Skip to main content
Egyptian Journal of Biological Pest Control
Download PDF

Characterization of alphabaculovirus: HearNPV-IIPR05 isolate infecting Helicoverpa armigera (Hubner) larvae

Egyptian Journal of Biological Pest Control volume 31, Article number: 18 (2021) Cite this article

Abstract

Background

The alphabaculoviruses are lethal pathogens of lepidopteran caterpillars including a polyphagous and globally recognized pest, Helicoverpa armigera (Hubner) infesting economically important agriculture crops worldwide. The biological and molecular characterizations of indigenous nucleopolyhedrovirus of the genus Alphabaculovirus isolated from H. armigera in chickpea fields are described.

Results

The virulence of virus isolate was tested in 3rd instar H. armigera larvae, and LC50 (median lethal concentration) was estimated to be 2.69 × 104 OBs ml−1. The ST50 (median survival time) was 4 days post-inoculation, when the 3rd instar H. armigera larvae were inoculated by OB (occlusion body) concentration equivalent to LC90. An average incubation period of the virus isolate in 3rd instar ranged between 4 and 6 days post-inoculation. The OBs of a virus isolate appeared irregular in shape and variable in size with diameter ranging from 0.57 to 1.46 μm on the longest edge and average of 1.071 ± 0.068 μm (mean ± SE). On the basis of phylogenetic analysis of polh, pif-1, and lef-8 genes, the isolate was found to be a member of the genus Alphabaculovirus. The isolate showed a genetic affinity with species of group II Alphabaculoviruses and appeared to be a group II NPV.

Conclusions

On the basis of molecular phylogeny and associated host insect, this indigenous isolate was designated as HearNPV-IIPR05 isolate, which could be a potential candidate for the biological control of H. armigera infesting legumes and other commercial crops.

Background

The baculoviruses are regarded as potential biological control agents because of their safety to vertebrates, high pathogenicity, host-specificity, and ability to cause epizootics in the insect population. They have been reported worldwide from over 600 host species (Martignoni and Iwai 1986), mostly from the insect species of orders Lepidoptera, Diptera, and Hymenoptera (Herniou et al. 2012). On the basis of genome sequence analysis and associated arthropod host, 4 genera, viz., Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus, were recognized within the family Baculoviridae (Herniou et al. 2012). The genus Alphabaculovirus that included lepidopteran-specific nucleopolyhedroviruses (NPVs) is further classified into group I and group II NPVs, based on the presence of envelope fusion proteins in budded viruses, viz., GP64 and F protein, respectively (Lauzon et al. 2006).

Nearly 38 genes are reported to be conserved among all sequenced genomes of baculovirus till date, and these genes are assigned as core genes (Williams et al. 2016; Blissard and Theilmann 2018). Degenerate primers amplifying conserved genomic regions of a broad phylogenetic range of baculoviruses were used to study the relationship between viruses based on nucleotide variations. Oligonucleotides that amplify partial sequences of baculovirus core genes, polyhedrin encoding a major matrix protein of OBs and late expression factors (lef-8 and lef-9) encoding subunits of baculovirus RNA polymerase from various baculovirus genomes, were commonly used for characterization of baculovirus species (Herniou et al. 2003; Lange et al. 2004; Jehle et al. 2006b; Jose et al. 2013). Few of the genes coding for ODV envelope protein, per os infectivity factors (pif-1 and pif-2) mediating oral infectivity were also reported to be conserved in baculoviruses (Herniou et al. 2003; Ohkawa et al. 2005; Okano et al. 2006), and used for species discrimination.

Baculovirus isolates from diverse ecological regions differed in their biological traits (Erlandson et al. 2007; Ali et al. 2018) due to their evolution over a period of time and space. The variations in their biological traits in terms of pathogenesis, virulence, and speed of kill were reported among the distinct geographical isolates of baculovirus (Rabindra 1992). The genotypic variations within or between baculovirus populations have been reported. Alphabaculoviruses comprised of lepidopteran-specific NPVs isolated from the same host species in different geographical regions indicated heterogeneity in DNA profiles (Gettig and McCarthy 1982). The genetic variants isolated from single host insect differed in their phenotypic traits for pathogenicity and speed of kill (Murillo et al. 2006). The intraspecific genetic diversity among the 8 strains of H. armigera NPV (HearNPV) was identified (Figueiredo et al. 2009), and coexistence of genotypic variants is frequently reported within the natural baculovirus populations (Cory and Myers 2003). The NPVs isolated from Helicoverpa armigera (Hubner) in different locations exhibited significant variations in virulence measured in terms of median lethal concentration (Rabindra 1992). Hence, development of viral biocontrol agents involves selection of naturally occurring region-specific isolates and their virulence against local host-insect population for the effective management of associated host-insect pest.

