Avian Paramyxovirus Type Research Articles

Characterization of ten paramyxovirus type 1 viruses isolated from pigeons in China during 1996–2019

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of avian paramyxovirus type 1, which mainly infects pigeons. Here, we characterized ten PPMV-1 viruses isolated from pigeons in China during 1996–2019. Phylogenetic analysis of available complete genomes, F and HN genes of PPMV-1 from China showed that multiple PPMV-1 genotypes (I, II, VI, and VII) exist in pigeons in China. Ten PPMV-1 viruses isolated in this study belonged to genotypes VI.1.2.2.2, VI.2.1.1.2.1, VI.2.1.1.2.2 and VII respectively. Genotype VI is predominant in pigeons. VI.2.1.1.2.2 contains most recently isolated PPMV-1 viruses, suggesting that VI.2.1.1.2.2 is a prevalent genotype in pigeons in China. In vitro and in vivo studies showed that four representative viruses from genotypes VI.2.1.1.2.1 (TA14), VI.2.1.1.2.2 (SD19), VI.1.2.2.2 (SD16), and VII (JN08) could replicate efficiently in chicken embryo fibroblasts, while the replication titer of JN08 (VII) virus was significantly lower than that of VI gene viruses in pigeon embryo fibroblasts. The TA14 (VI.2.1.1.2.1) and SD19 (VI.2.1.1.2.2) viruses caused 20 % and 30 % mortality in pigeons, respectively. No birds infected with SD16 (VI.1.2.2.2) died during the study period. JN08 (VII) virus did not cause obvious clinical signs in infected pigeons. All data indicated that VI.2.1.1.2.2 is the prevalent genotype circulating in China and poses a major threat to pigeons, suggesting that a matched vaccine is necessary to control the disease.

Retraction for Kumar et al., “Evaluation of the Newcastle Disease Virus F and HN Proteins in Protective Immunity by Using a Recombinant Avian Paramyxovirus Type 3 Vector in Chickens”

Volume 85, no. 13, p. 6521–6534, 2011, https://doi.org/10.1128/JVI.00367-11. It has been called to our attention that in Fig. 6C, lanes 4 and 10 (top blot) and lanes 9 and 10 (bottom blot) are duplicates. Since the samples used for this figure are no longer available, we are taking responsibility for the mistakes and retracting the paper.

Vaccination: A key way to prevent Newcastle disease in poultry of Nepal

Nepal is a country with variation in topography and diverse climatic zones. Agriculture is a major occupation for majority of Nepalese engaging 66% of total population in farming and Poultry farming is integral part of the agriculture in Nepal. Nepal has long history of traditional poultry keeping. Poultry are the important source of valuable dietary proteins and rural cash income. In recent data, about 72 million chickens and 400 thousand ducks are present which include commercial, small holder and backyard type of farm. Poultry sector is an emerging industry engaging and employing 70,000 people directly contributing about 4% in National GDP. In present days, the traditional poultry farming is replaced by commercial farming. But the challenge and constraints on poultry sector are increasing day by day. Outbreak and spread of emerging infectious disease is headache in poultry farming. Various disease like Avian influenza, Newcastle disease, IBD, IB, ILT, coccidiosis, Avian Mycoplasmosis, Fowl typhoid, Fowl pox etc. cause serious economic loss in poultry industry. Newcastle disease is common and spread throughout the country having higher incidence in the summer which is caused by avian paramyxovirus type-1 of paramyxoviridae. Vaccines are important tool for prophylactic measures. In many countries, vaccination against ND is routinely practiced for prevention and control of disease and types of vaccines and vaccination schedule vary according to potential threat and virulence property of the field challenge virus. There is big challenge for vaccine researchers, manufacturers and scientists for development of effective vaccine against Newcastle disease. In this paper, we will discuss about present status of Newcastle disease and Vaccine produced in Nepal and Vaccination protocol to prevent spread of Newcastle Disease in Nepal.

Genetic characterization of one duck-origin paramyxovirus type 4 strain in China.

