviruses reported previously to occur in Iran. Double or triple infections of CCYV and CYSDV occurred in 49 of 166 of the CVYV-infected plants. The CVYV and CCYV combined infections were more prevalent than CVYV and CYSDV combined infections. TAS-ELISA positive samples were used to mechanically inoculate healthy cucumber plants, and mild vein yellowing was observed on the inoculated leaves. Identical symptoms were also observed on whitefly inoculated healthy cucumber plants. The presence of CVYV in mechanically and whitefly inoculated plants was confirmed by TAS-ELISA and RT-PCR. Sequence analysis revealed that the Iranian isolate of CVYV was more closely related to Spanish isolates than to isolates from Jordan. Phylogenetic analysis showed that CVYV isolates can be divided into two phylogenetic groups (I and II). Despite the close vicinity of Jordan to Iran, the Iranian CVYV isolate clustered with Spanish isolates in group I and not with the Jordanian isolates of group II.

Characterization of Variations in PB2, NS1, M, Neuraminidase and Hemagglutinin of Influenza A (H3N2) Viruses in Iran.

Background:In the influenza A viruses, neuraminidase (NA), hemagglutinin (HA), PB2, NS1 and M are responsible for the disease pathogenicity. The mechanism of pathogenicity differs among these viruses. Binding of host proteases by the viral NA, sequence of HA in the cleavage and receptor-binding sites, number of oligosaccharide side chains of HA, shortening of NA, and substitutions in PB2, NS1 and M genes, all have been suggested as molecular correlates of pathogenicity of influenza viruses.Objectives:The goal of this study was to find the alterations in genes, which might be responsible in the virus pathogenesis.Materials and Methods:Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of HA, NA, PB2, NS and M genes were performed.Results:In the receptor binding site of HA, Ile-226, Pro-227, Ser-228, and Asp-190 were found. Arg was in the cleavage site of all viruses and 11-12 N-linked glycosylation sites were found. In NS1, Asp-92 and Ala-149 were detected and Lys-627 was found in PB2 of all viruses in this study. Val-15, Thr-139 and Ala-218 of M1 and Val-28, Leu-54 and His-57 were found in M2 gene. At residue 146 of NA, there was N-linked glycosylation, and Ile-222 was found in the enzyme active site.Conclusions:The changes found in these five genes, compared to other studies, suggest that viruses studied in this research had the ability to bind to Neu Acα2,6 Gal linkage and had low pathogenicity. It is important to mention that these changes were at the amino acid level and studies need to be performed on animals to investigate the significance of these findings.

Genotype circulation pattern of human respiratory syncytial virus in Iran

In order to have information on the molecular epidemiology and genetic circulation pattern of human respiratory syncytial virus (HRSV) in Iran, we studied the genetic variability of both group A and B HRSV strains during seven consecutive years by sequencing the hypervariable C-terminal domain of G protein. A total of 485 children <2years of age who were negative for influenza viruses, screened for the presence of HRSV in this research. HRSV was detected in 94 (19.38%) of the samples using nested RT-PCR. Group A viruses were isolated during each year, while group B viruses were isolated during 2009 and 2013. Phylogenetic analysis showed that all HRSV group A viruses belonged to three genotypes: GA1, GA2, GA5 and the group B viruses were in BA genotype.

Outbreak of contagious ecthyma in camels (&lt;em&gt;Camelus dromedarius&lt;/em&gt; and &lt;em&gt;Camelus bactrianus&lt;/em&gt;) in Southwest Iran

Camel contagious ecthyma (CCE) is a highly infectious viral skin disease of sheep, goats, and camels caused by a Parapoxvirus. This study reports an outbreak of CCE in a herd of 34 camels/dromedaries, with 24 infected animals. The infected animals exhibited severe papules on the lips and legs, increase in body temperature, profuse salivation, foul mouth smell, and facial edema. Staphylococcus aureus was identified in some samples. The morbidity and mortality rates were 70.6 and 6%, respectively. A supportive treatment was administered to the infected camels. Most of the camels recovered within three weeks. Skin analysis by the polymerase chain reaction revealed the pre- sence of the B2L gene of the CE Kerman/2000 strain. This is the second report on the presence of CCE in Iran. As there is no vaccination program for camels against contagious ecthyma virus in Iran, this study may form the basis for establishing such a program

Genetic diversity, host range, and distribution of tomato yellow leaf curl virus in Iran.