In the present study, an indigenous baculovirus was isolated from naturally infected H. armigera larvae considering its adaptability to given set of climatic conditions of agroecosystem and virus-host interaction influencing characteristics of concentration-response and survival time of infected host. Biological activity, OB morphology, and phylogeny of 3 core genes (polh, pif-1, and lef-8) of a virus isolate were described.

Methods

Test insect

The culture of H. armigera used in this study was established in the laboratory from the field collected larval population. Larval colonies were provided by chickpea-based semi-synthetic diet as described by Nagarkatti and Satyaprakash (1974). The moths obtained from the field collected larvae were examined by mother moth examination method for every generation to obtain disease-free colony (FAO 2011). The abdominal content from the spent moths was excised and homogenized in sterilized water. A droplet was smeared on a slide and examined under light microscope (× 100) for the presence of disease-causing organisms including baculovirus. All the progenies from infected moths, if any, were discarded. The eggs and larvae obtained from the healthy moths were raised carefully. The disease-free colonies were reared for 3 successive generations under controlled conditions (26 ± 1 °C and 70 ± 5% RH) and a photoperiod (14:10 h light:dark) in BOD incubator, before being used for virus multiplication.

Virus isolate

The virus was isolated from naturally infected larvae of H. armigera collected from chickpea fields in New Research Farm, IIPR, Kanpur (India) (26° 31′ 13.41″ N; 080° 14′ 49.69″ E) on the basis of typical baculovirus-induced symptoms described by Whitlock (1974). The presence of OBs of virus in the cadavers was confirmed by light microscopy. The virus was propagated in the healthy early instar (~ 3rd instar) larvae of H. armigera by establishing infection with OB contaminated artificial diet, and cadavers exhibiting baculovirus symptoms were harvested for extraction of OBs.

Purification of viral occlusion bodies

The OBs from the cadavers were recovered by 2 differential cycles of centrifugation following the procedures of Rabindra et al. (2003) with slight modifications as described below. Infected insect cadavers were homogenized in a small volume of sodium dodecyl sulfate (SDS) (0.1%) and maintained overnight at room temperature. The homogenate was filtered through 4 layers of muslin cloth to remove the insect tissue debris. The filtrate was initially centrifuged at 500 rpm for 1 min to remove larger contaminants, and supernatant was again spun at 5000 rpm twice for 15 min each to pellet the occlusion bodies. The pellet was washed 3 times by distilled water and re-suspended in double-distilled water, and aqueous suspension of virus was preserved at ≤ 4 °C. The OBs were enumerated, using improved Neubauer’s Hemocytometer (0.1 mm depth) (Fein-Optik Jena, Germany) at × 400 magnification for bioassay studies.

Electron microscopy

Morphology of the purified OBs extracted from individual larvae was studied under scanning electron microscope (SEM) (Model: FEI Quanta 250, The Netherlands) and transmission electron microscope (TEM) (Model: Jeol JEM 1400, Japan) at EM Unit-SAIF, CSIR-CDRI, Lucknow (India), and photographed at different magnifications. The shape and average size on the longest edge of OBs were recorded.

Virus bioassay

Biological activity of the virus isolate was tested in 3rd instar larvae of H. armigera infected per os by “diet surface contamination” method (Eberle et al. 2012). To study the concentration-mortality response, 6 concentrations ranging between 3 × 106 and 3 × 102 OBs ml−1 were prepared. About 50 μl of viral suspension was dispensed over the surface of chickpea-based semi-synthetic diet and spread uniformly over the surface using blunt glass rod, which was allowed to dry for about 10 min. Each treatment consisted of 15 pre-starved larvae (for 4 h) including the untreated control. The larvae were maintained individually, and each test was repeated thrice. The bioassay was carried out under controlled conditions at 26 ± 1 °C and 70 ± 5% RH. Observations on mortality were recorded at 24 h interval starting from 4th day post-inoculation, and viral infection was confirmed by smear test. The mortality data were corrected depending upon the mortality in the control following Abbott’s formula (Abbott 1925). Any assays with more than 10% larval mortality in controls were rejected. The corrected mean larval mortality for each concentration was subjected to probit analysis (Finney 1971) for determination of median lethal concentration (LC50) using SPSS statistical program 25.

The time-mortality response was studied in the 3rd instar larvae of H. armigera that was inoculated at 2 concentrations representing LC50 and LC90 previously determined in concentration-mortality assay. A group of 15 larvae, which were pre-starved for 4 h, were inoculated by 50 μl of viral suspension and incubated as described under the concentration-mortality assay. Larvae in controls that had not consumed viral inoculum were incubated at similar conditions, and larval mortality was recorded at every 24 h interval for 10 days. Time-mortality results of individual larvae that died due to viral infection were subjected to Kaplan-Meier estimate for survival analysis using SPSS statistical program 25. A test for significant variation between survival curves was performed by a log rank test (level of significance, P ≤ 0.05).