Avian paramyxovirus type 4 (APMV-4) has been frequently reported from wildfowl and waterfowl inrecentyear. However, few studies have reported on the molecular characteristics and regional transmission of APMV-4, knowledge of which is important for understanding the genetic diversity and epidemiology of avian paramyxovirus. Herein, we report the isolation of one APMV-4 strain, designated as QY17, from the duck in eastern China. The determined complete genome of the isolate with six gene segments 3'-N-P-M-F-HN-L-5' was 15,054nt in length. Genetic analysis of the whole-fusion gene of this isolate showed that QY17 was derived from a Eurasian lineage. Further phylogenetic analysis showed that the duck-origin strain QY17 had a highly genetic relationship with representative APMV-4 strains from wildfowl in neighbouring regions. These genetic results suggested that APMV-4 viral exchange may occur in wildfowl and poultry via wild bird migration.

Surveillance and investigative diagnosis of a poultry flock in Great Britain co-infected with an influenza A virus and an avirulent avian avulavirus type 1.

A detailed veterinary and laboratory investigation revealed an unusual case of concurrent avian avulavirus type 1 (AAvV-1, formerly called avian paramyxovirus type 1) and low pathogenicity avian influenza (LPAI) virus infections of chickens during March 2010 in a mixed poultry and livestock farm in Great Britain. Respiratory signs and daily mortality of 5-6 birds in a broiler flock 8-weeks of age prompted submission of two carcasses to an Animal and Plant Health Agency (APHA) regional laboratory. Infectious bronchitis virus infection was suspected initially and virus isolation in SPF embryonated fowls' eggs was attempted at APHA-Weybridge. Avirulent AAvV-1 was detected in the first sampling. Both invitro nucleotide sequencing of the fusion gene and invivo pathotyping by intracerebral pathogenicity index revealed an avirulent AAvV-1 not definitively ascribed to licensed vaccine. Upon initial detection of the AAvV-1 virus, statutory restrictions were placed on the farm, an official veterinary visit was performed and further samples were submitted to APHA-Weybridge for official statutory disease investigation. An H2N3 LPAI virus was subsequently isolated from tissue samples and swabs submitted from the follow-up statutory investigation. The subtype was confirmed by haemagglutination inhibition test (HAIT) and neuraminidase inhibition (NI) tests on egg-amplified virus. As neither virus was notifiable according to the internationally recognized EU and OIE standards, and/or definitions of disease, statutory farm restrictions were lifted. Veterinary investigations identified the broiler flock to be free-range, next to a river and duck pen, reinforcing the suspicion of wild bird origin for both viruses which may have been co-circulating in ducks. It could not, however, be established as to whether there were separate introductions of the two viruses or whether there had been a single co-introduction of the viruses. The described case highlights the value of integrated surveillance and laboratory approaches, including veterinary field investigations, international standards and definitions of notifiable avian disease, validated RRT-PCR assays, and virus isolation in achieving rapid and accurate diagnostic results.

Serologic Status of Newcastle Disease in Native Chickens by Hemagglutination Inhibition Test

Newcastle disease (NCD) is a poultry disease caused by avian Paramyxovirus type 1, characterized by gastrointestinal, respiratory and neurological symptoms. The study established the prevalence of NCD in native chickens and evaluated the protection levels of vaccinated chickens. Blood serum samples were subjected to hemagglutination inhibition test. A total of 75 blood samples were collected from five sites in Davao City: 60 samples from four unvaccinated native chicken farms, and 15 from a vaccinated broiler farm. Results showed seven (7) unvaccinated native chickens with positive titer levels ranging from 2 to 32, of which two(2) were considered significant, indicating protection even without an elicited immune response. This cannot be simply attributed to environmental factors considering uniform exposure of other individuals to similar conditions but exhibited no positive titers. The significant titer count of vaccinated samples ranging from 16 to 128 is attributed to their vaccination history. Differences in titer levels despite similar vaccine administration indicate a disparity in levels of protection due to different individual antibody immune responses, and efficacy of vaccines. Analysis by Chi-square goodness of fit test showed no difference in the titer levels of native chickens, which was expected as they did not have previous exposure to NCD and most had no titers.

Genotype Diversity of Newcastle Disease Virus in Nigeria: Disease Control Challenges and Future Outlook.

