Molecular epidem iology and phylogeny of N ipah virus infection: A m ini review

Silvia Angeletti， Alessandra Lo Presti， Eleonora Cella，3， Massimo Ciccozzi*

1Unit of Clinical Pathology and Microbiology， University Campus Bio-Medico of Rome， Italy2Department of Infectious Parasitic and Immunomediated Diseases， Istituto Superiore di Sanità， Rome， Italy3Public Health and Infectious Diseases， Sapienza University， Rome， Italy



Molecular epidem iology and phylogeny of N ipah virus infection: A m ini review

Silvia Angeletti1， Alessandra Lo Presti2， Eleonora Cella2，3， Massimo Ciccozzi2*

1Unit of Clinical Pathology and Microbiology， University Campus Bio-Medico of Rome， Italy
2Department of Infectious Parasitic and Immunomediated Diseases， Istituto Superiore di Sanità， Rome， Italy
3Public Health and Infectious Diseases， Sapienza University， Rome， Italy

AR T ICLE IN FO

Article history:

Received 15 April 2016

Received in revised form 16 May 2016 Accepted 15 June 2016

Available online 20 July 2016

Keywords:

Nipah virus

Phylogenetic analysis

Molecular epidemiology NiV reservoir

ABSTR ACT

Nipah virus （NiV） is a member of the genus Henipavirus of the fam ily Paramyxoviridae，characterized by high pathogenicity and endem ic in South Asia. It is classified as a Biosafety Level-4 （BSL-4） agent. The case-fatality varies from 40%-70% depending on the severity of the disease and on the availability of adequate healthcare facilities. At present no antiviral drugs are available for NiV disease and the treatment is just supportive. Phylogenetic and evolutionary analyses can be used to help in understanding the epidemiology and the temporal origin of this virus. This review provides an overview of evolutionary studies performed on Nipah viruses circulating in different countries. Thirty phylogenetic studies have been published from 2000 to 2015 years， searching on pub-med using the key words ‘Nipah virus AND phylogeny’ and twenty-eight molecular epidem iological studies from 2006 to 2015 have been performed， typing the key words ‘Nipah virus AND molecular epidem iology’. Overall data from the published study demonstrated as phylogenetic and evolutionary analysis represent prom ising tools to evidence NiV epidem ics， to study their origin and evolution and finally to act with effective preventive measure.

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E-mail: massimo.ciccozzi@iss.it

1. Introduction

Nipah virus （NiV） is member of the genus Henipavirus in the fam ily Param yxoviridae. Due to its highly pathogenicity and relative new finding， it is classified as a Biosafety Level-4 （BSL-4）agent. Moreover， the Centers for Disease Control and Prevention （CDC） and the National Institute of A llergy and Infectious Diseases （NIAID） have classified NiV as a Category C priority pathogen.

Nipah virus disease is a recently discovered zoonotic disease characterized by fever， constitutional symptoms， and encephalitis，sometimes accompanied by respiratory illness. NiV has an envelope with filamentous nucleocap- sids［1］， the genome consists of a single-stranded negative-sense RNA of approximately 18.2 kb. The genome encodes for six major structural proteins: nucleocapsid （N）， phosphoprotein （P）， ma- trix protein （M）， fusion protein （F），glycoprotein （G）， and large protein or RNA polymerase （L）［2］.

The name ‘Nipah virus’ originated from Sungai Nipah （Nipah River Village）， where the first isolates were obtained［3-5］. Bats of the genus Pteropus appear to be the natural reservoir of the virus. Nipah virus swept through numerous piggeries in Malaysia and killed 1 100 people during the period from 1998 through 1999.

NiV was identified as the etiological agent responsible of an outbreak， in pigs and humans， in Malaysia and Singapore. Transm ission may be from consumption of contam inated foodby bats secretion， or contact with infected pigs. Another way can be human-to-human spread. Since 1998 there have been several cases of infections in Bangladesh and India［7-19］. The case-fatality varies from 40%-70% depending on whether encephalitic or severe manifestations are noted and whether adequate healthcare facilities are available. At present there is no antiviral drug available for Nipah virus disease and the treatment is supportive. Ribavirin has been used in few patients but its efficacy for Nipah virus disease has not yet been determined. Because of the lack of effective vaccines or therapies and the fact that NiV can infects animals such as pigs， NiV infection can be considered an emerging disease and a public health issue［13，15］.

Since NiV is considered an important pathogen， especially in South Eastern regions， phylogenetic， and evolutionary analyses can be used to help in understanding the epidem iology and the temporal origin of this virus. This review provides an overview of evolutionary studies performed on Nipah viruses circulating in different countries.