Phylogenetic analysis of polh, pif-1, and lef-8 genes

Viral DNA was extracted from purified OB suspension according to the procedure described by Woo (2001) with some requisite modifications described hereafter. The purified OBs were re-suspended in 0.1 M sodium carbonate solution. About 0.5 mg ml−1 of proteinase K and 1% SDS was added to the final suspension and incubated at 37 °C for overnight. This was followed by extraction with phenol:chloroform:isoamylalcohol solution (25:24:1). The DNA was precipitated in ice cold absolute ethanol, and the pellet was re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The quality of extracted DNA was confirmed by electrophoresing in 0.8% agarose gel. The coding region of core genes, polyhedrin (polh), per os infectivity factor (pif-1), and late expression factor (lef-8), was targeted to amplify using specific primers. The primer pairs targeting polh and pif-1 genes were synthesized as described previously (Ashika et al. 2017). A set of 3 primer pairs amplifying 2.2 kb of lef-8 gene from HearNPV were designed with published sequences available in the GenBank database of National Center for Biotechnology Information (NCBI) using Primer3 tool (Untergasser et al. 2012) (Table 1). Each polymerase chain reaction (PCR) mixture consisted of 50 ng template DNA sample, 1 U of Taq DNA polymerase, 10× Taq buffer, 2.5 mM MgCl2, 2.5 mM of dNTP mix (GeneiTM), and 5 μl of each forward and reverse primer in a final volume of 50 μl reaction mixture. Amplification of partial sequences of polh, pif-1, and lef-8 genes was done using BioRad Thermal Cycler with an amplification program as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 48 to 60 °C (annealing temperature varied for primer sets based on Tm value and given in Table 1) for 30 s and extension at 72 °C for 45 s, final extension at 72 °C for 10 min, and storage at 4 °C till further usage. The amplicons were checked in 2% agarose gel for their target size amplification.

Table 1 Details of the baculovirus target core genes, primer sequences, and annealing temperature for amplification of DNA fragments
Full size table

The PCR amplicons were purified prior to sequencing and sequenced using both forward and reverse primers by Genei Laboratories (Bengaluru) and Bioserve Biotechnologies (Hyderabad). For each gene, 3 independent amplification and sequencing reactions were performed. The sequence data of polh, pif-1, and lef-8 were assembled into contigs by using contig assembly module present in the BioEdit Software (Hall 1999). The sequences obtained in this study were subjected to similarity search using NCBI BLAST programs. The sequences generated were used to determine the phylogenetic relationships between the isolate under study and other baculovirus species recognized by the International Committee on Taxonomy of Viruses (ICTV). The deduced amino acid sequences of core genes from the isolate and GenBank retrieved sequences were aligned by MUSCLE using MEGA6 software suite. Phylogenetic analysis of aligned amino acid sequences was performed to study the taxonomic relationship of the isolate under study using MEGA6 tool, following UPGMA method (Tamura et al. 2013) with bootstrap of 1000 replications.

Results

Virus isolate

The virus isolated from the naturally infected H. armigera larvae collected from chickpea plant manifested typical signs of virus infection (Fig. 1a). The presence of polyhedral occlusion bodies was detected in the discharged body fluid of cadavers under phase-contrast light microscope at × 400 magnification (Fig. 1b). These POBs exhibited Brownian movement in wet mounts. This isolate of virus established lethal infection when healthy and early instar larvae of H. armigera were fed with purified OB contaminated semi-synthetic diet in the laboratory (Fig. 1c).

Fig. 1
figure1

a Helicoverpa armigera larvae on chickpea showing baculovirus infection. b Phase-contrast image (× 400) of baculovirus OBs. c Virosed larvae of Helicoverpa armigera exhibiting symptoms of nucleopolyhedrovirus (NPV) infection

Full size image

Virulence of the virus isolate

Biological activity of the virus isolate was determined by measuring the concentration-mortality response (LC50) of 3rd instar larvae of H. armigera. The isolate of virus induced lethal infection in the inoculated 3rd instar larvae within 5 or rarely 6 days post-inoculation. Untreated larvae developed normally until pupation. The LC50 value of virus isolate was estimated to be 2.69 × 104 OBs ml−1 (95% fiducial limits, 2.83 × 103 to 2.34 × 105). The slope of the concentration-response line was 0.38 ± 0.12 (χ2 0.45) (Fig. 2).