Newcastle disease (ND) is one of the most important avian diseases with considerable threat to the productivity of poultry all over the world. The disease is associated with severe respiratory, gastrointestinal, and neurological lesions in chicken leading to high mortality and several other production related losses. The aetiology of the disease is an avian paramyxovirus type-1 or Newcastle disease virus (NDV), whose isolates are serologically grouped into a single serotype but genetically classified into a total of 19 genotypes, owing to the continuous emergence and evolution of the virus. In Nigeria, molecular characterization of NDV is generally very scanty and majorly focuses on the amplification of the partial F gene for genotype assignment. However, with the introduction of the most objective NDV genotyping criteria which utilize complete fusion protein coding sequences in phylogenetic taxonomy, the enormous genetic diversity of the virus in Nigeria became very conspicuous. In this review, we examine the current ecological distribution of various NDV genotypes in Nigeria based on the available complete fusion protein nucleotide sequences (1662 bp) in the NCBI database. We then discuss the challenges of ND control as a result of the wide genetic distance between the currently circulating NDV isolates and the commonest vaccines used to combat the disease in the country. Finally, we suggest future directions in the war against the economically devastating ND in Nigeria.

Chimeric Newcastle disease virus-vectored vaccine protects chickens against H9N2 avian influenza virus in the presence of pre-existing NDV immunity.

A chimeric Newcastle disease virus (NDV) vector (NDV/AI4-TFHN) was constructed with the replacement of the ectodomains of the fusion and hemagglutinin-neuraminidase proteins by those from avian paramyxovirus type 2. The chimeric virus induced high antibody response in chickens pre-immunized with NDV. A recombinant vaccine candidate, NDV/AI4-TFHN-H9, expressing the hemagglutinin of H9N2 avian influenza virus, was generated, on the basis of the chimeric NDV vector mentioned above. The NDV/AI4-TFHN-H9 vaccine elicited H9-specific hemagglutination inhibition antibodies in chickens pre-immunized with NDV vaccine, and reduced the numbers of chickens shedding virus after H9N2 challenge. NDV/AI4-TFHN-H9 could serve as an alternative vaccine for the prevention of H9N2 infection in commercial poultry flocks.

Genetic Analysis of a Commercial Egg Laying Line Challenged With Newcastle Disease Virus.

In low income countries, chickens play a vital role in daily life. They provide a critical source of protein through egg production and meat. Newcastle disease, caused by avian paramyxovirus type 1, has been ranked as the most devastating disease for scavenging chickens in Africa and Asia. High mortality among flocks infected with velogenic strains leads to a devastating loss of dietary protein and buying power for rural households. Improving the genetic resistance of chickens to Newcastle Disease virus (NDV), in addition to vaccination, is a practical target for improvement of poultry production in low income countries. Because response to NDV has a component of genetic control, it can be influenced through selective breeding. Adding genomic information to a breeding program can increase the amount of genetic progress per generation. In this study, we challenged a commercial egg-laying line with a lentogenic strain of NDV, measured phenotypic responses, collected genotypes, and associated genotypes with phenotypes. Collected phenotypes included viral load at 2 and 6 days post-infection (dpi), antibody levels pre-challenge and 10 dpi, and growth rates pre- and post-challenge. Six suggestive QTL associated with response to NDV and/or growth were identified, including novel and known QTL confirming previously reported associations with related traits. Additionally, previous RNA-seq analysis provided support for several of the genes located in or near the identified QTL. Considering the trend of negative genetic correlation between antibody and Newcastle Disease tolerance (growth under disease) and estimates of moderate to high heritability, we provide evidence that these NDV response traits can be influenced through selective breeding. Producing chickens that perform favorably in challenging environments will ultimately increase the supply of quality protein for human consumption.