2. Phylogenetic analysis

Evolutionary analysis， over the last three decades， has increasingly been applied to the study of m icrobial pathogens. Phylogenetic and phylodinamic analysis are the fundamental tools to investigate how the genealogy of a pathogen population is influenced by the interaction between pathogen’s demographic history and environmental， ecological and host immunological factors［20，21］. Phylogenetics and phylodinam ics are a branch of molecular biology evaluating taxonomy and species evolution［22］. These methodologies are used as a complement to the ‘classical epidem iology’［23］ and represent powerful tools w idely used to analyze epidemics especially，in particular settings such as in case of nosocom ial outbreaks. By phylogenetic and evolutionary analysis， factors contributing to the evolution of novel and emerging microbial variants can be identified. In recent years， a number of methods that infer phylogenetic trees based on genetic distances， evolutionary parsimony， Maximum-Likelihood and Bayesian theory， have been introduced［24-27］. Genetic distances and phylogenetic trees （coupled with a correct epidem iological design i.e.， cross sectional studies）， inferred via different sequence evolutionary models and model selection criteria，are normally used to assign the genotype［28］. Coalescent theory and the molecular clock hypothesis are instead used to study the ancestral relationships of individuals sampled from a population （i.e.，longitudinal studies） which can be inferred from a gene genealogy （phylogenetic tree）［29-34］.

A deductive and normally used cycle in phylogenetic analysis was started with m icroorganism isolation and sequencing， and an appropriate data set have to be built. The alignment with reference sequences， manual editing to delete ‘indels’ （insertions/deletions），

and the determ ination of the phylogenetic signal is required. Phylogenetic and/or phylodinam ic analyses represent the ‘core’of the data analysis and hypothesis testing. To test for the best substitution model， to infer phylogeny using different algorithms （e.g.， genetic distance， Maximum Likelihood， Bayesian methods），to test the trees reliability （e.g.， by bootstrapping and posterior probability）， are essential steps for evolutionary analyses［31］.

The analytical power of the phylogenetic and Bayesian methods available today should prompt the researchers to use dataset as large as possible to monitor the epidem iological changes of the m icroorganism over the time. Perform ing phylogenetic analyses on the gene region and sometimes， when available， on the whole genome， may result in a better identification of novel subtypes or recombinants. Moreover， whenever it is possible， combined sequencing and phylogenetic analysis should always be used in order to gain information about the starting of the epidem ic， its spread and the dynamics of viral strains.

Finally， phylogeographic methods can provide information about the spread of viral strains between different geographic regions. Phylogenetic analysis， can also be applied to define and characterize the possible viral vector， as it was identifying NiV in Bats so as in other hosts［35］.

Monitoring the genetic evolution of NiV represents an essential strategy to control the local as well as global epidem ic and to develop efficient preventive and therapeutic strategies with a great impact in clinical practice.

3. Phylogenetic studies

About thirty phylogenetic studies on NiV have been published from 2000 to 2015 years， with weak peak in 2012， typing on pub-med the key words ‘Nipah virus AND phylogeny’， as reported in Figure 1. About twenty-eight molecular epidem iological studies from 2006 to 2015 have been performed， with weak peak in 2010， typing the key words ‘Nipah virus AND molecular epidemiology’， as in Figure 1. The first phylogenetic studies were performed to investigate the similarity between NiV and Hendra virus， another member of the fam ily Param ixoviridae［13，31，36，37］. These studies demonstrated the strong sim ilarity betw een the two viruses， encouraging authors to include a new genus in the fam ily Param ixoviridae，named Henipavirus， consisting only of NiV and Hendra viruses. Phylogenetic studies were performed to follow and characterize the first important epidem ics， the Malaysia in the 1999 and the Bangladesh one in 2004. Interestingly， some authors sequenced the whole NiV genome from strains isolated in the Malaysian outbreak of the year 1999. NiV genome， although 12 nucleotide longer than the Hendra Virus genome， was identical w ithin the regulatory genetic regions and the predicted aminoacid sequence of structural proteins and RNA polymerase［38］.

Figure 1. Number of publications on phylogeny of NiV by year of publication.