Fig. 2
figure2

Log (conc.)-probit mortality curve for Helicoverpa armigera larvae at different occlusion body (OB) concentrations

Full size image

To test the time response of H. armigera, a survival time analysis was conducted on 3rd instar larvae inoculated with OBs at concentrations representing LC50 and LC90 values, in contrast to full-range bioassay. The survival rate of larvae differed significantly at different concentrations by log rank test (log rank test; χ2 = 4.32; P ≤ 0.05), and 2 patterns in Kaplan-Meier survival curves were visible (Fig. 3). The larval mortality did not occur before 3 dpi, and probability of survival started to decline at 3 dpi for both concentrations tested. The survival rate for larvae declined gradually, irrespective of OB concentrations received by larvae. The median survival time (ST50) was 4 and 6 dpi for larvae receiving OB concentration equivalent to LC90 and LC50 values, respectively, which differed significantly according to log rank test (P = 0.038). The survival rate of H. armigera larvae was 15 ± 0.08 and 40 ± 0.11% at LC90 and LC50, respectively.

Fig. 3
figure3

Survival function of Helicoverpa armigera larvae at different occlusion body (OB) concentrations

Full size image

OB morphology

A distinguishing feature of baculoviridae is the presence of OBs which are critical for transmission of virus in host insects and survival of the virus outside the host (Blissard and Rohrmann 1990). In the present study, the OBs of virus isolate exhibited irregular shape, a typical character of OBs of NPV belonging to Alphabaculovirus. The OBs in SEM micrograph measured between 0.57 and 1.46 μm in diameter on the longest edge, with an average of 1.071 ± 0.068 μm (mean ± SE, n = 20) (Fig. 4).

Fig. 4
figure4

a SEM and b TEM micrographs of viral occlusion bodies (OBs)

Full size image

Phylogeny of polh, pif-1, and lef-8 genes

Nucleotide sequences of baculovirus core genes, polh, pif-1, and lef-8, were generated to determine the taxonomic position of virus isolate. PCR amplification of polh, pif-1, and lef-8 genes from the present isolate using gene-specific primers resulted in amplicons of 596, 1375, and 2199 bp size, respectively (Fig. 5). The partial nucleotide sequences of 3 genes from the present isolate were submitted in GenBank (Accession Numbers MN603765, MN603764, and MN603763, respectively). The amplicon size obtained in the present study for each gene corroborated with published reports (Jose et al. 2013; Ashika et al. 2017). The full-length ORF of completely sequenced lef-8 genes varied between 2484 and 2757 bp (Pang et al. 2001).

Fig. 5
figure5

PCR amplification of polh, lef-8, and pif-1 gene coding regions. a Amplification of polh gene. M—1 kb ladder; 1 and 2—659 bp (2 replicas). b Amplifications of lef-8 gene. M—50 bp step up ladder; 1—940 bp; 2—855 bp; and 3—949 bp. c Amplification of pif-1 gene. M—50 bp step up ladder; 1 and 2—1392 bp (2 replicas)

Full size image

The top matches of BLASTx queries for the present isolate were species of alphabaculovirus infecting host species of the genus Helicoverpa, indicating genetic closeness with these viruses. The polh gene sequence exhibited more than 99% nucleotide identity with 17 isolates of HearNPV and 6 isolates of HzNPV available in GenBank. The sequences of lef-8 revealed more than 99% homology with 16 isolates of HearNPV and 4 isolates of HzNPV. The pif-1 gene analysis revealed greater than 99% sequence identity with 8 isolates of HearNPV and 3 isolates of HzNPV. All the GenBank isolates exhibiting maximum identity with our isolate belonged to the genus Alphabaculovirus.

The genetic relatedness of the isolate was determined by phylogenetic analysis of concatenated amino acid sequences from 3 core genes (polh, pif-1, and lef-8) of baculoviruses, following UPGMA analysis (Fig. 6). The phylogenetic tree of representative ICTV recognized baculovirus species and our isolate formed 4 distinct clades, viz., Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus, with high bootstrap support and two sub-groups (group I and II NPVs) within Alphabaculovirus. The UPGMA tree of concatenated sequences placed our isolate in a clade with ICTV recognized NPV species belonging to the genus Alphabaculovirus and appeared to be closely related with members of alphabaculovirus. Phylogenetic analysis of sequences further positioned the isolate among the species of group II NPVs. Within this group, the obtained isolate clustered with two NPV species infecting H. armigera belonging to lepidopteran family Noctuidae (with bootstrap support of 100%).

Fig. 6
figure6

Phylogeny of concatenated amino acid sequences of polh, pif-1, and lef-8 genes

Full size image

Discussion

The virus isolate established lethal infection in the healthy larvae of H. armigera when fed with purified OB contaminated semi-synthetic diet. The diseased larvae manifested typical signs of baculovirus infection. Similar symptomatology was documented by Whitlock (1974) and Rowley et al. (2011) in NPV infected H. armigera larvae.