Genotypic characterisation of Avian paramyxovirus type-1 viruses isolated from aquatic birds in Uganda

Avian paramyxovirus type-1 (APMV-1) viruses of the lentogenic pathotypes are often isolated from wild aquatic birds and may mutate to high pathogenicity when they cross into poultry and cause debilitating Newcastle disease. This study characterised AMPV-1 isolated from fresh faecal droppings from wild aquatic birds roosting sites in Uganda. Fresh faecal samples from wild aquatic birds at several waterbodies in Uganda were collected and inoculated into 9–10-day-old embryonated chicken eggs. After isolation, the viruses were confirmed as APMV-1 by APMV-1-specific polymerase chain reaction (PCR). The cleavage site of the fusion protein gene for 24 representative isolates was sequenced and phylogenetically analysed and compared with representative isolates of the different APMV-1 genotypes in the GenBank database. In total, 711 samples were collected from different regions in the country from which 72 isolates were recovered, giving a prevalence of 10.1%. Sequence analysis of 24 isolates revealed that the isolates were all lentogenic, with the typical 111GGRQGR’L117 avirulent motif. Twenty-two isolates had similar amino acid sequences at the cleavage site, which were different from the LaSota vaccine strain by a silent nucleotide substitution T357C. Two isolates, NDV/waterfowl/Uganda/MU150/2011 and NDV/waterfowl/Uganda/MU186/2011, were different from the rest of the isolates in a single amino acid, with aspartate and alanine at positions 124 and 129, respectively. The results of this study revealed that Ugandan aquatic birds indeed harbour APMV-1 that clustered with class II genotype II strains and had limited genetic diversity.

Molecular characterization and genetic diversity of avian paramyxovirus type 4 isolated in South Korea from 2013 to 2017

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013–2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe.

Genetic Diversity of Avian Paramyxovirus Type 6 Isolated from Wild Ducks in the Republic of Korea.

Eleven avian paramyxovirus type 6 (APMV-6) isolates from Eurasian Wigeon ( n=5; Anas penelope), Mallards ( n=2; Anas platyrhynchos), and unknown species of wild ducks ( n=4) from Korea were analyzed based on the nucleotide (nt) and deduced amino acid sequences of the fusion (F) gene. Fecal samples were collected in 2010-14. Genotypes were assigned based on phylogenetic analyses. Our results revealed that APMV-6 could be classified into at least two distinct genotypes, G1 and G2. The open reading frame (ORF) of the G1 genotype was 1,668 nt in length, and the putative F0 cleavage site sequence was 113PAPEPRL119. The G2 genotype viruses included five isolates from Eurasian wigeons and four isolates from unknown waterfowl species, together with two reference APMV-6 strains from the Red-necked Stint ( Calidris ruficollis) from Japan and an unknown duck from Italy. There was an N-truncated ORF (1,638 nt), due to an N-terminal truncation of 30 nt in the signal peptide region of the F gene, and the putative F0 cleavage site sequence was 103SIREPRL109. The genetic diversity and ecology of APMV-6 are discussed.

Engineered recombinant protein products of the avian paramyxovirus type-1 nucleocapsid and phosphoprotein genes for serological diagnosis

BackgroundVirulent Newcastle disease virus (NDV, avian Avulavirus-1, APMV-1) induces a highly contagious and lethal systemic disease in gallinaceous poultry. APMV-1 antibody detection is used for surveillance and to control vaccination, but is hampered by cross-reactivity to other subtypes of avian Avulaviruses. Data are lacking concerning the applicability of NDV V proteins as differential diagnostic marker to distinguish vaccinated from virus-infected birds (DIVA strategy).MethodsFull length and C-terminally truncated nucleocapsid (NP) protein, and the unique C-terminal regions of the phospho- (P) and V proteins of the NDV LaSota strain were bacterially expressed as fusion proteins with the multimerization domain of the human C4 binding protein, and used as diagnostic antigens in indirect ELISA.ResultsWhen used as diagnostic antigen in indirect ELISAs, recombinant full-length proved to be a sensitive target to detect seroconversion in chickens after APMV-1 vaccination and infection, but revealed some degree of cross reactivity with sera raised against other APMV subtypes. Cross reactivity was abolished but also sensitivity decreased when employing a C-terminal fragment of the NP of NDV as diagnostic antigen. Antibodies to the NDV V protein were mounted in poultry following NDV infection but also, albeit at lower rates and titers, after vaccination with attenuated NDV vaccines. V-specific seroconversion within the flock was incomplete and titers in individual bird transient.ConclusionsIndirect ELISA based on bacterially expressed recombinant full-length NP compared favorably with a commercial NDV ELISA based on whole virus antigen, but cross reactivity between the NP proteins of different APMV subtypes could compromise specificity. However, specificity increased when using a less conserved C-terminal fragment of NP instead. Moreover, a serological DIVA strategy built on the NDV V protein was not feasible due to reduced immunogenicity of the V protein and frequent use of live-attenuated NDV vaccines.