Advances from these studies gave the opportunity to evidence that， during the Malaysian epidem ic， at least two major strains of NiV were circulating in pigs， one strain from the initial outbreak in the north and the other strain from the subsequent outbreak，approximately 4 months later in the south. The isolates from the south had identical sequences to those detected from human infections， which confirmed that infections occurred in humans during the southern outbreak originated from infected pigs. This finding implied that the 1998 Malaysia outbreak was probably due at least to two different origins of Nipah virus infections［39］. The genome of NiV from the outbreak of Bangladesh was 6 nucleotide longer than the prototype Malaysian strain and the phylogenetic analysis demonstrated that this virus should represent a new strain of NiV， strictly correlated to the Malaysian strain， but exhibiting a higher interstrain nucleotide heterogeneity［40］. These findings could suggest differences in the way of transmission of the virus between the two countries: in Malaysia， the phylogenetic analysis suggested that at least two introductions of NiV into pigs have been occurred， whereas in Bangladesh the sequence heterogeneity observed should indicate multiple introductions of the virus in humans from different colonies of fruit bats. From this study emerged that NiV circulating in different areas have specific genetic characteristics and may have coevolved with the local natural reservoirs. This coevolution was further supported by Halpin et al in 2007. These authors demonstrated the phylogenetic relationship between bats and their associated virus suggesting an important role of bats as the reservoir hosts of new ly emergent viruses， such as Nipah virus， Hendra virus，and severe acute respiratory syndrome-like coronaviruses［41］.

In the year 2010， Rahaman et al.［42］ demonstrated that the putative reservoir for the 1998 NiV outbreak occurred in Malaysia was Pteropus vampyrus （P. vampyrus） bat. The virus isolated in bats resulted monophyletic with previous NiV and the phylogenetic analysis enforced the hypothesis that sim ilar strains were cocirculating in sympatric reservoir species.

In 2011， an intra-familial NiV outbreak in West Bengal region of India， was described by Arankalle et al.［43］. The full-genome sequence of the virus showed 99.2% of nucleotide and 99.8% am inoacid sim ilarity with the Bangladesh-2004 isolate， suggesting a probable common source of the virus. Phylogenetic analysis，interestingly， showed that viruses from Bangladesh and India clustered and diverged from the viruses of Malaysia.

Lo et al.［44］ in the year 2012 reported the molecular phylogenetic analysis of available complete NiV gene sequences including those from the outbreaks in Bangladesh during 2008 and 2010. These authors proposed a genotyping scheme based on a 729-nt sequence w indow， localized in the N term inal region of the genome， but with a sequence variability comparable to that observed using the complete genome. This genotyping method produced a phylogenetic tree with high bootstrap values and proved to be a relatively accurate indicator of overall nucleotide variability useful for NiV sequences classification.

One of the most recent phylogenetic study on NiV infection，was performed by Lo Presti et al.［45］ to investigate the genetic diversity of the virus， to estimate the date of origin and the spread of the infection. For the first time， these authors demonstrated，using the time-scaled phylogenetic analysis， with the root of the tree originated in 1947 when the virus entered in south eastern Asiatic regions. At the phylogenetic analysis the nucleocapsid gene sequences segregated in two main clades， indicating two different introductions: one in 1995 corresponding and the other in 1985. The phylogeographic reconstruction indicated that the epidemic followed two different routes spreading to the other locations facilitated by bats of the Pteropus genus that are able to travel to long distances. The molecular evolutionary approach was used by these authors to investigate also the presence of sites under positive and negative selection， using a selective pressure analysis method［45］. Only negatively selected sites were detected confirming the stability of theviral protein studied. Interestingly， some of these negatively selected sites were found in positions previously described as important interaction sites［46］. Even if a large proportion of am ino acids are invariable， the occurrence of adaptive at certain sites of the genome，over the time， cannot be excluded， especially if infected pigs trade and bats m igration are not adequately monitored. This situation is in analogy with Chikungunya virus where only one nutation determined a change of vector from Aedes aegypti to Aedes albopictus［47］.

4. Nipah virus reservoirs

Paramyxoviruses are characterized by broad host range and for this reason they show an important zoonotic potential， like Hendra and Nipah viruses originating from bats. Bats represent the most successful mammals on earth including about 1 200 chiropteran species distributed worldw ide. In the last decades Hendra virus，Nipah virus and other zoonotic viruses like Ebola， Marburg， and SARS virus， have been identified in various Pteropus spp. fruit bats［48-52］.

The route of infection of NiV from bats to humans is by ingestion and consumption of NiV-contaminated or partially eaten fruits， or by contact with infected animals such as pigs， cattle and goats.