Survival time and proportion of response in treated insects are critical parameters to assess the effectiveness of the virus isolate (van Beek et al. 1988). The average incubation period of virus in 3rd instar larvae was about 4 to 6 dpi. Whitlock (1974) stated 6–7 or rarely 4–9 days of incubation period in Heliothis NPV. The speed of killing a host insect is eventually an estimate of the virulence of virus. Hughes et al. (1983) noted mean survival time ranging from 77.8 to 116.6 h for H. zea infected with NPV of different country of origin. The survival time and survival function of H. armigera larvae were decreased at the higher OB inoculums. Previous studies reported a decrease in larval median survival time of Heliothis zea (van Beek et al. 1988) and H. armigera (Dasgupta et al. 2016) with increase in concentration of virus inoculums. The small slope value of concentration-response line for isolate indicated a great variability in response of larvae within the population in the rate of disease progression (Hughes et al. 1983; Teakle et al. 1986). Differences in biological activity were earlier reported among the baculovirus isolates from distinct geographical regions; however, isolates had overlapping fiducial limits of LC50 (Rabindra 1992; Somasekhar et al. 1993; Geetha and Rabindra 1999). Shapiro and Ignoffo (1970) noticed 56-fold differences in concentration-mortality response among the 34 Heliothis NPV isolates. Hence, the local geographical strain of baculovirus is always preferred due to its adaptability to local environmental conditions and efficacy under a given set of climatic conditions in an agroecosystem. It is well recognized that the geographic origins of both virus and its host insect can affect their interactions particularly the characteristics of concentration-response and survival time of infected host insect (Milks 1997). The difference in virulence among the strains of baculoviruses was reported to be attributed to structure of the virus and its genetic composition (Escribano et al. 1999).

In the present study, the irregularly spherical OBs and their variable diameter confirmed to be of NPVs rather than GVs. Previous investigations on OB morphology had revealed the similar reports. Hamm and Young (1974) noticed the variation in size and shape of polyhedral inclusion bodies of NPV from Heliothis zea. The OBs in the Baculoviridae family measured about 0.15–5 μm (Harrison et al. 2018). The OBs from virosed H. armigera were observed irregular, and diameter ranged between 0.5 and 2.5 μm (Kumar et al. 2011) with a mean diameter of 0.89 (Jacob and Subramanian 1972) and 1.35 μm (Sudhakar and Mathavan 1999).

The polh, pif-1, and lef-8 phylogeny strongly suggested that this isolate was found to be a species of group II Alphabaculovirus. The present results are in concordance with published reports, wherein the viruses infecting caterpillars of Noctuidae family were placed in group II NPVs on the basis of lef-8, lef-9, and polh gene phylogeny (Herniou et al. 2004; Lange et al. 2004; Jehle et al. 2006a, b). The amino acid-based phylogenetic analysis of baculovirus core genes is reported to be more consistent and robust than individual genes (Harrison et al. 2008). The polyhedrin was the first baculovirus gene ever sequenced and one of the most conserved genes of lepidopteran-specific baculoviruses, and sequence data for this gene are available for a large number of baculoviruses hence widely used in phylogenetic analysis (Lange et al. 2004). Pif-1 gene was also reported to be conserved in baculoviruses (Ohkawa et al. 2005). Similarly, lef-8 gene was identified in all sequenced baculoviruses (Herniou et al. 2003). Baculovirus genomics provided an approach for rapid and reliable identification of virus isolates (Herniou et al. 2001). Degenerate nucleotides from comparative analysis of lepidopteran-specific baculovirus genome sequences especially at gene levels such as lef-8, lef-9, and polh were well utilized to demarcate baculovirus species (Jehle et al. 2006a, b) and to classify the baculoviruses.

Conclusion

In the present study, an indigenous alphabaculovirus—HearNPV-IIPR05, specific to H. armigera, was isolated and identified. The molecular phylogeny of baculovirus core genes (polh, pif-1, and lef-8) indicated that the isolate belongs to the genus Alphabaculovirus of Baculoviridae. This isolate could be further utilized for the development of bio-pesticide for managing the H. armigera in legumes and other important host crops.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

NPV:

Nucleopolyhedrovirus

OBs:

Occlusion bodies

GV:

Granulovirus

LC:

Lethal concentration

ST:

Survival time

HearNPV:

Helicoverpa armigera NPV

Polh :

Polyhedrin

Pif :

Per os infectivity factor

Lef :

Late expression factor

ICAR:

Indian Council of Agricultural Research

IIPR:

Indian Institute of Pulses Research

SDS:

Sodium dodecyl sulfate

SEM:

Scanning electron microscope

TEM:

Transmission electron microscope

dpi:

Day post-inoculation

PCR:

Polymerase chain reaction

NCBI:

National Center for Biotechnology Information

BLAST:

Basic Local Alignment Search Tool

ICTV:

International Committee on Taxonomy of Viruses

References

  1. Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–267

    CAS  Google Scholar 

  2. Ali G, Abma-Henkens MHC, van der Werf W, Hemerik L, Vlak JM (2018) Genotype assembly, biological activity and adaptation of spatially separated isolates of Spodoptera litura nucleopolyhedrovirus. J Invertebr Pathol 153:20–29

    PubMed  Article  Google Scholar 

  3. Ashika TR, Ojha R, Jalali SK, Shivalingaswamy TM (2017) Comparative study of hot-start PCR characterized species specific conserved gene regions of a biocontrol agent Helicoverpa armigera nucleopolyhedrovirus with its whole genome. J Entomol Zool Stud 5(4):512–519

    Google Scholar 

  4. Blissard GW, Rohrmann GF (1990) Baculovirus diversity and molecular biology. Annu Rev Entomol 35:127–155

    CAS  PubMed  Article  Google Scholar 

  5. Blissard GW, Theilmann DA (2018) Baculovirus entry and egress from insect cells. Annu Rev Virol 5:113–139

    CAS  PubMed  Article  Google Scholar 

  6. Cory JS, Myers JH (2003) The ecology and evolution of insect baculoviruses. Annu Rev Ecol Evol Syst 34:239–372

    Article  Google Scholar 

  7. Dasgupta S, Singh SR, Das S, Pathak SK, Bhola RK (2016) Molecular detection and pathogenicity of a nucleopolyhedrovirus isolated from looper caterpillar (Hyposidra talaca), a tea pest. 3-Biotech 6(2):245

    PubMed  PubMed Central  Google Scholar 

  8. Eberle KE, Wennmann JT, Kleespies RG, Jehle JA (2012) Basic techniques in virus technology. In: Lacey LA (ed) Manual of techniques in invertebrate pathology, 2nd edn. Academic Press, Cambridge, pp 15–74

    Chapter  Google Scholar 

  9. Erlandson M, Newhouse S, Moore K, Janmaat A, Myers J, Theilmann D (2007) Characterization of baculovirus isolates from Trichoplusia ni populations from vegetable greenhouses. Biol Control 41:256–263

    Article  Google Scholar 

  10. Escribano A, Williams T, Goulson D, Cave RD, Chapman JW, Caballero P (1999) Selection of a nucleopolyhedrovirus for control of Spodoptera frugiperda (Lepidoptera: Noctuidae): structural, genetic and biological comparison of four isolates from the Americas. J Econ Entomol 92(5):1079–1085

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. FAO (2011) The Helicoverpa armigera NPV production manual (Grzywacz D, Rabindra RJ, Brown M, Jones KA, Parnell M). Retrieved from: http://www.fao.org/docs/eims/upload/aggrotech/2011/HaNPVmanual-pt1.pdf on 03 Mar 2019

  12. Figueiredo E, Munoz D, Murillo R, Mexia A, Caballero P (2009) Diversity of Iberian nucleopolyhedrovirus wild-type isolates infecting Helicoverpa armigera (Lepidoptera: Noctuidae). Biol Control 50:43–49

    Article  Google Scholar 

  13. Finney DJ (1971) Probit analysis. Cambridge University Press, Cambridge, p 333

    Google Scholar 

  14. Geetha N, Rabindra RJ (1999) Genetic variability and comparative virulence of some geographic isolates of nuclear polyhedrosis virus of Helicoverpa armigera Hub. In: Ignacimuthu S, Sen A, Janarthana S (eds) Biotechnological application for integrated pest management. Oxford and IBH Publ. Co. Pvt. Ltd., New Delhi, pp 65–78

    Google Scholar 

  15. Gettig RG, McCarthy WJ (1982) Genotypic variation among wild isolates of Heliothis spp. nuclear polyhedrosis viruses from different geographic regions. J Virol 43:174–181

    Article  Google Scholar 

  16. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acid Symposium Series No. 41:95-98.