Molecular detection and characterisation of avian paramyxovirus type 1 in backyard chickens and pigeons in Alzintan city of Libya.

Avian paramyxovirus-1 (APMV-1) is the causative agent of Newcastle Disease which affects many species of birds leading to high mortality and heavy economic losses among poultry industry worldwide. Newcastle disease is endemic in Libya with frequent outbreaks occurring in commercial and backyard poultry. APMV-1 was isolated and characterised during the outbreak in 2013. In current study, we report another Newcastle disease outbreak that emerged in backyard chickens and pigeons in Alzintan city on March 2015. Two viruses were detected in cloacal swabs from backyard chickens, namely APMV-1/Libya/15VIR5368/2015 and APMV-1/Libya/15VIR5371/2015. Genetic sequencing of these viruses revealed the presence of velogenic APMV-1 belonging to genotype VIIi genetically similar to the viruses isolated on 2013. During the same period, neurologic signs and mortality were noticed in pigeons. Samples of brain tissue were tested by rRT-PCR which revealed presence of velogenic APMV-1 belonging to lineage 4A (GKKRKR*F Lin.4A) or genotype VIb. To the best of our knowledge, this is the first report of the detection and molecular characterization of APMV-1 in a pigeon in Libya. The phylogenetic analysis of the F gene showed 86% identity to isolates from Iran and Egypt. This study may indicate the circulation of APMV-1 within backyard birds and pigeons which may present a threat to commercial poultry. Considering these findings, vaccination of backyard birds and pigeons and further epidemiological studies are strongly strongly recommended.

Pigeon paramyxovirus type 1 from a fatal human case induces pneumonia in experimentally infected cynomolgus macaques (Macaca fascicularis)

Although avian paramyxovirus type 1 is known to cause mild transient conjunctivitis in human beings, there are two recent reports of fatal respiratory disease in immunocompromised human patients infected with the pigeon lineage of the virus (PPMV-1). In order to evaluate the potential of PPMV-1 to cause respiratory tract disease, we inoculated a PPMV-1 isolate (hPPMV-1/Netherlands/579/2003) from an immunocompromised human patient into three healthy cynomolgus macaques (Macaca fascicularis) and examined them by clinical, virological, and pathological assays. In all three macaques, PPMV-1 replication was restricted to the respiratory tract and caused pulmonary consolidation affecting up to 30% of the lung surface. Both alveolar and bronchiolar epithelial cells expressed viral antigen, which co-localized with areas of diffuse alveolar damage. The results of this study demonstrate that PPMV-1 is a primary respiratory pathogen in cynomolgus macaques, and support the conclusion that PPMV-1 may cause fatal respiratory disease in immunocompromised human patients.

Avian Viral Pathogens in Swallows, Zimbabwe : Infectious Diseases in Hirundinidae: A Risk to Swallow?

We sampled 417 swallows in a wetland ecosystem of Zimbabwe in February 2010 and October 2011. RT-PCR tests revealed circulation of avian paramyxovirus type I, avian influenza and West Nile disease viruses in these populations. We discuss the relevance of these findings in relation to what is known on the epidemiology of these viruses in these hosts and in relation to the host ecology. We conclude with recommendations to focus more research on Passeriformes in disease ecology and in particular on the hirundinidae family.