Rahman et al. in 2010［53］， reported the results of a prospective cohort study focused on a group of P. vampyrus flying foxes captured in two different locations in Malaysia. Authors showed that NiV detected in P. vampyrus differs from all known isolates from Malaysia for the amino acid changes at 44 positions. The phylogenetic analyses unequivocally showed that NiV P. vampyrus forms a monophyletic clade with other NiV isolates from Malaysia，but it differs from human， pig， and Pteropus hypomelanus bat isolates. When 56 NiV sequences from Pteropus lylei bats isolated in Thailand were included， NiV P. vampyrus phylogenetically grouped most closely with NiV Pteropus lylei， and the monophyly of NiV sequences from Malysia was lost. This close homology suggested that NiV is naturally transm itted between these two species. From this study， the presence of NiV diversity in isolates from Pteropus lylei bats has also emerged. This diversity demonstrated that multiple strains co-circulate with in populations and that the ecology and sympatry of Pteropus spp.， not coevolutionary patterns， are determ inant for the NiV strain diversity observed in reservoir hosts. In 2012， Yadav et al.［54］ have surveyed the Indian states of Maharashtra and West Bengal to evaluate the presence of viral RNA and IgG against NiV in different bat populations belonging to the species Pteropus giganteus， Cynopterus sphinx and Megaderma lyra. Authors found NiV RNA in Pteropus bat thus suggesting it may be a reservoir for NiV in India. Furthermore， the phylogenetic analysis demonstrated that two phylogenetic lineages were formed for NiV sequences， one including Bangladesh and India sequences and the other Malaysia and Cambodia sequences. By phylogenetic analysis it was unm istakable confirmed that the same NiV strain circulates in India and Bangladesh and that it was different from that circulating in Malaysia and Cambodia. In the same period， a similar study was performed on free-ranging European insectivorous bats to assess the presence of paramyxovirus infection in these animals［55］. The study involved 120 deceased bats of 15 different European species. Bayesian reconstruction of phylogenetic trees was performed in concordance with the current proposals of Paramyxoviridae taxonomy. Interestingly， the phylogenetic analysis confirmed the presence of the first three paramyxoviruses in European insectivorous bats. The genetic distance between these three novel paramyxoviruses and the closest related member resulted higher than that observed in other members w ithin the paramyxovirus genera. This data suggested that all three viruses m ight be considered as new paramyxoviruses. Since， infected bats were found in close proximity to heavily populated human areas， a potential risk for a zoonotic paramyxovirus infection in Europe cannot be excluded.

Recently， the occurrence of Henipaviruses in fruit bat populations in the north of Australia was explored［56］. In particular， these Authors evaluated the possibility that NiV were restricted to the west of Wallace’s Line. This line represents the biogeographic barrier existing between the Australo-Papuan and Wallacean region on the one hand， and Southeast Asia on the other， with different distribution of vertebrates and invertebrates. Data from this study demonstrated the presence of Nipah virus in both P. vampyrus and Rousettus amplexicaudatus the fruit bat populations localized on the eastern side of Wallace’s Line.

5. Conclusions

Nipah virus causes a recently discovered zoonotic disease endemic in South Asia， where sporadic outbreaks have been reported in Malaysia， Singapore， India， and Bangladesh. The case-fatality varies from 40%-70% depending on the severity of the clinical manifestations， such as encephalitis， and on the availability of adequate healthcare facilities. At present there is no antiviral drug available for Nipah virus disease and the treatment is just supportive. NiV infection can be considered an emerging disease and a public health problem［13，14］ as a consequence of the lack of effective vaccines and therapies and of the evidence that NiV can infect pigs［13，14］. Phylogenetic and evolutionary analyses can represent very useful tools to elucidate the epidem iology and the temporal origin of this virus. Moreover， these analyses， especially the evolutionary analysis， could be advantageous to develop new therapy， vaccine and prevention strategies.

The circulation of NiV may be influenced by the presence of genetic polymorphisms along the virus genome. As a consequence，the antigenic variability is possible and may play an important role in the ability of the virus to escape the host immune response［44］.On this basis， monitoring w ill be important to implement possible intervention strategies. In Asiatic countries， there is a close contact between animals and humans， especially in rural settings. This aspect represents a vulnerability of Asia for outbreaks caused by zoonotic infections. This vulnerability is further increased by sociocultural beliefs and weak public health infrastructure［57］.

Consequently， the need of a multidisciplinary approach to prevent and control zoonotic infections in this country is evident. Phylogenetic and evolutionary analysis represent promising tools to evidence epidemics， to study their origin and evolution and finally to act with effective preventive measure.

Conflict of interest statement

We declare that we have no conflict of interest.

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doi:Document heading 10.1016/j.apjtm.2016.05.012

*Corresponding author:Massimo Ciccozzi， Department of Infectious， Parasite and Immunomediated diseases， Istituto Superiore di Sanità， Viale Regina Elena， Rome 299， Italy.