  17. Hamm JJ, Young JR (1974) Mode of transmission of nuclear-polyhedrosis virus to progeny of adult Heliothis zea. J Invertebr Pathol 24:70–81

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. Harrison RL, Herniou EA, Jehle JA, Theilmann DA, Burand JP, Becnel JJ, Krell PJ, van Oers MM, Mowery JD, Bauchan GR (2018) ICTV Report Consortium-2018. ICTV virus taxonomy profile: Baculoviridae. J Gen Virol 99:1185–1186

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. Harrison RL, Puttler B, Popham HJR (2008) Genomic sequence analysis of a fast-killing isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus. J Gen Virol 89:775–790

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. Herniou EA, Arif BM, Becnel JJ, Blissard GW, Bonning B, Harrison R, Jehle JA, Theilmann DA, Vlak JM (2012) Family: baculoviridae. In: Andrew MQ, Lefkowitz KE, Michael JA, Eric BC (eds) Virus taxonomy-ninth report of the International Committee on Taxonomy of Viruses 163–173, p 1327

    Google Scholar 

  21. Herniou EA, Luque T, Chen X, Vlak JM, Winstanley D, Cory JS, O’Reilly DR (2001) Use of whole genome sequence data to infer baculovirus phylogeny. J Virol 75:8117–8126

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Herniou EA, Olszewski JA, Cory JS, O’Reilly DR (2003) The genome sequence and evolution of baculoviruses. Annu Rev Entomol 48:211–234

    CAS  PubMed  Article  Google Scholar 

  23. Herniou EA, Olszewski JA, O’Reilly DR, Corey JS (2004) Ancient coevolution of baculoviruses and their insect hosts. J Virol 78(7):3244–3251

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Hughes PR, Getting RR, McCarthy WJ (1983) Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus. J Invertebr Pathol 41:256–261

    Article  Google Scholar 

  25. Jacob A, Subramanian TR (1972) Nuclear polyhedrosis on some Lepidoptera. Curr Sci 4:536

    Google Scholar 

  26. Jehle JA, Blissard GW, Bonning BC, Cory JS, Herniou EA, Rohrmann GF, Theilmann DA, Thiem SM, Vlak JM (2006a) On the classification and nomenclature of baculoviruses: a proposal for revision. Arch Virol 151(7):1257–1266

    CAS  PubMed  Article  Google Scholar 

  27. Jehle JA, Lange M, Wang H, Hu Z, Wang Y, Hauschild R (2006b) Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera. Virology 346:180–193

    CAS  PubMed  Article  Google Scholar 

  28. Jose A, Jalali SK, Shivalingaswamy TM, Krishna Kumar NK, Bhatnagar R, Bandyopadhyay A (2013) Molecular characterization of nucleopolyhedrovirus of three lepidopteran pests using late expression factor-8 gene. Indian J Virol 24(1):59–65

    PubMed  PubMed Central  Article  Google Scholar 

  29. Kumar SC, Rao GVR, Sireesha K, Kumar PL (2011) Isolation and characterization of baculoviruses from three major lepidopteran pests in the semi-arid tropics of India. Indian J Virol 22(1):29–36

    PubMed  PubMed Central  Article  Google Scholar 

  30. Lange M, Wang H, Zhihong H, Jehle JA (2004) Towards a molecular identification and classification system of lepidopteran-specific baculoviruses. Virology 325:36–47

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  31. Lauzon HAM, Garcia-Maruniak A, Zanotto PMA, Clemente JC, Herniou EA, Lucarotti CJ, Arif BM, Maruniak JE (2006) Genomic comparison of Neodiprion sertifer and Neodiprion lecontei nucleopolyhedron viruses and identification of potential hymenopteran baculovirus specific open reading frames. J Gen Virol 87:1477–1489

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. Martignoni ME, Iwai PJ (1986) A catalogue of viral diseases of insects, mites, and ticks, 4th edn. Gen. Tech. Rep. PNW-195. US Department of Agriculture, Forest Service, Pacific Northwest Research Station, Portland, p 51

    Book  Google Scholar 

  33. Milks LC (1997) Comparative biology and susceptibility of cabbage looper (Lepidoptera: Noctuidae) lines to a nuclear polyhedrosis virus. Environ Entomol 26:839–848

    Article  Google Scholar 

  34. Murillo R, Elvira S, Munoz D, Williams T, Caballero P (2006) Genetic and phenotypic variability in Spodoptera exigua nucleopolyhedrovirus isolates from greenhouse soils in southern Spain. Biol Control 38:157–165

    CAS  Article  Google Scholar 

  35. Nagarkatti S, Satyaprakash (1974) Rearing Heliothis armigera (Hub.) on artificial diet, vol 17. Technical Bulletin, Commonwealth Institute of Biological Control, Bangalore, pp 169–173

    Google Scholar 

  36. Ohkawa T, Washburn JO, Sitapara R, Sid E, Volkman LE (2005) Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115. J Virol 79(24):15258–15264

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Okano K, Vanarsdall AL, Mikhailov VS, Rohrmann GF (2006) Conserved molecular systems of the Baculoviridae. Virology 344:77–87