Respons Imun Ayam Petelur Pascavaksinasi Newcastle Disease dan Egg Drop Syndrome

Some viral diseases in poultry could lead to huge losses to the farmers. Newcastle Disease (ND) and Egg Drop Syndrome (EDS) are a group of infectious viral disease that can cause the decreasein egg production. Newcastle Disease is caused by Avian paramyxovirus type 1 (PMV-1) Paramyxoviridae family. The causative agent of EDS is Duck adenovirus-I Adenoviridae family. Both of these diseases affect the economic losses to the poultry. The main action to prevent hens from ND and EDS virus diseases is vaccination. The success ofvaccination can be tested by serology. ND and EDS virus characteristically agglutinate hen’s erythrocyte they have Hemagglutine protein on virus envelope so can be tested by hemagglutination. The study was conducted ona commercial poultry farm in order to determine the success of vaccination against ND and EDS. The hens were vaccinated by Newcastle Disease-Infectious Bronchitis- Egg Drop Syndrome (ND-IB-EDS) inactivated vaccines.Serological test was conducted in pre and post vaccination by using microtiter hemagglutination test. The antibody titre is expressed in units of HI log2. The results of the study, the mean antibody titer against ND pre vaccinationwas 4,53 ± 1,356 HI log2 and antibody titre in 2nd, 3rd and 4th week were 8,67 ± 0,617 HI log2, 7,73 ± 1,335 HI log2 and 5,20 ± 0,862 HI log2 post vaccination. Antibody titre against EDS pre vaccination was 0 ± 0,000 HI log2 and antibody titre post vaccination in 2nd, 3rd and 4th week were 7 ± 1,363 HI log2, 7,27 ± 1,438 HI log2 and 7,6 ± 1,056 HI log2. It showed that ND-IB-EDS inactivated vaccines is serological protective for ND and EDS titres.

Complete Genome Sequence of an Avian Paramyxovirus Type 4 Strain Isolated from Domestic Duck at a Live Bird Market in South Korea.

ABSTRACTWe report here the first full-genome sequence of an avian paramyxovirus type 4 (APMV-4) strain isolated from a domestic mallard duck at a live bird market in South Korea. Phylogenetic analyses provide genetic information on a new genetic clade, APMV-4, isolated from a domestic duck and evidence of APMV-4 exchange between poultry and wild birds.

Characterization of Colombian serotype 1 avian paramyxoviruses, 2008-2010.

Newcastle disease (ND) still remains one of the most important diseases affecting domestic poultry in Colombia. Here, for the first time, we report on the molecular characterization of 12 virulent and 12 avirulent or lentogenic avian paramyxovirus type 1 (APMV-1) strains that were isolated from commercial, backyard, and game poultry in Colombia from 2008 to 2010. The 12 virulent isolates had a fusion (F) protein cleavage site with basic amino acids at positions 113, 115, and 116 and a phenylalanine at position 117 (112RRQKR*F117), characteristic of virulent strains. The remaining 12 isolates had the F protein cleavage sites 112GKQGR*L117 or 112GRQGR*L117 typical of avirulent or lentogenic APMV-1 strains. Phylogenetic analysis of full-length F genes of all isolates was performed, and based on the recently proposed criteria for classification of APMV-1 strains, the 24 Colombian isolates were found to belong to class II viruses and clustered into four different genotypes. Ten virulent isolates clustered with genotype VII (sub-genotype VIId), seven lentogenic strains within genotype II, five lentogenic strains with genotype I (sub-genotype Ia), and two virulent isolates within genotype XII. Our data provide essential information on the genetic diversity of AMPV-1 isolates circulating in Colombia.

Emergence of a deviating genotype VI pigeon paramyxovirus type-1 isolated from India.

Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic variant of avian paramyxovirus type 1 (APMV-1), which infects pigeons. The virus causes high morbidity and mortality, creating an alarming state for the poultry industry. The present work describes the molecular and pathogenic characterization of a PPMV-1 strain isolated from pigeon in Bhopal, India. Complete genome sequence analysis revealed a genome of 15,192 nucleotides encoding six genes organized in the order 3'-N-P-M-F-HN-L-5'. The fusion gene sequence analysis showed the presence of multiple basic amino acids 112R-R-Q-K-R-F117 at the cleavage site corresponding to pathogenic strains. The mean death time and intracerebral pathogenicity index values indicated a mesogenic nature for the PPMV-1 isolate. On phylogenetic analysis, the strain clustered with genotype VI viruses, including isolates from pigeon and dove. The Bhopal strain showed significant intra and inter-genotype evolutionary distance, suggesting the emergence of a new sub-genotype, VIj.