Tomato yellow leaf curl virus (TYLCV) is considered one of the most important tomato pathogens in tropical and subtropical regions including Iran. During the years 2007 to 2009, a total number of 510 symptomatic and asymptomatic vegetable, ornamental and weed samples were collected from fields and greenhouses in ten provinces of Iran. Symptoms included stunting, yellowing, leaf curl and flower senescence. PCR with specific primers showed TYLCV infection in 184 samples (36%) such as cucumber, pepper, tomato and several weeds from seven provinces. Based on the geographical origin, host range and symptoms, twenty three representative isolates were selected for phylogenetic analysis. An amplicon with a size about 608 base pair (bp) comprising partial sequence of the coat (CP) and movement protein (MP) coding regions of the viral genome was sequenced and compared with the corresponding selected sequences available in GenBank for Iran and worldwide. Phylogenetic analyses on the basis of the nucleotide sequences indicated two geographically separated clades. Isolates collected from Hormozgan, Khuzestan and Kerman provinces were grouped together with other Iranian isolates including TYLCV-Ir2, TYLCV-Kahnooj, and an isolate from Oman. It was also revealed that isolates collected from Boushehr, Fars, Tehran, and Isfahan placed close to the Iranian isolate TYLCV-Abadeh and isolates from Israel and Egypt. No correlation was found between the genetic variation and the host species, but selected Iranian isolates were grouped on the basis of the geographical origins. Results of this study indicated a high genetic diversity among Iranian TYLCV isolates.

Detection and Molecular Variability of Fig Fleck‐Associated Virus and Fig Cryptic Virus in Iran

AbstractForty‐eight fig orchards were surveyed to determine the presence and incidence of Fig cryptic virus (FCV), Fig fleck‐associated virus (FFkaV), Fig leaf mottle‐associated virus 1 (FLMaV‐1), Fig leaf mottle‐associated virus 2 (FLMaV‐2) andFig mosaic virus(FMV) in four provinces of northeast, northwest and central regions of Iran. A total of 197 leaf samples from commercial and outdoor fig gardens were collected in April and September 2012 and tested by reverse‐transcription polymerase chain reaction (RT‐PCR). Approximately 14.7% of the tested fig trees were infected byFCV,FFkaV andFMVwith a peak of 18% in Tehran province. None of samples was found to be infected withFLMaV‐1 orFLMaV‐2.FFkaV was found in fig trees collected in all the four provinces, but noFCVinfection was found inSemnan province, andFMVwas just occurred inMarkai andTehran. Mixed infections ofFCVwithFFkaV andFMVwere detected in 2.5% of the samples. The sequence of theCPfragment of sixIranianFCVisolates showed 97–99% nucleotide (nt) identity with the referenceFCVisolate, while that of a RdRp and Gp fragments of nineFFkaV and twoFMVIranian isolates showed 87–91% nt and 89–90% identity with the reference isolates, respectively. Phylogenetic analysis grouped theFCVandFFkaV isolates in two distinct clusters, with the Iranian isolates included in both clusters. Results showed genetic diversity among Iranian viruses. Structure and diversity ofFCVandFFkaV populations are discussed.

Molecular detection of Acute Bee Paralysis Virus in Iran

Acute bee paralysis virus (ABPV) is a small single stranded RNA virus recently classified within the family Dicistroviridae, genus Cripavirus. Here, we describe the first study of ABPV in unhealthy bee colonies, which has been an unusual loss in adult bee population and significant honey bee mortality during the year. The aim of this study was evaluation of ABPV infection in honey bee colonies in apiaries with loss of population in different geographical provinces in Iran. Adult bee samples were collected between July - September 2011 and 2012 and were originated from 23 provinces with different geographic areas of Iran. Following the RT-PCR reaction with the specific primers on the isolated RNA, an approximately 618 bp product was detected. We demonstrated the presence of ABPV RNA in 9 (5.62 %) out of 160 samples collected from Iranian apiaries.

Genetic and phylogenetic analysis of the ribonucleoprotein complex genes of H9N2 avian influenza viruses isolated from commercial poultry in Iran

BACKGROUND: The H9N2 subtype of avian influenza viruses (AIVs) has been isolated in multiple avian species in many European, Asian, African and American countries. Since the first outbreak of H9N2 virus in Iran in 1998, this virus has widely circulated throughout the country, resulting in major economic losses in chicken flocks. Several amino acids in the virus ribonucleoprotein (RNP) complex including the nucleo- protein (NP) and polymerase (PB2, PB1 and PA) proteins are associated with host range and virulence. OBJECTIVES: Our aim was to understand the molecular characterization of RNP complex proteins of Iranian H9N2 subtype isolates. METHODS: The full length nucleotide sequences of RNP complex genes of two strains designated as Ck/IR/ZMT-101/98 and Ck/IR/EBGV- 88/10 were amplified and sequenced. RESULTS: The phylo- genetic analysis revealed that both strains were located in different sub-lineages. However, based on the genetic similari- ties, PB1, PA and NP genes of Ck/IR/EBGV-88/10 strain had a close relationship with a H7N3 subtype strain, isolated from Pakistan. Most positions of RNP proteins contained amino acids typical of avian determinants of host range. The results showed that the Iranian RNP complex genes have undergone genetic reassortment. CONCLUSIONS: Continuous AIV monitoring in poultry industry would help to obtain more information about genetic variation of H9N2 viruses and possible emergence of virulent and/or pandemic viruses.