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. Pang Y, Yu J, Wang L, Hu X, Bao W, Li G, Chen C, Han H, Hu S, Yang H (2001) Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287:391–404

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. Rabindra RJ (1992) Genetic improvement of baculoviruses. In: Ananthkrishnan TN (ed) Emerging trends in biological control of phytophagous insects. Oxford and FBH Publishing Cooperative Private Ltd., New Delhi, pp 183–186

    Google Scholar 

  40. Rabindra RJ, Swamiappan M, Parthasarathy R, Subramanian S, Kennedy JS, Sathiah N, Rajasekaran B (2003) Isolation and DNA characterization of a nuclear polyhedrosis virus from the looper Boarmia (=Ascotis) selenaria (Lepidoptera: Geometridae). Pest Manage Hort Ecosyst 9(1):49–63

    Google Scholar 

  41. Rowley DL, Popham HJR, Harrison RL (2011) Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests, Helicoverpa armigera, Helicoverpa zea and Heliothis virescens. J Invertebr Pathol 107:112–126

    PubMed  Article  Google Scholar 

  42. Shapiro M, Ignoffo CM (1970) Nuclepolyhedrosis of Heliothis: activity of isolates from Heliothis zea. J Invertebr Pathol 16:107–111

    CAS  PubMed  Article  Google Scholar 

  43. Somasekhar S, Jayapragasam M, Rabindra RJ (1993) Characterization of five Indian isolates of the nuclear polyhedrosis virus of H. armigera. Phytoparasitica 21:333–337

    Article  Google Scholar 

  44. Sudhakar S, Mathavan S (1999) Electron microscopical studies and restriction analysis of Helicoverpa armigera nucleopolyhedrosis virus. J Biosci 24(3):361–370

    CAS  Article  Google Scholar 

  45. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetic analysis version 6.0. Mol Biol Evol 30:2725–2729

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Teakle RE, Jensen JM, Giles JE (1986) Age-related susceptibility of Heliothis punctigera to commercial formulation of nuclear polyhedrosis virus. J Invertebr Pathol 47:82–92

    Article  Google Scholar 

  47. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3 – new capabilities and interfaces. Nucleic Acids Res 40(15):e115

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. van Beek NAM, Wood HA, Hughes PR (1988) Quantitative aspects of nuclear polyhedrosis virus infections in lepidopterous larvae: the dose-survival time relationship. J Invertebr Pathol 51:58–63

    Article  Google Scholar 

  49. Whitlock VH (1974) Symptomatology of two viruses infecting Heliothis armigera. J Invertebr Pathol 23:70–75

    Article  Google Scholar 

  50. Williams T, Bergoin M, Van Oers MM (2016) Diversity of large DNA viruses of invertebrates. J Invertebr Pathol 147:4–22

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  51. Woo SD (2001) Rapid detection of multiple nucleopolyhedroviruses using polymerase chain reaction. Mol Cell 11(3):334–340

    CAS  Google Scholar 

Download references

Acknowledgements

The authors are thankful to the Director, ICAR-IIPR, Kanpur (India), for providing facilities. We also acknowledge Dr. Kalyan Mitra (Principal Scientist) for providing SEM and TEM facilities at Electron Microscopy Unit (Sophisticated Analytical Instrument Facility) of CSIR-CDRI, Lucknow (India). Authors express special thanks to Dr. S.K. Jalali (Former Principal Scientist and Head, Division of GR, ICAR-NBAIR, Bengaluru, India) for his valuable inputs during the study and critical review of the manuscript.

Funding

Not applicable.

Author information

Affiliations

  1. ICAR-Indian Institute of Pulses Research, Kanpur, 208024, India

    Sanjay M. Bandi, P. S. Shanmugavadivel & A. Revanasidda

  2. ICAR-Indian Institute of Farming Systems Research, Modipuram, 250110, India

    Lalit Kumar

Authors
  1. Sanjay M. Bandi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. S. Shanmugavadivel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lalit Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Revanasidda
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SMB designed the study, conducted the experiments, and prepared the manuscript. PSS, LK, and R contributed in conducting the experiments and preparation of manuscript. All the authors read and approved the final manuscript for publication.

Corresponding author

Correspondence to Sanjay M. Bandi.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bandi, S.M., Shanmugavadivel, P.S., Kumar, L. et al. Characterization of alphabaculovirus: HearNPV-IIPR05 isolate infecting Helicoverpa armigera (Hubner) larvae. Egypt J Biol Pest Control 31, 18 (2021). https://doi.org/10.1186/s41938-021-00367-9

Download citation

Keywords

  • Alphabaculovirus
  • Nucleopolyhedrovirus
  • Helicoverpa armigera
  • Phylogeny
  • Virulence
  • Biological control