Phylogenetic relationships, recombination analysis, and genetic variability among diverse variants of tomato yellow leaf curl virus in Iran and the Arabian Peninsula: further support for a TYLCV center of diversity

The discovery of five strains of TYLCV in Iran, including the most well-known and widespread, TYLCV-IL, spurred a detailed study of the full-length genomes of additional TYLCV field isolates and an in-depth analysis of phylogenetic relationships, extent of recombination, and genetic variability of TYLCV isolates within Iran and throughout the Arabian Peninsula. Phylogenetic analysis of complete genome sequences of TYLCV isolates from Iran and other countries revealed four monophyletic clusters could be differentiated based on geographical origin, indicating that recent dispersal of these populations (by the vector or by humans) from these four regions has occurred minimally, or not at all. Genetic analysis revealed that TYLCV-IL isolates from southern Iran possessed greater genetic variability than the northeastern isolates, a pattern that may be reflective of evolution driven by geographically dependent isolation. Similarly, isolates of TYLCV-OM originating from Oman showed greater genetic variability than TYLCV-OM variants from Iran. Major recombination events, which were detected in all strains of TYLCV had breakpoints initiating in the C1, C1/C4, C2/C3 and V1 open reading frames (ORFs) and ending at the non-coding region and the C1, C1/C2 and C3 ORFs. Hence, these regions have consistently served as hot spots for recombination worldwide during the evolution of all currently recognized isolates and strains of TYLCV.

Prevalence of Tobacco mosaic virus in Iran and Evolutionary Analyses of the Coat Protein Gene.

The incidence and distribution of Tobacco mosaic virus (TMV) and related tobamoviruses was determined using an enzyme-linked immunosorbent assay on 1,926 symptomatic horticultural crops and 107 asymptomatic weed samples collected from 78 highly infected fields in the major horticultural crop-producing areas in 17 provinces throughout Iran. The results were confirmed by host range studies and reverse transcription-polymerase chain reaction. The overall incidence of infection by these viruses in symptomatic plants was 11.3%. The coat protein (CP) gene sequences of a number of isolates were determined and disclosed to be a high identity (up to 100%) among the Iranian isolates. Phylogenetic analysis of all known TMV CP genes showed three clades on the basis of nucleotide sequences with all Iranian isolates distinctly clustered in clade II. Analysis using the complete CP amino acid sequence showed one clade with two subgroups, IA and IB, with Iranian isolates in both subgroups. The nucleotide diversity within each sub-group was very low, but higher between the two clades. No correlation was found between genetic distance and geographical origin or host species of isolation. Statistical analyses suggested a negative selection and demonstrated the occurrence of gene flow from the isolates in other clades to the Iranian population.

Genetic diversity of early (1998) and recent (2010) avian influenza H9N2 virus strains isolated from poultry in Iran

Infection with avian influenza H9N2 virus is widespread in the Asian poultry industry, resulting in great economic losses due to mortality and a severe decline in egg production. To obtain more-comprehensive genomic data from circulating H9N2 viruses in Iran, we sequenced the whole genomes of early (Ck/IR/ZMT-101/98) and recent (Ck/IR/EBGV-88/10) isolates of this virus in Iran. The M and NS genes of Ck/IR/EBGV-88/10 shared a high level of similarity with a highly pathogenic H7N3 virus isolated from Pakistan. The cleavage site within the HA protein of these viruses contained two different motifs, RSSR and KSSR, which are similar to those found in low-pathogenic viruses. The deduced amino acid sequence of the new isolate contained the mutation Q226L, which is a characteristic of human-type sialic acid influenza receptor binding. An analysis of the viral amino acid sequence of the M2 protein of the recent strain revealed a V27A mutation, which is associated with amantadine resistance in avian influenza virus. The present results emphasize the need for continuous surveillance of H9N2 viruses in poultry and the human population to obtain more information about the nature and evolution of future pandemic influenza viruses.